15000 g for 10 min ) and the supernatant was kept at −80°C prior to analysis .","Three biological replicates were prepared for each cell line and treatment .","Samples were analysed by hydrophilic interaction liquid chromatography coupled to high resolution-mass spectrometry ( LC-MS ) according to a previously published method ( Stoessel et al . , 2016 ) .","All samples were analyzed as a single batch , in randomized order and pooled quality control samples were analyzed regularly throughout the batch to confirm reproducibility .","Approximately 250 metabolite standards were analyzed immediately preceding the batch run to determine accurate retention times to facilitate metabolite identification .","Additional retention times for metabolites lacking authentic standards were predicted computationally as previously described ( Creek et al . , 2011 ) .","Data was analysed using the IDEOM workflow ( Creek et al . , 2011 , 2012 ) .","Peak areas for significant metabolites were confirmed by manual integration with Tracefinder software ( Thermo Scientific ) .","Multivariate statistical analysis utilized principal component analysis ( PCA ) on log-transformed and auto-scaled metabolite peak intensity data using the web-based analytical tool , MetaboAnalyst ( Xia et al . , 2015 ) .","The IDEOM files containing all metabolomics data are uploaded on Figshare and can be accessed at https://figshare . com/s/c38c0a98fb01634677f6 ."]],"string":"[\n [\n \"Malaria is caused by infection of the blood with Apicomplexan parasites of the genus Plasmodium .\",\n \"Critical for the proliferation and survival of Plasmodium in the blood is their ability to quickly penetrate host erythrocytes and acquire nutrients required for rapid growth .\",\n \"To facilitate this , the invasive merozoite forms of Plasmodium spp .\",\n \"sequentially secrete proteins from their apical organelles , the micronemes , rhoptries and dense granules .\",\n \"Proteins localizing to the micronemes and rhoptry neck are implicated in the irreversible attachment of the parasite to the host cell and are critical for invasion ( reviewed in [Harvey et al . , 2012; Weiss et al . , 2016] ) .\",\n \"Dense granule proteins are secreted once Plasmodium parasites have invaded their host cell ( Riglar et al . , 2011 ) , contributing to remodeling of the host cell ( de Koning-Ward et al . , 2016 ) .\",\n \"However , the role of proteins that localize to the rhoptry bulb is less clear and although they have been implicated in roles ranging from rhoptry biogenesis , erythrocyte invasion , formation of the parasitophorous vacuole ( PV ) in which the parasite is encased , as well as modification of the host cell ( Kats et al . , 2006; Counihan et al . , 2013 ) , functional data supporting these roles is very limited .\",\n \"RhopH2 is one of ~15 known proteins that localize to the rhoptry bulb in Plasmodium merozoites ( Counihan et al . , 2013; Ling et al . , 2003 ) .\",\n \"It is found in a high molecular weight complex with RhopH1 and RhopH3 ( Cooper et al . , 1988 ) that is discharged from merozoites , associating with the erythrocyte surface upon merozoite contact ( Sam-Yellowe et al . , 1988; Sam-Yellowe and Perkins , 1991 ) .\",\n \"The localization of RhopH proteins in the newly-infected erythrocyte is less clear as multiple localizations , including the PV membrane ( PVM ) , Maurer’s clefts and the cytosolic face of the erythrocyte membrane have been described for its constituents using different experimental approaches ( Perkins and Ziefer , 1994; Ndengele et al . , 1995; Sam-Yellowe et al . , 2001; Hiller et al . , 2003; Vincensini et al . , 2005 , 2008 ) .\",\n \"RhopH2 and RhopH3 are each encoded by a single gene .\",\n \"In contrast , RhopH1 in P . falciparum , the most pathogenic of the species infecting humans , is encoded by a multi-gene family comprising five variant genes termed clag 2 , 3 . 1 , 3 . 2 , 8 and 9 ( with clag3 . 1 and 3 . 2 mutually exclusively transcribed ) ( Gupta et al . , 2015; Kaneko et al . , 2001 , 2005; Ling et al . , 2004 ) .\",\n \"Of all the RhopH proteins , putative functions have only been assigned for RhopH1/Clag3 and Clag9 ( Gupta et al . , 2015 ) , although there is conflicting evidence for the involvement of Clag9 in cytoadherence ( Trenholme et al . , 2000; Goel et al . , 2010; Nacer et al . , 2011 ) .\",\n \"Via a high throughput drug-screening approach Clag3 has been linked to plasmodial surface anion channel ( PSAC ) activity ( Nguitragool et al . , 2011 ) .\",\n \"PSAC is a type of new permeability pathway ( NPP ) induced in the erythrocyte membrane by Plasmodium spp .\",\n \"that increases the cell’s porosity to organic and inorganic solutes .\",\n \"P . falciparum Clag3 null-mutants exhibit delayed in vitro growth , although NPP activity has not been investigated ( Comeaux et al . , 2011 ) .\",\n \"Intriguingly , Clag3 exhibits no homology to known ion channel proteins and lacks conventional membrane spanning regions to form a pore through the erythrocyte membrane , although it exists as both an integral and peripheral membrane protein in the infected erythrocyte ( Nguitragool et al . , 2011; Zainabadi , 2016 ) .\",\n \"Thus whether Clag3 forms ion channels directly and exclusively or if other parasite proteins or host cell membrane components contribute to a functional NPP is unknown .\",\n \"Alternatively , Clag3 may participate indirectly , for example , by regulating NPP activity .\",\n \"Both the rhopH2 gene and rhopH3 gene are refractory to deletion ( Cowman et al . , 2000; Janse et al . , 2011 ) .\",\n \"As RhopH1 is encoded by a multi-gene family , it is difficult to establish without genetically disrupting all but one clag variant within a parasite , whether the clag genes serve complementary functions or play distinct roles , including in NPP activity .\",\n \"To address these questions , we characterized RhopH2 in P . falciparum and conditionally depleted its expression in P . falciparum and the rodent malaria parasite P . berghei to investigate its contribution to erythrocyte invasion , parasite growth and erythrocyte permeability .\",\n \"Depletion of RhopH2 in cycle one did not affect transition into cycle two , suggesting RhopH2 plays no direct role in invasion .\",\n \"However , NPP activity was greatly reduced and parasite growth slowed as parasites progressed into trophozoite stage in cycle two , possibly due to nutrient depravation and/or accumulation of waste products .\",\n \"Transition into cycle three was curtailed by interesting phenomena including reduced schizont rupture and merozoite malformation that may be linked to reduced de novo pyrimidine synthesis .\",\n \"Taken together , RhopH2 appears to be important for NPP activity and for the exchange of nutrients and wastes with the blood plasma to facilitate parasite growth and proliferation .\"\n ],\n [\n \"Conditional gene knockdown approaches were utilized herein to gain insight into the functional role of RhopH2 in Plasmodium parasites .\",\n \"This involved transfecting pRhopH2-HAglmS into P . falciparum that when correctly integrated into the rhopH2 locus , would lead to incorporation of a triple hemagglutinin ( HA ) and single strep II tag at the C-terminus of RhopH2 and the glucosamine ( GlcN ) -inducible glmS ribozyme ( Prommana et al . , 2013 ) within its 3' untranslated region ( UTR ) ( Figure 1a ) .\",\n \"Diagnostic PCR of transfectants resistant to WR99210 selection after three rounds of drug cycling confirmed that transgenic parasites , termed PfRhopH2-HAglmS , harbored the expected integration event ( Figure 1b ) .\",\n \"This was further validated by western blotting of parasite lysates from clonal PfRhopH2-HAglmS parasites using an anti-HA antibody; RhopH2 typically runs at 140 kDa by SDS-PAGE ( Cooper et al . , 1988; Ling et al . , 2003 ) and the observed 150 kDa band of RhopH2-HA is consistent with its anticipated size ( Figure 1c ) .\",\n \"Immunofluorescence analysis ( IFA ) confirmed RhopH2-HA localized to the rhoptry and co-localized with other rhoptry bulb proteins , RhopH1 , RhopH3 and RAMA but not with the rhoptry neck protein , RON4 , the micronemal marker , AMA-1 or the plasma membrane protein MSP1 ( Figure 1d ) .\",\n \"Comparison of the wildtype 3D7 and RhopH2-HAglmS parasite lines revealed that the addition of the epitope tags and ribozyme sequence did not impact on RhopH2-HAglmS to grow normally ( Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 23217 . 003Figure 1 . Generation of transgenic parasites in which RhopH2 is epitope-tagged .\",\n \"( a ) The P . falciparum RhopH2 targeting construct was designed to integrate into the endogenous locus by a single crossover recombination event .\",\n \"The predicted structure of the endogenous locus before and after integration is shown .\",\n \"Haemagglutinin ( HA ) and strep II ( Str ) epitope tags , selectable marker ( sm ) , glmS ribozyme and untranslated regions ( UTR ) are shown .\",\n \"Arrows indicate oligonucleotides used in diagnostic PCR analysis and indicative product size .\",\n \"( b ) Diagnostic PCR showing the PfRhoph2 gene contains the integrated sequence .\",\n \"Oligonucleotide pairs shown in\",\n \"( a ) were used on genomic DNA prepared from drug-resistant parasites after transfection with the targeting construct ( integrant ) or 3D7 ( WT ) .\",\n \"DO354 and DO228 oligonucleotides , which recognize the rhoph2 locus , serve as a positive control for the PCR .\",\n \"( c ) Western blot analysis showing the integrant line expresses the HA epitope tags .\",\n \"The predicted molecular mass of epitope-tagged RhopH2 is 164 kDa .\",\n \"PfHSP101-HA ( 101-HA ) serves as a positive control .\",\n \"( d ) Immunofluorescence analysis ( IFA ) on schizonts fixed with acetone/methanol and labelled with anti-HA antibody to detect RhopH2 and other antibodies , as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 00310 . 7554/eLife . 23217 . 004Figure 1—figure supplement 1 . Comparison of growth between P . falciparum wildtype ( 3D7 ) and RhopH2-HAglmS parasite lines . Parasite lines were grown in the absence of GlcN for three cycles and growth was measured by calculating the percentage parasitemia ( left panel ) or by lactate dehydrogenase assay ( LDH ) ( right panel ) .\",\n \"Shown is the mean ± SD fold-increase in parasitemia or LDH activity ( n = 6 independent biological replicates ) .\",\n \"An unpaired t-test was used to calculate statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 004 As RhopH2 has been described to reside at several different locations post-invasion , we took advantage of our RhopH2-HA line to characterize the expression and localization of RhopH2 at different times post-invasion using anti-HA antibodies .\",\n \"Western blot analysis revealed weak expression of RhopH2 during the ring and trophozoite stages , with a peak of expression at schizont stage ( Figure 2a ) , in keeping with when RhopH2 is maximally transcribed ( Ling et al . , 2003 ) .\",\n \"IFA confirmed RhopH2 synthesized during schizogony is carried in during invasion and localizes to the interface between the parasite and host cell ( Figure 2b ) .\",\n \"Weak labeling could also be observed at the erythrocyte membrane .\",\n \"As the parasite matured , RhopH2 could be detected in the erythrocyte cytoplasm , often exhibiting distinct punctate labeling , and the intensity of labeling at the erythrocyte membrane became more pronounced .\",\n \"RhopH2 did not co-localize with SBP1 , a Maurer’s cleft resident protein , indicating RhopH2 is not trafficked to the erythrocyte membrane via these membranous structures as previously suggested ( Sam-Yellowe et al . , 2001; Vincensini et al . , 2005 ) ( Figure 2c ) . 10 . 7554/eLife . 23217 . 005Figure 2 . Expression , localisation and solubility profile of P . falciparum RhopH2 .\",\n \"( a ) Western blot analysis of RhopH2-HA expression across the erythrocytic cycle .\",\n \"Immunoblots were probed with the antibodies as indicated .\",\n \"( b ) Immunofluorescence analysis ( IFA ) on erythrocytes infected with PfRhopH2-HAglmS and fixed with acetone/methanol .\",\n \"RhopH2 is labeled with the anti-HA antibody .\",\n \"The bars represent 5 µm .\",\n \"( c ) IFA on erythrocytes infected with PfRhopH2-HAglmS , fixed with acetone/methanol and probed with anti-HA ( for RhopH2 ) and antibodies to the Maurer’s cleft protein SBP1 show that RhopH2 and SBP1 do not co-localise .\",\n \"( d ) Solubility of RhopH2-HAglmS .\",\n \"Upper panel: Infected erythrocytes were synchronized and saponin-lysed when parasites reached ring ( R ) or schizont ( S ) stage and the pelleted material was sequentially dissolved in the buffers as indicated in the order of left to right ( upper panel ) .\",\n \"Supernatant fractions were analysed by western blotting with the indicated antibodies .\",\n \"Insoluble material represents protein remaining in the pellet fraction after 1% Triton X-100 treatment .\",\n \"Lower panel: Alternatively , infected erythrocytes were saponin-lysed when parasites were at ring stages , split into equal portions and pelleted before dissolving in one of the indicated buffers .\",\n \"Both supernatant ( Sn ) and pellet ( P ) fractions were analysed by western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 005 Although RhopH2 has been shown to be present in detergent resistant membranes at schizont stages ( Sanders et al . , 2007; Hiller et al . , 2003 ) , localizes to the erythrocyte cytosolic face of the PVM ( Hiller et al . , 2003 ) and is present at the erythrocyte membrane , it is unclear how RhopH2 associates with these membranes .\",\n \"The hydrophobic region at I739-H759 is not universally predicted as a conventional transmembrane domain bioinformatically ( eg . TMHMM , SOSUI , TMPred ) .\",\n \"Therefore , we examined the solubility profile of RhopH2 at both schizont and ring-stages .\",\n \"We found that in contrast to EXP2 , which is a component of the Plasmodium translocon of exported proteins ( PTEX ) that resides at the PVM and requires Triton X-100 to be extracted from the membranes ( de Koning-Ward et al . , 2009 ) , the majority of RhopH2 could already be extracted with carbonate when sequential solubility assays were conducted ( Figure 2d , top panel ) .\",\n \"This indicates that RhopH2 is peripherally associated with membranes and is not an integral membrane protein .\",\n \"However , when erythrocytes infected with ring-stage parasites were saponin-lysed , pelleted by centrifugation and resuspended directly ( rather than sequentially ) in various detergents/buffers , RhopH2 could be extracted with carbonate and mostly with urea ( which also extracts peripheral membrane proteins ) , whereas it remained in the Triton X-100 pellet fraction ( Figure 2d , bottom panel ) .\",\n \"Combined , this data indicates that while RhopH2 predominantly has a peripheral association with the membranes at its respective locations , RhopH2 may be interacting with erythrocyte cytoskeletal proteins or is present in lipid rafts during the ring-stages , leading to its insolubility in Triton X-100 when resuspended in this buffer directly .\",\n \"To gain insight into proteins that interact with RhopH2 post-invasion , we next investigated the interactome of RhopH2 in ring and trophozoite stages by immunoprecipitating RhopH2 from PfRhopH2-HAglmS lysates using anti-HA antibodies and identifying proteins that had been affinity purified by mass-spectrometry ( Figure 3a ) .\",\n \"Bead-only and irrelevant protein controls ( Elsworth et al . , 2016 ) were used to identify non-specific interactions including ribosomal , nuclear and cytosolic proteins , which were subtracted to attain a list of likely specific interactions . 10 . 7554/eLife . 23217 . 006Figure 3 . The RhopH2 interactome .\",\n \"( a ) Coomassie-stained SDS-PAGE gel of elution fractions from immune-precipitations performed with HA antibodies on lysates made from erythrocytes infected with RhopH2-HAglmS parasites at ring ( R ) or trophozoite stage ( T ) .\",\n \"( b ) Bar graph showing the total number of peptides of particular subclasses of proteins that were affinity purified with PfRhopH2-HA .\",\n \"( c ) Pie charts showing the number of peptides from the respective RhopH proteins that affinity purified with RhopH2-HA .\",\n \"The numbers of peptides identified are indicated in brackets .\",\n \"Note RhopH1 includes all CLAG peptides .\",\n \"( d ) Western blot of blue-native PAGE performed on erythrocytes infected with trophozoite stage RhopH2-HAglmS parasites that had been solubilized in either 0 . 25% Triton X-100 or 1% ASB detergent reveal RhopH2 is present in ~670 and ~410 kDa species .\",\n \"( e ) Pie chart showing the numbers of the most abundant peptides from PEXEL proteins that affinity purified with PfRhopH2-HA from trophozoite stage parasites .\",\n \"( f ) Pie chart showing the numbers of the most abundant peptides from host erythrocyte proteins that affinity purified with PfRhopH2-HA in ring stage parasites . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 006 In both parasite stages , RhopH2 was pulled down as well as other members of the RhopH complex , relatively few PV proteins such the PTEX complex , many exported PEXEL proteins , especially in trophozoites , and a large number of erythrocyte cytoskeletal proteins , particularly in the ring stages ( Figure 3b ) .\",\n \"That RhopH2 was interacting with the other members of the RhopH complex ( Figure 3c ) is consistent with an earlier report demonstrating the RhopH complex persists intact for at least 18 hr post-invasion ( Lustigman et al . , 1988 ) .\",\n \"There were more peptides recovered for RhopH1 ( particularly Clags3 . 1 , 3 . 2 and 9 ) and RhopH3 than there were for RhopH2 at the ring and trophozoite stages ( Figure 3c ) .\",\n \"Given that predicted molecular weights for the Clags ( 160–171 kDa ) and RhopH2 ( 163 kDa ) are similar but that of RhopH3 is somewhat smaller ( 104 kDa ) , this indicates that each component of the RhopH complex may not be in a 1:1:1 stoichiometry .\",\n \"Blue-Native PAGE gel analysis revealed that RhopH2 is present in a ~670 kDa complex that has a molecular mass larger than the predicted ~425 kDa ( Figure 3d ) .\",\n \"Apart from not being in an equimolar ratio , other non-RhopH proteins may also be present in the ~670 kDa complex .\",\n \"We also observed a smaller ~410 kDa complex when using the zwitterionic detergent 3- ( tetradecanoylamidopropyl dimethylammonio ) propane 1-sulfonate ( ASB-14 ) , which could contain a subset of the RhopH proteins and/or other proteins ( Figure 3d ) .\",\n \"Almost as abundant as RhopH peptides identified from the trophozoite-stage immunoprecipitations were exported proteins ( Figure 3b and\",\n \"e ) .\",\n \"The most predominant peptides from known exported proteins included those of mature parasite-infected erythrocyte surface antigen ( MESA; a protein that interacts with host protein 4 . 1 ) ( Waller et al . , 2003 ) , small exported membrane protein 1 ( SEMP1; a non-essential protein that localizes to the Maurer’s clefts and is partially translocated to the erythrocyte membrane ) ( Dietz et al . , 2014 ) , glycophorin-binding protein 130 ( GBP130; an exported soluble protein ) ( Maier et al . , 2008 ) , a variety of Plasmodium helical interspersed subtelomeric proteins ( PHIST; some of which have been shown to interact with PfEMP1 ) ( Proellocks et al . , 2014; Oberli et al . , 2014 , 2016 ) and HSP70-x ( localizes to J-dots ) ( Külzer et al . , 2012 ) .\",\n \"Peptides from exported proteins were more abundant in the pull-down performed on lysates from trophozoites compared to ring-stages , in keeping with protein export peaking during the trophozoite stage .\",\n \"The exception was ring erythrocyte surface antigen ( RESA ) in which more peptides were observed in the ring-stage pull-down .\",\n \"RESA is one of the first proteins exported into the erythrocyte that ultimately localizes to the ankyrin-band 3 complex at the erythrocyte cytoskeleton .\",\n \"A large number of peptides to erythrocyte cytoskeletal proteins were also identified , including tubulin , spectrin , ankyrin , protein 4 . 1 , band 3 and actin ( Figure 3f ) .\",\n \"Whether RhopH2 is indirectly interacting with these cytoskeletal components via exported proteins , or specifically interacting with all or a subset of these cytoskeletal proteins is unknown , especially since many cytoskeletal elements are bound together in the cell .\",\n \"Taken together , these results indicate that after invasion , the RhopH complex traffics from the PVM to the erythrocyte membrane and either en route or when it reaches its final destination , RhopH2 interacts with a number of exported parasite proteins that also bind to components of the host cytoskeleton .\",\n \"As the epitope-tagged RhopH2 line harbors a glmS riboswitch sequence , the ability to regulate RhopH2 expression in parasites via the addition of GlcN was investigated to gain functional insight into this protein .\",\n \"Erythrocytes infected with synchronized ring-stage parasites were treated for up to two cycles with 2 . 5 mM GlcN and assessed for protein knockdown via western blot ( Figure 4a ) and parasite growth via Giemsa-stained smears relative to parasites grown in the absence of GlcN ( Figure 4b–c ) .\",\n \"RhopH2 is normally transcribed around the onset of schizogony ( Ling et al . , 2003; Bozdech et al . , 2003; Le Roch et al . , 2004 ) and the addition of 2 . 5 mM GlcN resulted in knockdown of RhopH2 expression in schizonts by 84% within the first cycle .\",\n \"By late in the second cycle , RhopH2 protein levels were reduced by 92% in those parasites that made it to schizont stage ( Figure 4a ) .\",\n \"RhopH2-HAglmS parasites ( +GlcN ) appeared morphologically normal by the end of the first cycle ( Figure 4b ) .\",\n \"In separate experiments whereby RhopH2-HAglmS parasites expressing GFP at the end of cycle one were incubated with donor erythrocytes , the conversion of schizonts to ring stage parasites and therefore invasion efficiency was not specifically affected by the knockdown of RhopH2 ( Figure 4c ) .\",\n \"In contrast , a striking growth defect in RhopH2-HAglmS ( +GlcN ) parasites was observed in the second cell cycle around the ring to trophozoite transition stage , with late-ring stage parasites appearing irregular in shape and trophozoites exhibiting an abnormal stunted phenotype rather than progressing to mature trophozoites ( Figure 4b ) .\",\n \"In addition , RhopH2-HAglmS ( +GlcN ) parasites that transitioned to schizonts at the end of the second cycle harboured significantly lower numbers of merozoites per schizont ( mean of 19 merozoites cf 12 merozoites for –GlcN and +GlcN cultures , respectively p<0 . 0001 ) ( Figure 4d ) .\",\n \"Moreover , the time required to complete the second cycle and commence the third cycle was delayed ( ~92 hr cf ~108 hr for -GlcN and +GlcN cultures , respectively ) ( Figure 4b , e ) .\",\n \"This all translated to ~4 fold reduction in the number of ring-stage parasites observed at the beginning of the third cycle when compared to parasites not exposed to GlcN ( Figure 4e ) . 10 . 7554/eLife . 23217 . 007Figure 4 . Reduction in PfRhopH2 expression leads to altered growth phenotypes in vitro .\",\n \"( a ) Effect of glucosamine on PfRhopH2 protein expression .\",\n \"Upper panel: overview of experiment .\",\n \"Synchronised cultures of PfRhopH2-glmS were treated with glucosamine ( GlcN ) at the indicated time and material harvested , as indicated .\",\n \"Lower panels: infected erythrocytes were harvested by saponin lysis and subject to SDS-PAGE and western blotting .\",\n \"PfRhopH2-HA was detected using an anti-HA antibody and EXP2 ( used as a loading control ) detected with a specific polyclonal EXP2 antibody .\",\n \"Right panel: Densitometry performed on bands observed in western blot using ImageJ was performed to calculate the ratio of EXP2 or RhopH2 protein levels in parasite lines grown in the presence ( + ) or absence ( - ) of GlcN ( n = 3 independent experiments ) .\",\n \"Shown is the mean ± SEM ( n = 3 ) .\",\n \"( b ) Representative Giemsa-stained smears parasites depleted of RhopH2 progress to schizont stage in cycle one but parasite growth is slowed around the trophozoite stage ( n = 3 independent experiments ) .\",\n \"( c ) Analysis of the number of schizonts in cultures of wildtype ( 3D7 ) and RhopH2-HAglmS parasites grown in the absence ( − ) or presence ( + ) of 2 . 5 mM GlcN that invaded donor erythrocytes within 3 or 5 hr post-incubation ( hpi ) , as measured by FACS ( n = 3 ) .\",\n \"Shown is the mean ± SEM .\",\n \"( d ) Box plot indicating the number of merozoites formed per schizont in cultures of RhopH2-HAglmS grown in 0 mM ( 35 schizonts examined ) or 2 . 5 mM ( 51 schizonts examined ) GlcN .\",\n \"The central bar in the box plot denotes the median whilst the whiskers delineate the 10th and 90th percentiles .\",\n \"p<0 . 0001 by unpaired t-test .\",\n \"( e ) Parasitemias of cultured PfRhopH2-HAglmS parasites grown in 0 mM or 2 . 5 mM GlcN , determined by counting a minimum of 1000 erythrocytes .\",\n \"Depletion of PfRhopH2 expression increases the length of the cell cycle and has a marked effect on the numbers of parasites progressing to cycle 3 .\",\n \"Shown is the mean ± SEM ( n = 3 ) .\",\n \"( f ) Growth of 3D7 and PfRhopH2-HAglmS parasites when cultured in various concentrations of GlcN , as measured by lactate dehydrogenase assay ( LDH ) .\",\n \"The LDH activities of 3D7 and RhopH2-HAglmS cultured in the absence of GlcN at cycle three were normalized to 100% , and activity of all lines ( ± GlcN ) across the three cycles was measured relative to this .\",\n \"Shown is the mean ± SD ( n = 3 ) .\",\n \"An unpaired t-test revealed RhopH2-HAglmS parasites grew significantly slower than 3D7 in all concentrations of GlcN by 36 hpi ( p<0 . 01 )\",\n \"( g ) Measurement of nanoluciferase ( Nluc ) released into the culture media and in pelleted erythrocytes infected with 3D7 or RhopH2-HAglmS parasites expressing Hyp1-Nluc .\",\n \"Measurements commenced around the time 3D7 parasites were starting to egress and invade new erythrocytes .\",\n \"The data represents the mean ± SD of one biological replicate completed in triplicate , with results expressed as percentage Nluc activity in the media relative to the pellet fraction . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 007 In separate experiments , parasite lactate dehydrogenase ( pLDH ) activity was also measured on trophozoite stages of RhopH2-HAglmS- or 3D7-parasitized erythrocytes grown in the presence of increasing concentrations of GlcN as a surrogate for parasite proliferation ( Figure 4f ) .\",\n \"The pLDH activities of 3D7 ( -GlcN ) and RhopH2-HAglmS ( -GlcN ) at cycle three were normalized to 100% , with activity of all parasite lines ( ± GlcN ) across the three cycles measured relative to this .\",\n \"While 3D7 parasite growth only begun to be affected by the addition of 2 mM GlcN by cycle 3 , in strong contrast , growth of RhopH2-HAglmS parasites was majorly reduced at all GlcN concentrations and also relative to 3D7 cultured in the same GlcN concentrations .\",\n \"The results also concur with the experiments above in that the effect of GlcN on pLDH activity could already be seen at cycle two and was drastically amplified when parasites transitioned into cycle three ( Figure 4f ) .\",\n \"To further validate the effects of knocking down RhopH2 upon parasite maturation and transition into cycle three , erythrocytes infected with RhopH2-HAglmS and the 3D7 parental line at trophozoite stage were transfected with an exported nanoluciferase fusion protein ( Hyp1-Nluc ) .\",\n \"This enabled schizont rupture and merozoite egress at the end cycle two to be monitored via measuring the amount of nanoluciferase released into the culture media compared to the cell pellet .\",\n \"Infected erythrocytes were supplemented with GlcN in cycle one when the parasites were at trophozoite stage and when the 3D7 line had grown to late-schizont stage in cycle two and new ring-stage parasites were beginning to be visible in Giemsa stain ( indicative of the start of merozoite egress and commencement of transition into cycle 3 ) , the media and cell pellets were harvested and every two hours thereafter for eight hours .\",\n \"The percentage ratio of nanoluciferase activity of media/pellet was then determined .\",\n \"This revealed that egress of RhopH2-HAglmS ( +GlcN ) line was markedly delayed compared to 3D7 ( +GlcN ) ( p<0 . 001 for 2 . 5 mM GlcN ) ( Figure 4g ) .\",\n \"These results validate that the slower growth of RhopH2-HAglmS ( +GlcN ) observed in cycle 2 is due to the specific effect of depletion of RhopH2 expression .\",\n \"Since the growth experiments revealed a defect in parasite transition from cycle two to cycle three , video microscopy of live schizont-stage parasites at the end of the second cycle on GlcN was performed to visualize whether knockdown of RhopH2 was impacting on erythrocyte egress and invasion .\",\n \"No obvious differences were observed in general schizont morphology or in the ability of the erythrocyte to burst , indicating egress per se was not actually affected .\",\n \"Rather , instead of the merozoites dispersing rapidly after egress , GlcN treatment caused the merozoites to remain clumped together ( Figure 5a , Videos 1–2 ) , a phenotype not observed in 3D7 ( +GlcN ) parasites ( not shown ) .\",\n \"Occasionally remnants of membranes could be observed around the merozoites , but even when these broke down , the merozoites remained clumped ( Figure 5a , see 3 mM 5s versus 29 . 1s ) .\",\n \"Nine and eleven schizont ruptures were observed ± GlcN treatment respectively , and as a consequence of merozoite clumping following GlcN treatment , less than two merozoites per rupture were released and able to contact new erythrocytes compared with six merozoites without GlcN ( Figure 5b ) .\",\n \"The net effect was fewer average invasions per rupture , with only 0 . 25 after GlcN treatment compared to 2 . 6 without treatment ( Figure 5c ) .\",\n \"Whilst 28 out of the 63 merozoites that made erythrocyte contact without GlcN went on to invade erythrocytes ( Figure 5d ) , only two out of the 14 merozoites treated with GlcN invaded erythrocytes , indicating a success rate of 0 . 44 and 0 . 14 invasions per contact , respectively ( Figure 5d ) .\",\n \"From these results , it was inferred that the lower fold-increase in parasitemia from cycle two to the next after RhopH2 knockdown stemmed from a combined effect of reduction in the number of parasites reaching schizogony in cycle two and a reduced invasion rate .\",\n \"The latter most likely stems from an indirect effect of RhopH2 knockdown that results in a clumping of merozoites incapable of breaking free to invade a new host cell and a reduced competency of merozoites forming at the end of cycle two to successfully invade an erythrocyte . 10 . 7554/eLife . 23217 . 008Figure 5 . Merozoites depleted of PfRhopH2 show defect in parasite invasion the following cycle .\",\n \"( a ) Panel of images from videos of PfRhopH2-HAglmS schizonts observed rupturing and releasing merozoites at the end of cycle 2 , post-addition of 0 or 3 mM GlcN .\",\n \"The number of seconds post-rupture is indicated .\",\n \"( b ) The number of merozoites contacting nearby erythrocytes per schizont rupture following GlcN treatment is shown .\",\n \"( c ) The number of erythrocyte invasions per schizont rupture is shown .\",\n \"( d ) The proportion of merozoite-erythrocyte contacts that successfully result in invasion are indicated .\",\n \"For\",\n \"( b ) and\",\n \"( c ) , the central bar denotes median , the box denotes 25–75th percentile and the whiskers the data range .\",\n \"****p<0 . 0001 by unpaired t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 00810 . 7554/eLife . 23217 . 009Video 1 . Plasmodium falciparum RhopH2-HAglmS schizont rupturing and releasing merozoites which invade nearby human erythrocytes . Successful invasions are indicated with white arrows .\",\n \"Time in seconds from egress is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 00910 . 7554/eLife . 23217 . 010Video 2 . A rupturing Plasmodium falciparum RhopH2-HAglmS schizont that had been treated with 3 mM glucosamine for 2 cell cycles to knockdown RhopH2-HA expression . At 0 s the erythrocyte membrane surrounding the schizont begins to break down but the merozoites do not disperse until about 68 s later .\",\n \"None of the merozoites appeared to invade neighbouring erythrocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 010 To unequivocally show that the growth defects in P . falciparum were a consequence of RhopH2 knockdown , conditional regulation of RhopH2 in P . berghei was also performed .\",\n \"This also provided insight into the consequences of depleting RhopH2 expression on parasite growth in vivo .\",\n \"In this case , the P . berghei rhoph2 locus was modified to insert an anhydrotetracycline ( ATc ) -regulated transactivator element ( TRAD ) downstream of the endogenous rhoph2 promoter and a minimal promoter with TRAD binding sites upstream of the rhoph2 coding sequence .\",\n \"P . berghei ANKA parasites transfected with linearized pTRAD4-RhopH2ss and surviving pyrimethamine drug pressure were analyzed by diagnostic PCR and Southern blot , confirming that the targeting construct had integrated correctly into the rhoph2 locus and the line was clonal ( Figure 6—figure supplement 1a–c ) .\",\n \"Transcription of rhoph2 in this line , termed PbRhopH2-iKD , was highly responsive to ATc , with ~11-fold reduction of rhoph2 mRNA in schizont stages as determined by qRT-PCR and RT-PCR ( Figure 6—figure supplement 1d ) .\",\n \"The growth of the PbRhopH2-iKD line was specifically sensitive to ATc treatment .\",\n \"PbRhopH2-iKD parasites grew poorly in mice that had been pre-exposed to ATc 24 hr prior to infection ( Figure 6a ) .\",\n \"Conversely , growth of parental P . berghei parasites was unaffected by the presence of ATc ( Figure 6a ) as has been shown previously ( Pino et al . , 2012; Elsworth et al . , 2014 ) .\",\n \"The slower growth of PbRhopH2-iKD exposed to sucrose compared to parental P . berghei parasites exposed to ATc is most likely due to the transactivator not being able to induce transcription of rhopH2 to the same level as the native promoter . 10 . 7554/eLife . 23217 . 011Figure 6 . Depletion of RhopH2 in P . berghei leads to altered growth phenotypes in vivo and in vitro .\",\n \"( a ) Representative growth curve ( n = 2 ) of P . berghei iRhopH2 and wildtype ( WT ) parasites .\",\n \"Groups of 5 mice were pre-treated for 24 hr with either 0 . 2 mg/ml ATc or sucrose ( vehicle control ) , then infected with the PbiRhopH2 iKD line or WT PbANKA .\",\n \"Parasitaemia was calculated at the indicated timepoints .\",\n \"Error bars represent standard error of the mean .\",\n \"An unpaired t-test revealed growth of RhopH iKD +ATc was significantly impaired at all time points ( p<0 . 0001 ) and that of RhopH2 iKD + sucrose was slower that PbAWT +ATc by day five post infection ( p=0 . 026 )\",\n \"( b ) Representative Giemsa-stained smears showing effect of RhopH2 knockdown with ATc on parasite growth and schizont formation .\",\n \"Schematic shows experimental outline .\",\n \"( c ) Depletion of RhopH2 protein levels also impacts on the number of merozoites formed per schizont ( n = 59 and 55 schizonts examined for parasites grown in the absence and presence of ATc , respectively , and taken from three individual experiments ) .\",\n \"The central bar in the box plot denotes the median whilst the whiskers delineate the 10th and 90th percentiles .\",\n \"p<0 . 0001 by unpaired t-test .\",\n \"( d ) Representative invasion assay ( n = 2 ) performed with merozoites from mechanically ruptured schizonts cultured in vitro ± ATc showing percentage of parasites from n = 50–100 that were at ring ( R ) , early trophozoite ( ET ) , late trophozoite ( LT ) or schizont ( S ) stage of development . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 01110 . 7554/eLife . 23217 . 012Figure 6—figure supplement 1 . Characterization of inducible P . berghei RhopH2 parasites .\",\n \"( a ) The P . berghei inducible RhopH2 targeting construct was designed to integrate into the endogenous locus by double-crossover recombination .\",\n \"The predicted structure of the endogenous locus before and after integration is shown .\",\n \"Green and pink bars indicate regions used to generate probes for Southern blot , arrows indicate oligonucleotides used in diagnostic PCR analysis and indicative product size .\",\n \"( b ) Diagnostic PCR showing PbRhopH2 iKD parasites have integrated the targeting sequence .\",\n \"Oligonucleotide pairs shown in A were used on genomic DNA prepared from drug-resistant parasites after transfection with the targeting construct ( integrant ) or P . berghei ANKA ( WT ) .\",\n \"A product should only be observed for WT with DO291 and DO67 oligonucleotides .\",\n \"( c ) Southern blot showing homologous integration into Pbrhoph2 gene as predicted .\",\n \"Plasmid DNA from the targeting construct and genomic DNA from integrant and WT were digested with restriction enzymes ( RE ) and probed with the 3' targeting sequence or TRAD sequence .\",\n \"In both cases , the endogenous locus ( E ) has been modified and integration ( In ) bands of the predicted size are seen .\",\n \"( d ) RT-PCR showing reduced expression of PbRhopH2 in the presence of ATc .\",\n \"Upper panel: experimental outline .\",\n \"Mice infected with inducible RhopH2 line ( PbiRhopH2 ) were treated with ATc or vehicle control ( -ATc ) for 24 hr prior to harvest and overnight culture in vitro ± ATc , upon which RNA was extracted from the schizont stages .\",\n \"Lower panel: Diagnostic PCR using oligonucleotides specific for rhoph2 or exp2 .\",\n \"RhopH2 cDNA is only detected in the absence of ATc .\",\n \"Amplification products using gDNA as a template are shown in the last two lanes as controls . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 012 Since the more mature stages of P . berghei sequester in vivo , erythrocytes infected with PbRhopH2-iKD parasites ( 1 × 107 ) were inoculated into ATc-pretreated mice and harvested the following cycle when the parasites were at ring-stage .\",\n \"They were then cultured ex vivo in the presence of ATc to examine the development of parasites across the entire cell cycle .\",\n \"Parasites in which RhopH2 had been depleted , exhibited delayed progression to trophozoite stage and the schizont stages displayed aberrant morphology , often appearing vacuolated and containing fewer merozoites ( Figure 6b–c ) .\",\n \"A synchronous in vitro invasion and growth assay using merozoites that had been generated from cultured schizonts confirmed these findings , showing that parasites depleted of RhopH2 could invade erythrocytes but exhibited a delay in the transition from the early to more mature trophozoite forms ( Figure 6d ) , consistent with our findings in P . falciparum .\",\n \"RhopH2 is localized on the host cytosolic side of the PVM immediately after invasion and was found to affinity purify some components of the PTEX and a variety of exported proteins .\",\n \"This raised the question of whether the RhopH complex helps traffic proteins that exit PTEX through the erythrocyte cytoplasm , particularly as protein export is required to support parasite growth ( Elsworth et al . , 2014; Dietz et al . , 2014; Beck et al . , 2014 ) .\",\n \"However , no defect in the export of PfEMP1 , or trafficking of either RESA to the erythrocyte membrane or SBP1 to the Maurer’s clefts was evident after knocking down RhopH2 expression with GlcN ( Figure 7a ) .\",\n \"In contrast , the localization of RhopH3 and to a lesser extent RhopH1/clag3 was affected when RhopH2 expression was knocked down ( Figure 7—figure supplement 1 ) .\",\n \"Moreover , RhopH2-HAglmS parasites supplemented with a reduced concentration of 0 . 5 mM GlcN that still gave efficient RhopH2 knockdown ( Figure 7b ) and which were harvested at mid-trophozite stage before parasites growth was impaired ( Figure 7c ) , could similarly export a nanoluciferase reporter ( Hyp1-NLuc ) as RhopH2-HAglmS ( -GlcN ) or 3D7 parasites ( ± ) GlcN ( Figure 7d ) . 10 . 7554/eLife . 23217 . 013Figure 7 . RhopH2 is not involved in the trafficking of exported proteins in the erythrocyte cytoplasm .\",\n \"( a ) Representative IFAs of erythrocytes infected with RhopH2-HAglmS parasites grown in 0 mM or 2 . 5 mM GlcN using the indicated antibodies show trafficking of RESA , SBP1 and PfEMP1 is unaffected upon RhopH2 knockdown .\",\n \"Scale bar = 5 µm\",\n \"( b ) Western blots of the parasites probed with an anti-HA antibody indicate that PfRhopH2 has been substantially knocked down with 0 . 5 mM GlcN relative to an EXP2 loading control .\",\n \"( c ) Giemsa stained images of the trophozoites that were analysed .\",\n \"( d ) Proportion of luciferase activity exported into the erythrocyte cytosol , secreted into the parasitophorous vacuole or present in the parasite cytoplasm of RhopH2-HAglmS and 3D7 wildtype parasites transfected with Hyp1-Nluc and grown in ± GlcN .\",\n \"Bars denote mean ± SD ( n = 3 ) .\",\n \"An unpaired t-test revealed there was no significance different in the exported NLuc fractions ± GlcN for 3D7 ( p=0 . 8579 ) and RhopH2-HAglmS ( p=0 . 1801 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 01310 . 7554/eLife . 23217 . 014Figure 7—figure supplement 1 . Localization of RhopH1/clag3 and RhopH3 in infected erythrocytes when RhopH2 expression is knocked down . Representative immunofluorescence analysis of erythrocytes infected with RhopH2-HAglmS parasites grown in 0 mM or 2 . 5 mM GlcN .\",\n \"Cells fixed with acetone/methanol and labelled with anti-HA antibody to detect RhopH2 IFAs and other antibodies , as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 014 Since RhopH2 forms a complex with RhopH1 , a protein implicated in NPP activity , and depletion of RhopH2 leads to growth defects around the time that NPPs are active in the infected erythrocyte , we next assessed whether RhopH2 contributes to NPP function .\",\n \"Sorbitol transport into infected erythrocytes requires NPP activity , resulting in hypotonicity-induced cell lysis ( Wagner et al . , 2003; Nguitragool et al . , 2011 ) .\",\n \"Thus RhopH2-HAglmS-parasitized erythrocytes transfected with the Hyp1-Nluc reporter were treated with sorbitol buffer containing NanoGlo .\",\n \"The degree of lysis and hence channel activity could be quantified by measuring the amount of NanoGlo hydrolysed by Hyp1-Nluc which is released during cell lysis ( Azevedo et al . , 2014 ) .\",\n \"We established that GlcN-mediated knockdown of RhopH2 dramatically reduced the capacity of infected erythrocytes to be lysed by sorbitol , suggesting RhopH2 contributes to NPP activity ( Figure 8a ) .\",\n \"In contrast , 3D7-parasitized erythrocytes treated with GlcN were not affected in their ability to be lysed by sorbitol indicating that depletion of RhopH2 and not treatment with GlcN was responsible for inhibition of NPP function ( Figure 8a ) .\",\n \"As the ability of parasitized erythrocytes to be lysed only commences >24 hpi , Giemsa smears of parasites used in the sorbitol assays were examined but this revealed the parasites were all similarly aged ( Figure 8b ) .\",\n \"When an iso-osmotic solution of alanine was used instead of sorbitol , similar results were obtained , with increasing concentrations of GlcN reducing the capacity of RhopH2-HAglmS parasitized erythrocytes to be lysed ( Figure 8c ) .\",\n \"More lysis inhibition was also observed in 32 hr trophozoites compared to 24 hr trophozoites , consistent with the NPPs being more developed in older parasites . 10 . 7554/eLife . 23217 . 015Figure 8 . Knockdown of RhopH2 impairs sorbitol and alanine uptake .\",\n \"( a ) GlcN-mediated knockdown of RhopH2 in PfRhopH2-HAglmS parasites expressing an exported Hyp1-Nluc reporter leads to a dramatic reduction in the capacity of infected erythrocytes to be lysed by the addition of sorbitol .\",\n \"In contrast erythrocytes infected with 3D7 parasites expressing Hyp1-Nluc are sensitive to sorbitol-mediated lysis .\",\n \"The % lysis was determined by the amount of NanoGlo substrate hydrolysed by Hyp1-Nluc , with 100% lysis defined as the Nluc activity ( RLU/min ) in parasites incubated in 280 mM sorbitol buffer with no GlcN .\",\n \"Data represents mean ± SD of three biological replicates completed in triplicate .\",\n \"( b ) Giemsa stained images of the trophozoites analysed in the sorbitol uptake assays .\",\n \"( c ) Analysis of sorbitol and alanine-mediated lysis of erythrocytes infected with PfRhopH2-HAglmS parasites at 24 and 32 hr post infection ( hpi ) at various concentrations of GlcN .\",\n \"The % lysis was determined by the amount of NanoGlo substrate hydrolysed by Hyp1-Nluc .\",\n \"Data represents mean ± SD of one biological experiment completed in triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 015 Given that RhopH2 depletion appeared to affect NPP activity , we next examined the effect of depleting RhopH2 on the metabolism of P . falciparum-infected erythrocytes .\",\n \"This was undertaken by conducting comparative untargeted metabolomics on 3D7 and RhopH2-HAglmS parasites incubated in the presence and absence of 2 . 5 mM GlcN .\",\n \"Overall , ~1000 metabolites from diverse pathways were detected and assigned putative identities based on accurate mass , and confirmed using retention time where standards were available ( Creek et al . , 2012 ) .\",\n \"A Principal Component Analysis ( PCA ) of all metabolite features across the four sample groups , 3D7 ±GlcN ) and RhopH2-HAglmS ( ±GlcN ) showed that replicates from groups 3D7 ( ±GlcN ) and RhopH2-HAglmS ( -GlcN ) clustered together , and that these were metabolically different to the induced RhopH2 knockdown , RhopH2-HAglmS ( +GlcN ) ( Figure 9a ) .\",\n \"This indicates that knockdown of RhopH2 causes a reproducible metabolic shift in the parasites .\",\n \"The heat map demonstrates a substantial impact of GlcN on global metabolite levels , even in wild-type 3D7 parasites ( Figure 9b ) .\",\n \"Nevertheless , as indicated in the PCA analysis , the inclusion of the 3D7 ( +GlcN ) control allowed detection of several metabolites that were specifically perturbed in response to RhopH2 knockdown , including selected vitamins/cofactors , nucleotides , amino acids and glycolytic metabolites ( Figure 9b ) .\",\n \"A detailed scrutiny of individual metabolites showed that while glucosamine treatment appeared to elevate metabolite levels in general , the RhopH2 knockdown resulted in decreased levels of folate and thiamin phosphates , which are essential vitamins and cofactors for cellular growth ( Figure 9c ) .\",\n \"The other class of metabolites to significantly decrease upon RhopH2 depletion were intermediates in the de novo pyrimidine synthesis pathway , N-carbamoyl L-aspartate , dihydroorotate and orotate .\",\n \"These metabolites are essential nucleotide precursors in P . falciparum , however , levels of downstream nucleotides were not affected at this time-point ( Supplementary file 1 ) .\",\n \"Few other metabolites were significantly and specifically depleted in the RhopH2 knockdown , with the exception of the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate ( Supplementary file 1 ) .\",\n \"The only putatively identified metabolite to extensively accumulate ( >5-fold higher than all controls ) in the RhopH2 knockdown was the urea cycle intermediate argininosuccinate , however , the other urea cycle intermediates were not significantly perturbed .\",\n \"Interestingly , a general increase in amino acid levels was also observed in the RhopH2 knockdown ( Figure 9d ) . 10 . 7554/eLife . 23217 . 016Figure 9 . Metabolomics analysis of 3D7 and RhopH2-HAglmS parasites +/- GlcN treatment .\",\n \"( a ) Principal Component Analysis scores plot of the first two principal components based on all metabolite features across the four sample groups .\",\n \"( b ) Heat map of relative abundance of all the putative metabolites detected in this study grouped according to metabolite classes .\",\n \"( c ) Fold change of metabolites showing a decrease in abundance , involved in vitamin and co-factor metabolism , de novo pyrimidine synthesis and glycolysis in the RhopH2-HAglmS ( +GlcN ) and 3D7 ( +Furosemide ) parasites compared to 3D7 ( untreated ) represented by the dotted vertical line .\",\n \"Error bars indicate relative standard deviation from n = 3 independent biological replicates .\",\n \"Thiamine monophosphate and orotate were not detected in the furosemide treatment experiment .\",\n \"( d ) Fold change of metabolites ( amino acids and a urea cycle intermediate ) showing an increase in abundance in the RhopH2-HAglmS ( +GlcN ) and 3D7 ( +Furosemide ) parasites compared to 3D7 ( untreated ) represented by the dotted vertical line .\",\n \"Error bars indicate relative standard deviation from n = 3 independent biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 016 In order to compare these metabolic perturbations to the effect of pharmacological NPP inhibition , erythrocytes infected with 3D7 were treated with furosemide and metabolite levels compared to untreated controls .\",\n \"Consistent with the RhopH2 knockdown , levels of folate and phosphoenolpyruvate were significantly lower in furosemide-treated parasites , and threonine , histidine , asparagine , serine and argininosuccinate levels all increased ( Figure 9d ) .\",\n \"Interestingly , the general depletion of de novo pyrimidine synthesis intermediates was not observed with furosemide , with N-carbamoyl L-aspartate levels found to be significantly higher following furosemide treatment .\"\n ],\n [\n \"A manuscript utilizing the same strategy to deplete RhopH2 in Plasmodium falciparum was also published by Ito and colleagues ( Ito et al . , 2017 ) .\",\n \"These authors derive very similar conclusions about the role of RhopH2 in nutrient uptake . 10 . 7554/eLife . 23217 . 017Figure 10 . Scheme illustrating how RhopH2 knockdown effects blood stage development .\",\n \"( a ) Knockdown of RhopH2 expression in cycle one appears to impair uptake of plasma nutrients in cycle two which delays development and replication in cycles 2 and 3 .\",\n \"( b ) ( 1 ) The RhopH complex is probably introduced onto the surface of the parasitophorous vacuole membrane ( PVM ) during merozoite invasion .\",\n \"( 2 ) Shortly after invasion the PTEX complex begins exporting parasite-synthesised proteins secreted into the parasitophorous vacuole ( PV ) , out into the erythrocyte cytoplasm .\",\n \"Some of the exported proteins such as PHISTs , MESA , LyMP , GBP130 and SEMP1 travel and bind to the erythrocyte cytoskeleton .\",\n \"The RhopH complex either ( 3 ) binds to these exported proteins at the erythrocyte surface or ( 4 ,\",\n \"5 ) assembles with these proteins en route to the surface .\",\n \"( 6 ) Once at the surface , the RhopH/exported protein complex forms NPPs either by forming their own membrane-spanning pore or by ‘opening up’ an erythrocyte pore .\",\n \"The NPPs function to permit the entry of nutrients and to dispose of parasite waste products . DOI: http://dx . doi . org/10 . 7554/eLife . 23217 . 017\"\n ],\n [\n \"Experiments involving the use of animals were performed in accordance with the recommendations of the Australian Government and the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes .\",\n \"The protocols were approved by the Deakin University Animal Welfare Committee ( approval number G37/2013 ) .\",\n \"To create a transgenic P . falciparum line in which RhopH2 expression could be knocked down , the transfection construct pRhopH2-HAglmS was created .\",\n \"This construct contains 1035 bp of sequence immediately upstream of the stop codon of RhopH2 ( Pf3D7_0929400 ) that had been PCR amplified from P . falciparum 3D7 genomic DNA ( gDNA ) with the primers DO227 and DO228 ( see Supplementary file 2 for oligonucleotide sequences ) and cloned into the BglII and PstI sites of pPfTEX88-HAglmS ( Chisholm et al . , 2016 ) .\",\n \"To engineer the PbRhopH2 inducible knockdown ( iKD ) line , the first 1477 bp of the PbRhopH2 coding sequence ( PbANKA_0830200 ) that had been PCR amplified with the primers DO291F and DO67R was cloned into the PstI and NheI sites of the modified pPRF-TRAD4-Tet07-HAPRF-hDHFR ( Pino et al . , 2012 ) described in Elsworth et al . ( 2014 ) .\",\n \"Also cloned into the NheI and BssHII sites of this vector were 1279 bp of the rhoph2 5' UTR sequence immediately upstream of the RhopH2 start codon , which had been PCR amplified using the primers DO62F and DO63R .\",\n \"Before transfection into P . berghei ANKA parasites , pTRAD4-iRhopH2ss was linearized with NheI .\",\n \"Blood-stage P . falciparum strain 3D7 was cultured continuously ( Trager and Jensen , 1976 ) and transfected as previously described ( Fidock and Wellems , 1997 ) .\",\n \"Transgenic parasites were selected with 2 . 5 nM WR99210 ( Jacobus ) or 5 μg/mL blasticidin S ( Sigma-Aldrich , Australia ) .\",\n \"P . berghei transgenic parasites were generated using the reference clone 15cy1 from the P . berghei ANKA strain .\",\n \"Transfection of parasites and selection of the transgenic parasites intravenously injected into 6- to 8-week-old female BALB/c mice was performed as previously described ( Janse et al . , 2006 ) .\",\n \"Erythrocytes infected with PfRhopH2-HAglmS parasites were treated at ring stage with 2 . 5 mM glucosamine ( GlcN ) or 0 mM GlcN as a control ( day 1 ) .\",\n \"Parasites were harvested at schizont stage , or in the following cycle at mid ring stage or schizont stage and treated with 0 . 05% saponin to remove haemoglobin .\",\n \"Western blots of parasite proteins fractionated on 8% Bis-Tris gels ( Life Technologies , Carlsbad , CA , USA ) were blocked in 5% skim milk in PBS and then incubated with mouse anti-HA ( 1:1000; Roche , Indianapolis , IN , USA ) for detection of RhopH2 and rabbit anti-EXP2 ( 1:1000 ) as a loading control .\",\n \"After washing , the membranes were probed with horseradish peroxidase-conjugated secondary antibodies ( 1:5000; Thermo Scientific , Waltham , MA , USA ) and detection was performed using the Clarity ECL Western blotting substrate ( Biorad , Hercules , CA , USA ) .\",\n \"The membrane was imaged using a Fujifilm LAS-4000 Luminescent Image Analyzer and ImageJ software ( NIH , version 1 . 46r ) was used to measure intensity of bands .\",\n \"Mice infected with erythrocytes infected with the PbRhopH2 iKD line were administered drinking water containing 0 . 2 mg/ml ATc ( Sigma ) made in 5% sucrose or 5% sucrose only as vehicle control when the parasitemia reached ~1% .\",\n \"After 24 hr when the parasites were predominantly at ring stage , mouse blood was harvested by cardiac bleed and cultured in vitro until parasites reached schizont stage ( ~16 hr ) in RPMI 1640 medium containing L-glutamine ( Life Technologies ) supplemented with 25 mM HEPES , 0 . 2% bicarbonate , 20% fetal bovine serum and 1 µg/ml ATc ( or vehicle as a control ) at 36 . 5°C .\",\n \"Experiments were performed on two independent occasions .\",\n \"The infected erythrocytes were lysed with 0 . 05% saponin prior to RNA extraction .\",\n \"To detect transcripts in P . berghei parasites by qRT-PCR , RNA was extracted from blood stage parasites using TRIsure reagent ( Bioline , UK ) .\",\n \"cDNA was then made using the iScript reverse transcription supermix ( Biorad ) according to the manufacturer’s instructions .\",\n \"cDNA ( or gDNA as a control ) was used in PCR reactions using oligonucleotides to rhopH2 ( O614F/O615R and O605F/O616R ) or gapdh ( O567F/O568R ) .\",\n \"The expression levels of rhopH2 were normalized against the gapdh house-keeping gene , with gene expression values calculated based on the 2ΔΔCt method .\",\n \"For analysis of P . falciparum growth , erythrocytes infected with PfRhopH2-HAglmS parasites at ring stage were sorbitol synchronized twice and the following cycle ( Cycle 1 ) , 2 . 5 mM GlcN was added to ring stage parasites , with 0 mM GlcN serving as the negative control .\",\n \"Parasitemias in Giemsa-stained smears were determined by counting a minimum of 1000 erythrocytes and comparative growth analysis was performed using a student’s t-test .\",\n \"Parasite growth of triplicate samples was also assessed using a modified Malstat assay protocol ( Makler and Hinrichs , 1993 ) .\",\n \"For this , GlcN ( Sigma ) was added to blood cultures of synchronized PfRhopH2-HAglmS ring stage parasites ( ~5% parasitemia and 2% hematocrit ) in cycle\",\n \"1 . In cycle 1 and cycle 2 , when parasites were at trophozoite stage , three aliquots were removed for subsequent proliferation assays and the cultures then diluted 1/5 to 1/10 and seeded into new plates with fresh erythrocytes and GlcN .\",\n \"A final three aliquots were removed in cycle three when parasites were at trophozoite stage .\",\n \"To quantitate parasite biomass , 30 µL of culture was mixed with 75 µL Malstat reagent ( 0 . 1 M Tris pH 8 . 5 , 0 . 2 g/mL lactic acid , 0 . 2% v/v Triton X-100 and 1 mg/mL acetylpyridine adenine dinucleotide ( Sigma ) , 0 . 01 mg/ml phenozine ethosulfate ( Sigma ) and 0 . 2 mg/mL nitro blue tetrazolium ( Sigma ) .\",\n \"Once the no drug control wells had developed a purple color the absorbance was measured at 650 nm in a spectrophotometer .\",\n \"The cumulative absorbance values were calculated by subtracting the absorbance of uninfected erythrocytes from infected erythrocytes and multiplying by the combined dilution factor .\",\n \"The pLDH activities of 3D7 and RhopH2-HAglmS cultured in the absence of GlcN at cycle three were normalized to 100% , and activity of all lines ( ± GlcN ) at each day was measured relative to this .\",\n \"Data was analysed using a student’s t-test .\",\n \"Female Balb/c mice at 6 weeks of age were randomized into groups of five mice per experiment and then given drinking water containing either 0 . 2 mg/mL ATc ( Sigma ) made in 5% ( w/v ) sucrose or 5% sucrose only as a vehicle control .\",\n \"After 24 hr pre-treatment , mice were infected intraperitoneally ( i . p ) with 1 × 106 PbRhopH2 iKD parasitized erythrocytes .\",\n \"From 3 days post infection , parasitemias were monitored daily by Giemsa-stained tail blood smears , with mice humanely culled once the parasitemias reached >20% .\",\n \"Parasitemias in Giemsa-stained smears were determined by counting a minimum of 1000 erythrocytes .\",\n \"Comparative growth experiments were analyzed using a students t-test , with p<0 . 05 considered significant .\",\n \"To establish synchronous P . berghei infections , blood was harvested from donor mice infected with PbRhopH2 iKD when the parasitemia was ~3% .\",\n \"The blood was then cultured overnight in vitro in RPMI/20% FCS in the presence or absence of 1 µg/mL ATc until parasites reached schizont stage .\",\n \"The schizonts were purified on Nycodenz ( ELITech Group , Australia ) and isolated merozoites were incubated with uninfected erythrocytes in vitro as previously described and invasion allowed to proceed for 30 min ( Matthews et al . , 2013 ) .\",\n \"Following merozoite invasion , parasites were maintained in culture for a further 36 hr , with smears made at intervals and stained with Giemsa to monitor parasite growth .\",\n \"Erythrocytes infected with PfRhopH2-HAglmS at either ring or schizont stage were lysed with 0 . 05% ( w/v ) saponin in PBS .\",\n \"For sequential solubility assays , the pelleted parasite material was resuspended in a hypotonic lysis buffer ( 1 mM HEPES , pH 7 . 4 ) and after a 30 min incubation on ice , the material was centrifuged at 100 , 000 g for 30 min at 4°C .\",\n \"The supernatant , which contains soluble proteins , was removed and kept for analysis .\",\n \"The pellet was then resuspended in 0 . 1 M Na2CO3 ( pH 11 . 5 ) to extract proteins peripherally-associated with membranes .\",\n \"After another 30 min incubation on ice and centrifugation step , the pellet was resuspended in 1% ( w/v ) Triton X-100 in PBS and incubated at room temperature for 30 min to extract integral membrane proteins and re-centrifuged .\",\n \"The starting material , soluble fractions and the Triton X-100 insoluble fraction were electrophoresed by SDS-PAGE and transferred to nitrocellulose membrane for Western blotting .\",\n \"In an alternative approach , parasitized erythrocytes that had been hypotonically lysed with 1 mM HEPES , pH7 . 4 to remove soluble proteins were split into five equal fractions and resuspended in either 10 mM Tris-HCl , 0 . 1 M Na2CO3 ( pH 11 . 5 ) , 2% Triton X-100 , 6 M urea ( extracts peripheral and soluble proteins ) or 2% SDS ( solubilizes membrane proteins ) .\",\n \"Samples were incubated on ice for 1 hr and then centrifuged at 100 , 000 g for 30 min at 4°C .\",\n \"Pellet fractions were washed in 10 mM Tris-HCl .\",\n \"Both the soluble and insoluble fractions were analysed by Western blotting using mouse anti-HA ( 1:1000 ) , rabbit anti-EXP2 ( 1:1000 ) , rabbit anti-HSP101 ( 1:1000 ) and rabbit anti-SERA5 ( 1:1000 ) .\",\n \"Immunoprecipitations were performed on synchronised ring stage and trophozoite P . falciparum RhopH2-HAglmS-infected erythrocytes harvested with 0 . 05% ( w/v ) saponin in PBS .\",\n \"Parasite pellets were solubilized in 1% ( w/v ) Triton X-100 containing Complete protease inhibitors ( Roche ) .\",\n \"After a 30 min incubation on ice , the material was centrifuged at 17 , 000 g for 10 min at 4°C and supernatants were added to 100 μl PBS-washed anti-HA-agarose beads ( mAb clone HA-7 ) ( Sigma ) and mixed overnight at 4°C .\",\n \"The beads were washed in 0 . 5% Triton X-100 in PBS plus protease inhibitors .\",\n \"Bound proteins were eluted with 100 μL 1x non-reducing sample buffer ( 50 mM Tris-HCl pH 6 . 8 , 10% glycerol , 2 mM EDTA , 2% SDS , 0 . 05% bromophenol blue ) , then reduced and electrophoresed by SDS-PAGE .\",\n \"After staining the gel with Imperial Protein Stain ( Thermo Scientific ) , protein bands were manually excised and subjected to manual in-gel reduction , alkylation , and tryptic digestion , and extracted peptides were analysed by LC-MS/MS using an Orbitrap Lumos mass spectrometer ( Thermo Scientific ) fitted with nanoflow reversed-phase-HPLC ( Ultimate 3000 RSLC , Dionex , Australia ) .\",\n \"The nano-LC system was equipped with an Acclaim Pepmap nano-trap column and an Acclaim Pepmap RSLC analytical column .\",\n \"1 μL of the peptide mix was loaded onto the enrichment ( trap ) column at an isocratic flow of 5 μL/min of 3% CH3CN containing 0 . 1% formic acid for 6 min before the enrichment column was switched in-line with the analytical column .\",\n \"The eluents used for the LC were 0 . 1% v/v formic acid ( solvent B ) and 100% CH3CN/0 . 1% formic acid v/v .\",\n \"The gradient used was 3% B to 20% B for 95 min , 20% B to 40% B in 10 min , 40% B to 80% B in 5 min and maintained at 80% B for the final 5 min before equilibration for 10 min at 3% B prior to the next sample .\",\n \"The mass spectrometer was equipped with a NanoEsi nano-electrospray ion source ( Thermo Fisher ) for automated MS/MS .\",\n \"The resolution was set to 120000 at MS1 with lock mass of 445 . 12003 with HCD Fragmentation and MS2 scan in ion trap .\",\n \"The top 3 s method was used to select species for fragmentation .\",\n \"Singly charged species were ignored and an ion threshold triggering at 1e4 was employed .\",\n \"CE voltage was set to 1 . 9 kv .\",\n \"Late trophozoite-stage ( 24–36 hr post invasion [hpi] ) P . falciparum-infected erythrocytes were lysed in 0 . 09% saponin in 5 mM Tris pH 7 . 5 and washed three times in PBS to remove haemoglobin .\",\n \"Following centrifugation , the parasite pellet was solubilized by sonication in 0 . 25% ( v/v ) Triton X-100 or 1% ( v/v ) ASB-14 ( 3- ( tetradecanoylamidopropyl dimethylammonio ) propane 1-sulfonate ) , the latter because it is often used for solubisation of proteins for 2D electrophoresis ) , then incubated with mixing at 4°C for 30 min .\",\n \"Insoluble material was pelleted ( 14 000 g for 30 min at 4°C ) .\",\n \"The supernatants were electrophoresed on NativePAGE Novex 3–12% Bis-Tris protein gels as per manufacturer’s instructions ( Invitrogen ) and transferred to PVDF for Western blotting .\",\n \"Bound antibody probes were detected with LiCor Odyssey Fc infrared imager followed by analysis with ODYSSEY v1 . 2 software .\",\n \"IFA was performed on thin smears of infected erythrocytes fixed with ice cold 90% acetone/10% methanol for 2 min .\",\n \"Cells were blocked in 1% ( w/v ) BSA/PBS for 1 hr .\",\n \"All antibody incubations were performed in 0 . 5% ( w/v ) BSA/PBS .\",\n \"Primary antibodies for P . falciparum were used at the following concentrations: rat anti-HA ( 1:100 , Life Technologies ) , mouse anti-HA ( 1:250 , Life Technologies ) , chicken anti-HA ( 1/200 , Abcam ) , rabbit anti-RhopH1/clag3 ( 1:200 ) ( Kaneko et al . , 2005 ) , rabbit anti-RhopH3 ( 1:250 ) , rabbit anti-RAMA-D ( 1:1000 ) , rabbit anti-AMA1 ( 1:300 ) , rabbit anti-RON4 ( 1:300 ) , rabbit anti-SBP1 ( 1:200 ) , mouse anti-RESA ( 1:1000 ) and mouse anti-MSP1-19 mAb 17B6 ( 20 µg/mL ) .\",\n \"After a one-hour incubation in primary antibody , cells were washed three times in PBS and incubated with the appropriate AlexaFluor 488/568-conjugated secondary antibodies ( 1:2000 ) for 1 hr .\",\n \"Cells were washed three times in PBS , and mounted with Prolong Gold Antifade reagent ( Life Technologies ) containing 4' , 6-diamidino-2-phenylindole ( DAPI ) ( VectorLabs , Australia ) .\",\n \"Images were taken on an Olympus IX71 microscope and processed using ImageJ v1 . 46r .\",\n \"Ring stage PfRhopH2-HAglmS infected erythrocytes ( cycle\",\n \"1 ) were cultured at 4% hematocrit in the presence of 3 mM GlcN ( or 0 mM GlcN as a control ) until parasites reached the late schizont stage of cycle\",\n \"2 . The culture was then diluted to 0 . 16% in RPMI media and 2 mL of this was allowed to settle to produce a monolayer onto a 35 mm Fluorodish ( World Precision Instruments , Sarasota , FL , USA ) .\",\n \"Live parasite imaging was performed at 37°C on a Zeiss AxioObserver Z1 fluorescence microscope equipped with humidified gas chamber ( 90% N2 , 1% O2 , and 5% CO2 ) .\",\n \"Late stage schizonts were observed until they looked ready to rupture and time-lapse videos were recorded with an AxioCam MRm camera at four frames per second .\",\n \"ImageJ and Prism ( Graphpad , La Jolla , CA , USA ) were used to perform image and statistical analyses .\",\n \"Quantitation of invasion was performed using an unpaired student’s t-test .\",\n \"PfRhopH2-HAglmS and 3D7 parasites transfected with pHGBHRB ( a plasmid encoding a GFP reporter under the expression of under the HSP86 5' UTR ) ( Wilson et al . , 2010 ) , were used for invasion assays .\",\n \"Tightly synchronized parasitized erythrocytes purified using a VarioMACS magnetic cell separator were mixed with erythrocytes ( 1:50 ratio ) that had been stained with 10 µM amine-reactive fluorescent dye 7-hydroxy-9H- ( 1 , 3-dichloro-9 , 9-dimethylacridin-2-one ) succinimidyl ester ( Cell Trace Far Red DDAO-SE ) ( Invitrogen , Carlsbad , CA , USA ) in RPMI-1640 for 1 hr at 37°C according to manufacturer’s protocol .\",\n \"At designated time points , erythrocytes were harvested and stained for 20 min at room temperature in the dark with the DNA dye Hoechst 34580 ( 2 μM ) ( Invitrogen ) made in RPMI-1640 .\",\n \"Following a washing step , stained samples were examined using a BD FACS Canto II flow cytometer ( BD Biosciences , Australia ) with 100 , 000 events recorded .\",\n \"Experiments were carried out in triplicate .\",\n \"The collected data was analysed with FlowJo software ( Tree Star , Ashland , Oregon ) .\",\n \"Data was analysed for statistical significance using an unpaired student’s t-test .\",\n \"Wildtype P . falciparum 3D7 and RhopH2-HAglmS-infected erythrocytes were transfected with a Nanoluciferase ( Nluc ) ( Hall et al . , 2012 ) protein N-terminally appended with the N-terminus of the PEXEL protein Hyp-1 as described in ( Azevedo et al . , 2014 ) but containing the blasticidin deaminase gene instead of the hDHFR gene .\",\n \"The infected erythrocytes were sorbitol synchronized and when the parasites reached late trophozoite stage , the cultures were treated with either 0 . 5 mM GlcN or no GlcN for 48 hr .\",\n \"Infected erythrocytes were subsequently transferred to a 96 well plate at 1% hematocrit , 1% parasitemia and the GlcN concentration was maintained prior to measurement of Nluc signal .\",\n \"A series of wells containing infected erythrocytes lacking exported Nluc were used to control for background luminescence .\",\n \"When performing the assay , the control well lacking Hyp1-Nluc was spiked with recombinant Nluc ( 1 ng/µL ) to control for Nluc quenching by haemoglobin .\",\n \"Subsequently 5 µL of resuspended culture was added to Greiner Lumitrac 96 well microplate in duplicate before adding 90 µL of either Background buffer ( 10 mM Tris phosphoric acid pH7 . 4 , 127 mM NaCL , 5 mM Na2EDTA , 5 mM DTT ) , Equinotoxin ( EQT ) buffer ( Background buffer with EQT ( 5 µg/mL ) prepared in house as per [Jackson et al . , 2007] ) , EQT/saponin buffer ( EQT buffer with 0 . 03% ( w/v ) saponin ) or hypotonic buffer ( 10 mM Tris phosphoric acid pH 7 . 4 , 5 mM Na2EDTA , 0 . 2% NP40 , 5 mM DTT ) , which allow differential fractionation of the infected erythrocyte .\",\n \"The cells were incubated for 10 min at RT to allow for lysis to occur .\",\n \"Following this , 5 µl of diluted NanoGlo ( Promega , Australia , diluted 1: 500 in background buffer ) was injected to each well , the plate shaken ( 700 rpm/30 s ) and relative light units were then measured with CLARIOstar plate reader ( BMG Labtech , Australia ) .\",\n \"Experiments were repeated on three independent occasions and two technical replicates were completed per biological replicate .\",\n \"The export of Nluc was calculated as follows: the mean ( X¯ ) was calculated before adjusting for the spike in control ( His-Nluc ) and subtracting the background ( buffer one ) .\",\n \"Error was estimated with standard deviation ( SD ) and coefficient of variation ( CV ) .\",\n \"Subsequently , percentage of Nluc was calculated for each compartment using the new mean:% Exported fraction=X¯EQTX¯ Hypox100 Standard deviation for exported fraction was calculated as follows:CVexported fraction=CVEQT 2+CVHypo2SDexported fraction=CV x % Exported fraction% Secreted fraction= ( X¯EQT+X¯SAP ) −X¯EQTX¯Hypox100 Standard deviation for secreted fraction was calculated as follows:CVsecreted fraction=SDEQT2+SDEQT+SAP2X¯EQT+SAP−X¯EQTSDsecreted fraction=CVsecreted fraction2+CVHypo2 x %secreted fraction% Parasite fraction=X¯Hypo− ( X¯EQT+X¯SAP ) X¯Hypox100 Standard deviation for parasite cytosol fraction was calculated as follows:CVparasite fraction=SDEQT+SAP2+SDHypo2X¯Hypo−X¯EQT+SAPSDparasite fraction=CVparasite fraction2+CVHypo2 x %parasite fraction Experiments were then combined depending on the weight of data reliability with error weighted mean and error weighted standard deviation .\",\n \"Weight of data depending reliability .\",\n \"Error weighted mean was calculated as follows:X¯weigthed=Value1Abs ( SD1 ) +Value2Abs ( SD2 ) +Value3Abs ( SD3 ) Abs ( 1SD1 ) +Abs ( 1SD2 ) +Abs ( 1SD3 ) Error weighted standard deviation was calculated as follows:SDweighted = ( Value1−X¯Weighted ) 2Abs ( SD1 ) + ( Value2−X¯Weighted ) 2Abs ( SD2 ) + ( Value3−X¯Weighted ) 2Abs ( SD3 ) 1SD1+1SD2+1SD3 Value : Percentage in relative compartment , where numbers refer to three biological replicates .\",\n \"SD: Standard deviation calculated for each compartment , where numbers refer to three biological replicates .\",\n \"Data were analysed for statistical significance using a two-tailed , unpaired student’s t test with equal variances .\",\n \"P . falciparum RhopH2-HAglmS and 3D7 parasites expressing Hyp1-Nluc were treated with 0–3 mM GlcN when parasites were at trophozoite ( 28–36 hr post invasion ) stage and parasites were then grown for a further 48 hr until trophozoites stage .\",\n \"After washing the parasitized erythrocytes twice in PBS , 10 µL at 1% hematacrit and 1% parasitemia ( or PBS as a control ) was dispensed in triplicate into a Thermo Scientific 96 well U bottom microplate and loaded into a Clariostar luminometer ( BMG labtech ) .\",\n \"To each well , 40 µL of sorbitol or alanine lysis buffer containing the NanoGlo substrate ( 280 mM sorbitol or 280 mM L-alanine , 20 mM Na-HEPES , 0 . 1 mg/ml BSA , pH 7 . 4 , Nano-Glo [1:1000 dilution] ) was added and the relative light units ( RLU ) measured every 3 min with gain set to 2500 .\",\n \"The percent lysis was determined by non-linear regression , exponential growth equation as analysed by GraphPad Prism software .\",\n \"The PBS control was subtracted and the value multiplied by 100 to get a percentage lysis .\",\n \"A value of 100% lysis is defined as the Nluc activity in relative light units ( RLU/min ) of parasites in 280 mM sorbitol or alanine buffer with no GlcN .\",\n \"A value of 0% lysis is defined as the Nluc activity of parasites in PBS containing nanoglo substrate .\",\n \"The rate of lysis was derived from a kinetic assay measuring the increase in RLU per minute ( Dickerman et al . , 2016 ) .\",\n \"Data was analysed for statistical significance using an unpaired student’s t-test .\",\n \"Erythrocytes infected with RhopH2-HAglmS and 3D7 parasites expressing NLuc ( as described above ) were sorbitol synchronized and subsequently GlcN-treated at trophozoite stage ( cycle 1 ) .\",\n \"Heparin was added ( 100 μg/ml ) to prevent any early invasion events and was subsequently removed when schizonts were observed ( GlcN concentrations were maintained ) .\",\n \"Parasites were allowed to invade over a six-hour window ( cycle 2 ) and were subsequently sorbitol synchronized prior to seeding into 96 well plates ( 100 µl/1% Hematocrit/1% parasitemia ) .\",\n \"Giemsa smears were taken at late schizont stage ( end of cycle 2 ) and when rings were observed , the cultures were pelleted ( 500g/3 min ) and 50 µl of supernatant containing released Nluc was removed for analysis ( media fraction ) .\",\n \"Infected erythrocyte cell pellets were also collected .\",\n \"Fractions were collected every two hours for a total of 8 hr .\",\n \"Prior to analysis of total Nluc content , 50 μl of media containing 1% hemocrit was added to each media fraction and 50 μl of media was added to each pellet sample to maintain equivalent volumes .\",\n \"Each fraction was fully re-suspended and 10 µl was added to 90 µl lysis buffer ( 10 mM tris phosphoric acid , 5 mM Ka2EDTA , 0 . 2% NP40 , 5 mM DTT , Nano-Glo ( 1:1000 dilution ) ) in a Greiner Lumitrac 96 well microplate prior to shaking ( 700 pm/30 s ) .\",\n \"Relative light units were measured with a ClARIOstar multimode plate reader ( BMG Labtech ) and data was subsequently analysed using GraphPad PRISM software .\",\n \"Tightly synchronized cultures of P . falciparum 3D7 or RhopH2-HAglmS ring stage parasites were exposed to either 0 mM or 2 . 5 mM GlcN in cycle one and they were harvested in the second cycle when they had sufficient haemazoin pigment ( ~24 hr post-invasion ) to facilitate magnetic purification using a VarioMACS magnetic cell separator .\",\n \"For furosemide treatment , 500 µM of furosemide was added to the cultures shortly after invasion when parasites were in the early ring stages of cycle two and the cultures were harvested at ~24 hr post-invasion .\",\n \"Morphology of parasites was monitored by light microscopy to obtain developmentally similar stages of parasites under GlcN and furosemide treatment and untreated control cultures .\",\n \"Metabolism was quenched by rapidly cooling down the cultures to 4°C , culture medium was removed following centrifugation at 3000 g for five minutes ) and metabolites were extracted from 4 . 5 × 107 cells using 150 µl of extraction buffer consisting of chloroform/methanol/water ( 1:3:1 v/v ) ( spiked with 1 µM PIPES , CHAPS and Tris as internal standards ) followed by vortex mixing for 1 hr at 4°C .\",\n \"After mixing , cellular debris was removed by centrifugation at 4°C ( >15000 g for 10 min ) and the supernatant was kept at −80°C prior to analysis .\",\n \"Three biological replicates were prepared for each cell line and treatment .\",\n \"Samples were analysed by hydrophilic interaction liquid chromatography coupled to high resolution-mass spectrometry ( LC-MS ) according to a previously published method ( Stoessel et al . , 2016 ) .\",\n \"All samples were analyzed as a single batch , in randomized order and pooled quality control samples were analyzed regularly throughout the batch to confirm reproducibility .\",\n \"Approximately 250 metabolite standards were analyzed immediately preceding the batch run to determine accurate retention times to facilitate metabolite identification .\",\n \"Additional retention times for metabolites lacking authentic standards were predicted computationally as previously described ( Creek et al . , 2011 ) .\",\n \"Data was analysed using the IDEOM workflow ( Creek et al . , 2011 , 2012 ) .\",\n \"Peak areas for significant metabolites were confirmed by manual integration with Tracefinder software ( Thermo Scientific ) .\",\n \"Multivariate statistical analysis utilized principal component analysis ( PCA ) on log-transformed and auto-scaled metabolite peak intensity data using the web-based analytical tool , MetaboAnalyst ( Xia et al . , 2015 ) .\",\n \"The IDEOM files containing all metabolomics data are uploaded on Figshare and can be accessed at https://figshare . com/s/c38c0a98fb01634677f6 .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Plasmodium falciparum parasites , the causative agents of malaria , modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste .","Here we investigate the contribution of the rhoptry protein RhopH2 , in the formation of new permeability pathways ( NPPs ) in Plasmodium-infected erythrocytes .","We show RhopH2 interacts with RhopH1 , RhopH3 , the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling .","Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two .","There is also a significant impact on parasite growth , replication and transition into cycle three .","The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown .","These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival ."],"string":"[\n \"Plasmodium falciparum parasites , the causative agents of malaria , modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste .\",\n \"Here we investigate the contribution of the rhoptry protein RhopH2 , in the formation of new permeability pathways ( NPPs ) in Plasmodium-infected erythrocytes .\",\n \"We show RhopH2 interacts with RhopH1 , RhopH3 , the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling .\",\n \"Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two .\",\n \"There is also a significant impact on parasite growth , replication and transition into cycle three .\",\n \"The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown .\",\n \"These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Malaria is a life-threatening disease that affects millions of people around the world .","The parasites that cause malaria have a complex life cycle that involves infecting both mosquitoes and mammals , including humans .","In humans , the parasites spend part of their life cycle inside red blood cells , which causes the symptoms of the disease .","In order to thrive , malaria parasites need to make the red blood cell more permeable so that they can absorb nutrients from the blood stream and get rid of toxic waste products they generate .","Previous research has shown that the parasites can produce a protein that makes red blood cells more permeable to a range of nutrients .","Understanding how the parasites can do this , and if they could change the permeability of their host red blood cell to prevent anti-malaria drugs from entering may help researchers to identify new approaches to starve the parasite .","Counihan et al . investigated whether the parasites also use other proteins to modify red blood cells and demonstrated that a protein called RhopH2 can make the blood cells more permeable .","The experiments used a genetically modified version of the parasite that lacked RhopH2 .","Counihan et al . show that essential nutrients and vitamins were depleted in these parasites and that the parasites were much slower to grow and reproduce .","The next important step would be to identify all proteins that are involved in making red blood cells more permeable and how they achieve this , and use this knowledge to help generate anti-malarial drugs ."],"string":"[\n \"Malaria is a life-threatening disease that affects millions of people around the world .\",\n \"The parasites that cause malaria have a complex life cycle that involves infecting both mosquitoes and mammals , including humans .\",\n \"In humans , the parasites spend part of their life cycle inside red blood cells , which causes the symptoms of the disease .\",\n \"In order to thrive , malaria parasites need to make the red blood cell more permeable so that they can absorb nutrients from the blood stream and get rid of toxic waste products they generate .\",\n \"Previous research has shown that the parasites can produce a protein that makes red blood cells more permeable to a range of nutrients .\",\n \"Understanding how the parasites can do this , and if they could change the permeability of their host red blood cell to prevent anti-malaria drugs from entering may help researchers to identify new approaches to starve the parasite .\",\n \"Counihan et al . investigated whether the parasites also use other proteins to modify red blood cells and demonstrated that a protein called RhopH2 can make the blood cells more permeable .\",\n \"The experiments used a genetically modified version of the parasite that lacked RhopH2 .\",\n \"Counihan et al . show that essential nutrients and vitamins were depleted in these parasites and that the parasites were much slower to grow and reproduce .\",\n \"The next important step would be to identify all proteins that are involved in making red blood cells more permeable and how they achieve this , and use this knowledge to help generate anti-malarial drugs .\"\n]","isConversational":false},"year":{"kind":"string","value":"2017"}}},{"rowIdx":283,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["plant biology"],"string":"[\n \"plant biology\"\n]","isConversational":false},"title":{"kind":"string","value":"KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis"},"id":{"kind":"string","value":"elife-57282-v1"},"sections":{"kind":"list like","value":[["Following deposition of dehydrated pollen grains on the receptive surface of the female organ , the stigma , pollen rehydrates , germinates and produces a pollen tube that carries the male gametes toward the ovules where the double fertilization takes place .","This long itinerary through the different tissues of the pistil is finely controlled , avoiding misrouting of the pollen tube and hence assuring proper delivery of the sperm cells to the female gametes .","How the pollen germinates a tube and how pollen tube growth is regulated have been the object of many investigations ( Palanivelu and Tsukamoto , 2012; Mizuta and Higashiyama , 2018 ) .","The use of in vitro pollen germination as well as semi-in vivo fertilization assays , together with the analysis of mutants defective in pollen or ovule functions , have rapidly expanded our knowledge of the mechanisms that sustain pollen tube growth , its guidance toward the ovule and the final delivery of male gametes within the embryo sac ( Dresselhaus and Franklin-Tong , 2013; Higashiyama and Yang , 2017; Cameron and Geitmann , 2018 ) .","At the cellular level , cytoskeleton has been extensively studied during pollen tube elongation ( Fu , 2015 ) , highlighting the critical role played by the actin microfilaments in pollen-tube tip growth through delivery of materials for the biosynthesis of the plasma membrane and cell wall .","By contrast , CMTs seem to have a lower importance in the pollen tube elongation process , drugs affecting CMT polymerisation having no significant effects on pollen-tube growth rate , yet altering the capacity of pollen tubes to change their growth direction ( Gossot and Geitmann , 2007 ) .","In Arabidopsis thaliana , pollen tubes grow within the cell wall of papillae of the stigmatic epidermis , and then through the transmitting tissue of the style and ovary ( Lennon and Lord , 2000 ) .","The transmitting tissue has an essential function in pollen tube guidance , providing chemical attractants and nutrients ( Crawford and Yanofsky , 2008; Higashiyama and Hamamura , 2008 ) .","In contrast to these accumulating data showing the existence of factors mediating pollen tube growth in the pistil , whether guidance cues exist at the very early stage of pollen tube emergence and growth in the papilla cell wall remains largely unknown .","The cell wall constitutes a stiff substrate and hence a mechanical barrier to pollen tube progression .","There are numerous examples in animal cells demonstrating that mechanical properties of the cellular environment , and in particular rigidity , mediate cell signalling , proliferation , differentiation and migration ( Discher et al . , 2005; Fu et al . , 2010; Ermis et al . , 2018 ) .","In plant cells , cell wall rigidity depends mainly on its major component , cellulose , which is synthesized by plasma membrane-localized cellulose synthase complexes ( CSCs ) moving along cortical microtubule ( CMT ) tracks ( Paredez et al . , 2006 ) .","While penetrating the cell wall , the pollen tube exerts a pressure onto the stigmatic cell ( Sanati Nezhad and Geitmann , 2013 ) .","Physical forces are known to reorganise the cortical microtubules ( CMTs ) , which by directing CSCs to the plasma membrane , reinforce wall stiffness by novel cellulose microfibril ( CMF ) synthesis ( Paredez et al . , 2006; Sampathkumar et al . , 2014 ) .","Hence , there is an intricate interconnection between CMT organisation , CMF deposition and cell wall rigidity ( Xiao and Anderson , 2016 ) .","A major regulatory element of CMT dynamics is the KATANIN ( KTN1 ) microtubule-severing enzyme , which allows CMT reorientation following mechanical stimulation ( Uyttewaal et al . , 2012; Sampathkumar et al . , 2014; Louveaux et al . , 2016 ) .","Here we investigated whether the CMT network of papilla cells might contribute to pollen tube growth and guidance in stigmatic cells by combining cell imaging techniques , genetic tools , chemical analysis of the cell wall and atomic force microscopy .","We show that isotropic reorientation of CMTs occurs in aged Col-0 and ktn1-5 papilla cells , which is accompanied by a change in the growth direction of pollen tubes that tend to make coils on papillae .","We show that CMT reorganisation is associated with isotropic rearrangement of CMFs , high content of crystalline cellulose and particular cell wall mechanics of ktn1-5 stigmas .","Altogether , our results indicate that cytoskeleton dynamics and mechanical properties of the cell wall , which both depend on KTN1 activity , have a major role in guiding early pollen tube growth in stigma papillae ."],["To assess the functional role of stigmatic CMTs in pollen-papilla cell interaction , we first analysed their organisation in papillae at stages 12 to 15 of stigma development as described ( Smyth et al . , 1990; Figure 1A , B ) .","We generated a transgenic line expressing the CMT marker MAP65 . 1-citrine under the control of the stigma-specific promoter SLR1 ( Fobis-Loisy et al . , 2007 ) .","Before ( stage 12 ) and at anthesis ( stage 13 ) , where flowers are fully fertile ( Kandasamy et al . , 1994 ) , the CMTs were aligned perpendicularly to the longitudinal axis of papilla cells and were highly anisotropic ( median value of 0 . 40 ) ( Figure 1C , D ) .","At stage 14 , when anthers extend above the stigma , the CMT pattern became less organised , with a higher variability in anisotropy values .","Finally , at stage 15 when the stigma extends above anthers , CMT anisotropy had a median value of 0 . 09 indicative of an isotropic orientation of CMTs ( Figure 1C , D ) .","These findings reveal that the papilla CMT cytoskeleton is dynamic during development , with a change of CMT array orientation from anisotropy to isotropy .","We then wondered whether this change in CMT organisation could be correlated with pollen tube growth .","To this end , we self-pollinated Col-0 papillae from stages 12 to 15 and examined pollen tube growth one hour after pollination by scanning electron microscopy ( SEM ) ( Figure 2A ) .","At stage 12 and 13 , we found most ( ~60% ) pollen tubes grew straight in the papillae , whereas about 30% and 10% of tubes made half-turn or one turn around stigmatic cells , respectively ( Figure 2B ) .","At later stages of development , the tendency to coil around the papillae increased , with 3 . 5-fold more pollen tubes at stage 14 and 5 . 5-fold more at stage 15 making one or more than one turn around papillae .","These results suggest that CMT organisation in the papilla impacts the direction of growth of pollen tubes and that loss of CMT anisotropy is associated with coiled growth .","We next asked whether the pressure exerted by the growing pollen tube in the cell wall may induce changes in the CMT organisation of the papilla cell .","To this end , we set up a live imaging system where pollinated stigmas were maintained in air so as to prevent immediate hydration and burst of pollen grains .","During the time course of the experiment , starting at the beginning of pollen tube emergence on the stigma papilla at stage 13 ( t0 ) up to 13 min after germination , we found no major alteration of CMT arrays from early to late penetration stages ( Figure 3A ) .","This result was in contradiction with previous immunostaining analysis that showed fragmentation of CMTs following compatible pollination in Brassica napus ( Samuel et al . , 2011 ) .","To confirm our result and gain better insight into CMT organisation , we then observed pollinated stigmas at a single time point , that is 30 min after pollination , using a mounting medium to get higher resolution images .","Again , we did not detect any reorganisation or degradation of CMTs ( Figure 3B ) .","However , we found the stability of CMTs to be very sensitive to strong pressure , as when the coverslip was applied not cautiously on the stigma , a break-down of CMT arrays occurred ( Figure 3—figure supplement 1 ) .","These results indicate that the pressure exerted by the pollen tube while growing in the papilla cell wall does not alter the organisation of stigmatic CMTs .","To test the direct impact of stigma CMTs on pollen tube growth direction , we examined whether the destabilization of CMTs in Col-0 papillae could affect pollen tube growth .","To this end , we treated stigmas by local application of the depolymerizing microtubule drug oryzalin in lanolin pasted around the style .","After 4 hr of drug treatment , no more CMT labelling was detected in papillae , while CMTs were clearly visible in mock-treated ( DMSO ) stigmas ( Figure 4A ) .","Stigmas were then pollinated with Col-0 pollen and one hour later observed by SEM .","Pollen tubes were found to make one turn more frequently on drug-treated than on control papillae , with more than a 3-fold increase of tubes making one turn ( Figure 4B , C ) .","To confirm the relation between stigma CMTs and pollen behaviour , we examined pollen tube growth on stigmas of the katanin1-5 mutant , which is known to exhibit reduced CMT array anisotropy in root cells ( Bichet et al . , 2001; Burk et al . , 2001; Burk and Ye , 2002 ) .","Because the CMT organisation in ktn1-5 papillae is unknown , we crossed ktn1-5 with the MAP65-1-citrine marker line and found that CMT arrays were more isotropic in ktn1-5 papillae when compared with those of the WT ( Figure 5A , C ) .","We then analysed Col-0 pollen behaviour on ktn1-5 stigmatic cells at stage 13 ( Figure 5B ) .","We observed that Col-0 pollen tubes acquired a strong tendency to coil around ktn1-5 papillae , with more than 60% of tubes making one or more than one turn around papillae , sometimes making up to six turns , before reaching the base of the cell ( Figure 5D; Figure 5—figure supplement 1A–C ) .","In some rare cases , pollen tubes even grew upward in the ktn1-5 mutant and appeared blocked at the tip of the papilla ( Figure 5—figure supplement 1B , D ) .","The number of coils was significantly higher than on oryzalin-treated papillae .","Indeed , 25% of the pollen tubes made at least two coils in ktn1-5 papillae whereas this percentage represented only 4% on the oryzalin treated stigmas ( Figure 4C; Figure 5D ) .","To check whether papilla receptivity to pollen might be impaired in ktn1-5 stigmas , we examined hydration and tube growth of Col-0 pollen deposited on stage-13 Col-0 or ktn1-5 stigmas .","We found no significant differences in pollen hydration related to the stigma type ( Figure 5—figure supplement 2A ) and pollen tubes grew in the transmitting tract and reached the ovules in ktn1-5 pistils like in Col-0 ( Figure 5—figure supplement 2B ) .","We then wondered whether alteration of the KATANIN function in pollen might affect pollen tube growth directionality on Col-0 or ktn1-5 stigmas .","Cross-pollination revealed that ktn1-5 pollen tubes grew like Col-0 pollen tubes on stage-13 Col-0 papillae , while they coiled on ktn1-5 papillae ( Figure 5—figure supplement 2C ) .","Seed counts showed that fertility slightly decreased when ktn1-5 pollen was used to pollinate Col-0 stigmas , and was strongly affected when ktn1-5 was used as female , irrespective of whether the pollen was Col-0 or ktn1-5 ( Figure 5—figure supplement 2D ) .","This indicates that the fertilisation process is impaired in ktn1-5 ovules .","Altogether , these results show that the coiled phenotype observed on ktn1-5 stigmas is only dependent on defects occurring at the papilla level , that the stigma receptivity of ktn1-5 mutant is not altered and confirm that stigmatic CMTs , somehow , contribute to the directional growth of pollen tubes in papilla cells .","The katanin1/fra2 mutant was initially described as a mutant impaired in cell wall biosynthesis and CMT array organisation ( Burk et al . , 2001 ) .","This prompted us to investigate whether other mutants affected in cell wall biogenesis might exhibit the coiled pollen tube phenotype .","We selected mutants impaired in the cellulose synthase complex ( kor1 . 1 , prc1 and any1 ) , hemicellulose biosynthesis ( xxt1 xxt2 , xyl1 . 4 ) and pectin content ( qua2 . 1 ) , for which expression of the corresponding genes in stigma was confirmed ( Figure 5—source data 2 ) .","Strikingly , none of the 6 cell wall mutants displayed the coiled pollen tube phenotype ( Figure 5—figure supplement 3A–F ) .","To ascertain that the cell wall composition was altered in the stigma papillae of ktn1-5 and cell wall mutants , we undertook sugar analysis of ktn1-5 and xxt1 xxt2 stigmas and compared them with that of Col-0 .","We chose the xxt1 xxt2 mutant because its cell wall composition had been already well described ( Cavalier et al . , 2008; Xiao et al . , 2016 ) .","The relative abundance of monosaccharides in pistil cell walls varied between the three genotypes with major differences detected in the ktn1-5 cell wall composition that had an increased abundance of glucose , this latter deriving either from hemicellulose and amorphous cellulose or from crystalline cellulose ( Figure 5—figure supplement 4A ) .","ktn1-5 cell wall was characterized by the highest relative abundance of crystalline cellulose , which was 25% higher than in Col-0 and xxt1 xxt2 .","To explore whether side chains of hemicellulose might also be affected in the katanin mutant , we performed an enzymatic fingerprinting of xyloglucans from dissected stigmas of Col-0 and ktn1-5; xxt1xxt2 was not analysed as it lacks xyloglucan side chains .","We found in ktn1-5 a decrease in relative abundance of short side chains with terminal xylose ( XXG and XXXG ) and an increase in the longer ones with terminal fucose ( e . g XXFG ) ( Figure 5—figure supplement 4B ) .","The chemical analysis confirms that the cell wall composition is altered in ktn1-5 stigmas and presents some specific features compared with Col-0 and xxt1 xxt2 stigmas .","However , combined with the results of pollination on the six cell wall mutants , we cannot establish a causal effect of chemical alterations on the pollen tube coiled phenotype .","This suggests that the relation between CMT organisation , cell wall composition and pollen tube trajectory is more complex than anticipated .","Because CMTs guide cellulose deposition , they also control the directional elongation of plant cells ( Baskin , 2005 ) .","Indeed , katanin1/fra2 cells are wider and shorter than wild type ( Bichet et al . , 2001; Burk et al . , 2001 ) , and we may predict that the shape of papilla cells in the mutant might be similarly altered .","The contribution of stigmatic CMTs to pollen tube growth may thus be mediated by papilla cell shape only; for instance , wider papilla cells in ktn1-5 would promote pollen tube coiling .","To check that possibility , we measured the length and width of papilla cells in Col-0 ( at stage 13 and 15 ) , ktn1-5 , xxt1 xxt2 and any1 ( Figure 5—figure supplement 5 ) .","We did not find any correlation between papilla length and the coiled phenotype .","Indeed , coiled phenotype was observed in stage-15 WT papillae that were longer than those at stage-13 , as well as in ktn1-5 papillae that had a length similar to stage-13 WT papillae .","The correlation between papilla width and coiled phenotype was also not clear-cut .","As anticipated , ktn1-5 mutant exhibited wider papillae than Col-0 .","However , xxt1 xxt2 and any1 papillae were also wider than the WT but did not display the coiled phenotype .","Altogether , this suggests that papilla morphology is not sufficient to explain the coiled phenotype .","Because the main role of CMTs in plant cells is to guide the trajectory of CSCs , thereby impacting the mechanical anisotropy of the cell wall , we analysed the cell walls of Col-0 and ktn1-5 papillae following pollination .","First , using Transmission Electron Microscopy ( TEM ) , we found Col-0 pollen tubes penetrated the cuticle and grew between the two layers of the papilla cell wall , as previously described ( Kandasamy et al . , 1994 ) , in both Col-0 and ktn1-5 papilla cells ( Figure 6A ) .","We did not detect any significant difference in the ultrastructure of cell walls ( Figure 6—figure supplement 1 ) .","Interestingly , as the pollen tube progresses through the papilla cell wall , it generates a bump ( external deformation ) and an invagination ( internal deformation ) in the cell wall ( Figure 6A ) .","As such deformations could reflect differences in wall properties , we quantified the external ( extD ) and internal ( intD ) deformation following pollination of Col-0 and ktn1-5 stigmas .","To visualize more clearly this deformation , we pollinated stigmas expressing the plasma membrane protein LTI6B fused to GFP ( LTI6B-GFP ) with pollen whose tube was labelled with the red fluorescent protein RFP driven by the ACT11 promoter ( Rotman et al . , 2005; Figure 6B ) .","We found that Col-0 pollen tubes grew with almost equal extD and intD values in Col-0 cell wall .","However , the ratio between extD and intD was about three when Col-0 pollen tubes grew in ktn1-5 papilla cells ( Figure 6B–D ) .","These quantitative data were consistent with the observation that pollen tubes appeared more prominent on ktn1-5 stigmatic cells using SEM ( Figure 5B; Figure 5—figure supplement 1B ) .","Assuming that stigmatic cells are pressurized by their turgor pressure , this may reflect the presence of softer walls in ktn1-5 papillae .","To test this hypothesis , we assessed the stiffness of Col-0 and ktn1-5 papilla cell walls using Atomic Force Microscopy ( AFM ) with a 200 nm indentation .","We found that cell wall stiffness in ktn1-5 papilla cells was about 30% lower than that in stage 13 WT cells ( Figure 6E , F ) .","To confirm this result , we investigated the stiffness of the papilla cell wall on WT stigmas at stage 15 , where increased coiled pollen tubes were detected ( Figure 2A , B ) .","We found the cell wall to be softer than that of papillae at stage 13 but stiffer than that of the ktn1-5 ( Figure 6E , F ) .","We then reasoned that the presence of softer walls should also affect the pollen tube growth rate , stiffer walls reducing growth .","Thus , measuring the growth rate would provide an indication of the resistance force encountered by the tube while growing in the papilla cell wall .","We thus monitored the growth rate of Col-0 pollen tubes in Col-0 and ktn1-5 papillae .","We found that pollen tubes grew faster ( ~x 1 . 8 ) within ktn1-5 papillae ( Figure 6G ) .","Similarly , we found that pollen tube growth on stage 15 papillae was faster ( ~x 1 . 6 ) than on stage 13 papillae ( Figure 6H ) .","These results suggest that the coiled phenotype is associated with a higher pollen tube growth rate and hence , that papilla cell walls of ktn1-5 and stage-15 papillae exhibit less resistance to tube penetration .","Altogether , these data suggest that KATANIN-dependent mechanical properties of papilla cell wall play a role in pollen tube guidance .","Because CMT disorganisation in papillae affects both wall stiffness and mechanical anisotropy , we next investigated the relative contribution of these two parameters in pollen tube growth .","Hypocotyl cells of the xxt1 xxt2 double mutant were reported to display CMT orientation defects and reduced stiffness of the cell wall ( Xiao et al . , 2016 ) , features that we found in ktn1-5 papilla cells .","However , growing pollen tubes on xxt1 xxt2 stigmas did not coil and grew like on WT stigmas , questioning whether the cell wall stiffness of stigmatic cells was actually affected in the mutant .","Using AFM , we found the cell wall of xxt1 xxt2 papillae to be about 30% softer than that of Col-0 papillae , i . e . very similar to that of ktn1-5 ( Figure 6F ) .","Despite this similarity , the pollen tube growth rate in xxt1 xxt2 stigmas was identical to Col-0 stigmas at stage 13 ( Figure 6H ) .","In addition , contrary to ktn1-5 , pollen tubes were not prominent while growing in xxt1 xxt2 papilla cell walls ( Figure 5—figure supplement 3G ) .","These results suggested that although ktn1-5 and xxt1 xxt2 papillae had similar cell wall stiffness as inferred from AFM measurements , they exhibited different mechanical constraints to pollen tube growth .","Because of the close relationship between CMT organisation , CMF deposition and cell wall rigidity ( Xiao et al . , 2016; Xiao and Anderson , 2016 ) , we suspected that CMT and CMF organisation might be different between the two mutants and be the possible causal agent of the mechanical differences and related tube coiled phenotype observed in ktn1-5 .","To check this possibility , and to gain insight into the predominant cellulose pattern in the stigmatic cell walls , we stained CMFs by using Direct Red 23 .","We found cellulose fibres to be highly aligned and slanted to the longitudinal axis of the papilla in stage 13 Col-0 stigmas , whereas ktn1-5 and stage 15 Col-0 papillae both displayed a clearly disordered CMF pattern ( Figure 7A ) with CMFs forming thicker and more spaced bundles ( Figure 7B ) .","By contrast , CMFs in xxt1 xxt2 papillae at stage 13 organised in a dense and well oriented pattern of fibres , mostly perpendicular to the papilla long axis , which resembled Col-0 stage 13 CMF organisation ( Figure 7A , B ) .","These results suggest that KTN1 loss-of-function alters mechanical properties of the papilla cell wall in a complex manner , lowering both its stiffness to pressure applied perpendicularly to its surface ( e . g . , by the AFM indenter ) and its resistance to pollen tube progression .","This latter modification appears as the main cause of the pollen tube coiled phenotype .","By contrast , loss of XXT1 and XXT2 functions , by perturbing cell wall stiffness to indenter pressure only , does not induce change in pollen tube growth .","Altogether , our data suggest that the mechanical anisotropy of papilla cell walls plays a key role in pollen tube trajectory ."],["Previous work reported that rearrangements of actin microfilaments ( Iwano et al . , 2007; Rozier et al . , 2020 ) and destabilization of CMTs ( Samuel et al . , 2011 ) occur in stigmas following compatible pollination in Brassicaceae species .","However , CMT pattern and dynamics during papilla development have never been described .","Our data show that during the course of stigma maturation , which is associated with papilla cell elongation , CMT bundles progressively move from perpendicular ( anisotropic ) to the elongation axis at stage 12 to disorganised ( isotropic ) at stage 15 .","This correlates with the known CMT dynamics during elongation in plant cells , where CMT arrays are highly anisotropic in young cells and become more isotropic as cells differentiate ( Baskin , 2005; Landrein and Hamant , 2013 ) .","During invasion of plant cells by fungal or Oomycete pathogens or upon mechanical stimulation , the network of microtubules is rearranged , generally associated first with the depolymerization of CMTs at the contact site and later with the formation of an array of microtubules surrounding the invading organism or microneedle ( Hardham , 2013 ) .","Interestingly , penetration of the pollen tube in the papilla cell wall does not lead to such microtubule reorganisation ( Figure 3 ) .","This suggests that at the cellular level , the invading pollen tube does not elicit a defence-like response in the papilla , which accepts and supports pollen tube growth .","In addition , we show that when the stigma is manually pressed between slide and coverslip , the mechanical stimulation applied perpendicularly to the wall leads to CMT fragmentation ( Figure 3—figure supplement 1 ) .","This indicates that although the papilla microtubule network is sensitive to mechanical forces , the pressure exerted by the pollen tube is not strong enough to perturb CMT organisation .","One possible explanation is that the penetration force developed by the growing pollen tube acts mainly in plane to the cell wall surface and hence would only have a little effect on the papilla cell , contrary to a pressure applied perpendicularly to the wall .","Our data are in contradiction with the earlier observation that growing pollen tube provokes fragmentation of CMTs in the papilla ( Samuel et al . , 2011 ) .","This discrepancy is likely to relate to the microscopic approaches used between the two studies , live imaging and a fluorescent microtubule probe in our case , chemically fixed material and immunostaining of microtubules in Samuel et al . , 2011 .","The progressive randomisation of CMT orientation we observed in papilla cells during ageing is accompanied by an increased coiled growth of pollen tubes in papillae .","Similarly , when CMTs are destabilized by the microtubule depolymerizing drug oryzalin , coiled pollen tubes are more frequently observed compared with untreated control stigmas .","These results reveal a link between the stigmatic CMT cytoskeleton organisation and the trajectory that pollen tube takes while growing in the papilla cell wall , although the effects of oryzalin on cell wall properties remain unknown .","The fact that the most striking effect on pollen tube growth was found on ktn1-5 mutant suggests that the coiled phenotype depends not only on the CMT organisation but implicates other factors .","Among various phenotypic alterations described in loss-of-function mutants for KTN1 , are the impaired cell mechanical properties and cell elongation , defects in CMT organisation , cell wall composition and CMF orientation ( Burk et al . , 2001; Burk and Ye , 2002; Ryden et al . , 2003 ) .","Interestingly , we found that the protuberance of pollen tubes at the surface of ktn1-5 papillae was associated with a faster growth rate and a lower rigidity of the cell wall compared with Col-0 ( Figure 6 ) .","This places mechanics of the cell wall as a likely component involved in the coiled phenotype .","Surprisingly , of the six cell wall mutants we analysed , including xxt1 xxt2 and prc1 known to exhibit both abnormal CMT organisation and softer cell walls , none induced the coiled phenotype displayed by ktn1-5 papillae .","More remarkably , despite the similar stiffness of xxt1 xxt2 and ktn1-5 papilla cell walls as measured by AFM , pollen tubes behaved differently on these two stigma types , pollen tubes not only coiling but also growing faster on ktn1-5 stigmas .","This observation is in accordance with the recent findings that changes in indentation properties of plant cell walls do not correlate with changes in tensile stiffness as described in cell-free strips of onion epidermal walls ( Zhang et al . , 2019 ) .","In our experiments , indentations tell us about the elasticity of the wall to a pressure normal to the plane of the wall surface ( i . e . out-of-plane mechanics ) , while pollen tube growth rates inform about the resistance of the wall to a force in-plane to the papilla wall ( i . e . in-plane mechanics ) .","Several lines of evidence lead us to suggest that mechanical properties of the cell wall are not identical in the two mutants .","Previous work showed that cellulose crystallinity is one factor implicated in cell wall mechanics .","Indeed , the epidermal cells of the inflorescence stem in the any1 mutant have the relative amount of crystalline cellulose significantly reduced , while the total content of cellulose in the cell wall is unaltered , and this is accompanied by reduced stiffness of the cell wall ( Fujita et al . , 2013; Altartouri et al . , 2019 ) .","Our chemical analysis reveals that the cell wall composition is different between xxt1 xxt2 and ktn1-5 stigmas , with this latter having 25% more crystalline cellulose , and hence should exhibit stiffer cell walls ( Figure 5—figure supplement 4 ) .","This data is at odds with AFM measurements but a possible explanation would be that the two layers detected by TEM in the papilla cell wall ( Figure 6—figure supplement 1 ) display different mechanical properties , the inner layer being stiffer than the outer one in the ktn1-5 mutant .","This may explain the protruding growth of pollen tubes observed in ktn1-5 papillae , the tube pushing away the softer outer layer but not the inner ( Figure 6A–D ) .","Interestingly , Zhang et al . , 2019 reported that cellulose networks largely determine in-plane mechanics whereas out-of-plane mechanics depends on both homogalacturonan ( pectin ) and cellulose networks .","Cell walls of the qua2-1 mutant are specifically affected in pectins , with a 50% reduction in homogalacturonan content compared with Col-0 without observable changes in other polysaccharides ( Mouille et al . , 2007 ) .","In light of these data , it is noteworthy that papilla cells of the qua2-1 mutant do not induce pollen tube turns ( Figure 5—figure supplement 3 ) , which suggests that pectins play no significant role in the pollen tube phenotype and that the main factor contributing to pollen tube guidance is in-plane mechanics , and hence relies on the cellulose network .","The orientation of the rigid CMFs is another determinant of cell wall mechanics .","The ktn1-5 mutant was described to have a severe reduction in cell length and an increase in cell width in all organs ( Burk et al . , 2001 ) .","This cellular phenotype was attributed to the distorted deposition of CMFs correlated with the isotropic orientation of CMTs , whereas in WT cells , CMFs like CMTs are oriented perpendicularly to the elongation axis ( Burk and Ye , 2002 ) .","Similarly , we found that orientation of CMFs is also altered in ktn1-5 papillae ( Figure 7 ) and this was associated with larger papilla cells ( Figure 5—figure supplement 5 ) .","Hence , we may assume that mechanical anisotropy , described as the cell wall anisotropy made by the orientation of the rigid CMFs ( Sassi et al . , 2014 ) , is impaired in ktn1-5 papillae .","Contrary to the ktn1-5 mutant , papillae of the xxt1 xxt2 double mutant display an anisotropic CMF pattern with parallel fibres approximatively oriented transversally to the papilla axis .","These data are in agreement with those reported for etiolated hypocotyl cells , where CMFs are largely parallel to one another , straighter than in WT and oriented almost transversely to the long axis of the cell ( Xiao et al . , 2016 ) .","In addition , we found the papilla cell shape of xxt1 xxt2 stigmas to be closer to that of Col-0 than that of ktn1-5 papillae , which is consistent with an anisotropic growth of the papillae in the xxt1 xxt2 mutant compared with ktn1-5 .","Apart from the similar stiffness of papilla cell walls as deduced from AFM measurements , the main differences between ktn1-5 and xxt1 xxt2 cell walls are the cell wall composition , orientation of CMFs , and possible changes in molecular connections between cell wall components and plasma membrane and/or cytoskeleton proteins .","Indeed , a recent proteomic study revealed that loss of KATANIN function is associated with the decrease in abundance of several cytoskeleton proteins , such as profilin 1 , actin-depolymerizing factor 3 and actin 7 ( Takáč et al . , 2017 ) , whereas targeted quantitative RT-PCR showed that several Microtubule-associated protein ( MAP ) and wall signal receptor genes showed lower expression levels in xxt1 xxt2 ( Xiao et al . , 2016 ) .","In this latter study , KTN1 expression level was shown to be unchanged compared with Col-0 .","Altogether , our study suggests that KTN1 , by maintaining the papilla mechanical anisotropy , has a key function in mediating early pollen tube guidance on stigma papillae .","The coiled phenotype was not only observed in ktn1-5 but also in the WT Col-0 papillae at stage 15 .","Remarkably , several lines of evidence reveal that papilla cells from stage 15 share common features with ktn1-5 papillae , such as CMT and CMF increased isotropy , decreased stiffness of the cell wall as deduced from AFM and less resistance of the wall to pollen tube growth .","Isotropic orientation of the cytoskeleton at stage 15 is likely to relate to cell elongation , which is known to be accompanied by cytoskeleton reorganisation ( Crowell et al . , 2011; Zhang et al . , 2014 ) .","At the organ level , it has been suggested that the mechanical anisotropy of the wall restrains organ emergence ( Sassi et al . , 2014 ) .","The authors propose that for the same wall stiffness , a cell wall with isotropic properties would lead to larger outgrowth than a wall with anisotropic properties .","Our data are consistent with this hypothesis , albeit at the subcellular scale , since large protuberance of papilla wall following pollen tube growth is observed in ktn1-5 papilla cells , exhibiting walls with isotropic properties .","It remains unclear how mechanical anisotropy guides pollen tube growth .","We can hypothesise that as pollen tube grows inside the wall , it encounters recently deposited CMFs on the inner side of the wall ( facing the cytoplasm ) and older CMFs on the outer side .","It is likely that these layers have different mechanical properties related to CMF orientation ( Baskin , 2005 ) .","KATANIN , by acting on CMT dynamics and CMF organisation , would fine-tune the mechanical properties of the matrix through which the pollen tube grows .","It is worth noting that growth rate of pollen tubes germinated in vitro is slowed down when pollen tubes pass through a microgap of a microfluidic device , the tubes adapting their invasive force to the mechanical constraints ( Sanati Nezhad et al . , 2013 ) .","Based on measurements of pollen tube growth rates , our data show that the tube tip , while progressing in the papilla wall , senses the mechanical features of its environment and reacts accordingly .","Hence , it reveals some unanticipated internal and hidden properties of the cell wall .","Our study shows that mechanics plays a key role in early pollen tube guidance in the papilla cell .","This role is mediated by a specific CMT/CMF organisation and mechanical anisotropy of the papilla cell , which both are dependent on KTN1 .","Importantly , these specific mechanical properties of the stigmatic cells prevent emerging pollen tubes to grow upward on papillae and straighten pollen tube direction , helping the tube to find its correct path to the stylar transmitting tract .","Our findings also raise the question about the biological importance of the mechanical changes that occur during stigma development .","Indeed , for aged stigmas , modifications of the mechanical properties of the cell wall , accompanied by an acceleration of pollen tube growth , could be seen as beneficial for the plant by favouring ultimate fertilization and seed set on old flowers , and hence supporting dissemination of the species .","This assumption is particularly relevant given that the pistil length increases with ageing and similarly the journey the pollen tube has to travel to reach the ovules .","A recent work showed that ageing is associated with decreased fertility due to stigma senescence , which is initiated at stage 16–17 ( Gao et al . , 2018 ) .","The faster the pollen tube will grow in old stigmas , the more chance it will have to circumvent papilla cell death and hence to fertilize the ovule .","We may suggest that KTN1 , by mediating mechanical anisotropy of stigmatic cells and promoting pollen tube growth in old stigmas , has played an evolutionary role in the success of fertilization .","To conclude , we uncovered a yet unexpected function for KTN1 in early pollen tube guidance on the stigma .","In addition , our study also clearly unveils that the mechanical properties of one single cell ( e . g . , the stigmatic papilla ) impact the behaviour of its neighbouring cell ( e . g . , the pollen tube ) ."],["Arabidopsis thaliana , ecotype Columbia ( Col-0 ) , Arabidopsis transgenic plants generated in this study and Arabidopsis mutants were grown in soil under long-day conditions ( 16 hr of light/8 hr of dark , 21 °C / 19°C ) with a relative humidity around 60% .","ktn1-5 ( SAIL_343_D12 ) , xxt1xxt2 , prc1 . 1 , qua2 . 1 , xyl1 . 4 , kor1 . 1 and any1 mutant lines were described previously ( Cavalier et al . , 2008; Fagard et al . , 2000; Fujita et al . , 2013; Lin et al . , 2013; Mouille et al . , 2007; Nicol et al . , 1998; Sampedro et al . , 2010; Shoji et al . , 2004 ) .","All mutants were in Col-0 background except kor1 . 1 which was in WS .","All stigmas were analysed at stage 13 of flower development ( Smyth et al . , 1990 ) except when specified .","We used the Gateway technology ( Life Technologies , USA ) and two sets of Gateway-compatible binary T-DNA destination vectors ( Hellens et al . , 2000; Karimi et al . , 2002 ) for expression of transgenes in A . thaliana .","The DNA fragment containing the Brassica oleracea SLR1 promoter was inserted into the pDONP4-P1R vector .","The 165 bp-LTI6B fragment was introduced into the pDONR207 vector .","MAP65 gene spanning the coding region from start to stop codons was introduced into the pDONR221 vector .","CDS from citrine or GFP were cloned into the pDONP2R-P3 vector .","Final constructs , pSLR1::MAP65-citrine and pSLR1::LTI6B-GFP were obtained by a three-fragment recombination system ( Life Technologies ) using the pK7m34GW and the pB7m34GW destination vectors , respectively .","We generated a pACT11::RFP construct by amplifying the promoter of the A . thaliana ACTIN-11 gene and cloning it into the pGreenII gateway vector in front of the RFP coding sequence .","Transgenic lines were generated by Agrobacterium tumefaciens-mediated transformation of A . thaliana Col-0 as described ( Logemann et al . , 2006 ) .","Unique insertion lines , homozygous for the transgene were selected .","We introduced the pSLR1::LTI6B-GFP or pSLR1::MAP65-citrine construct in ktn1-5 background by crossing and further selecting the progeny on antibiotic containing medium .","Flowers at stage 13 and 15 were emasculated and pollinated on plants with mature pollen from the pACT11::RFP line .","Immediately after pollination , stigmas were mounted between two coverslips maintained separated by four grease plugs placed at each coverslip corner .","To maintain a constant humidity without adding liquid directly on the stigma surface , we used a wet piece of tissue in contact with the base of the stigma .","Pollinated stigmas were observed under a Zeiss microscope ( AxioObserver Z1 ) equipped with a spinning disk module ( CSU-W1-T3 , Yokogawa ) using a 40x Plan-Apochromat objective ( numerical aperture 1 . 1 , water immersion ) .","Serial confocal images were acquired in the entire volume of the stigma every 1 µm and every minute .","Images were processed with Image J software and pollen tube lengths were measured .","Pistils were placed straight in a 2% agar MS medium and 0 . 8% low-melting agarose was added up to a certain level where the papilla cells were maintained well immobilised in agarose while leaving the top accessible to the indenter .","This set up allowed accurate measurements of cell wall stiffness on the dome-shaped top of papilla cells .","AFM indentation experiments were carried out with a Catalyst Bioscope ( Bruker Nano Surface , Santa Barbara , CA , USA ) that was mounted on an optical microscope ( MacroFluo , Leica , Germany ) equipped with a x10 objective .","All quantitative measurements were performed using standard pyramidal tips ( RFESP-190 ( Bruker ) ) .","The tip radius given by the manufacturer was 8–12 nm .","The spring constant of the cantilever was measured using the thermal tune method and was 35 N/m .","The deflection sensitivity of the cantilever was calibrated against a sapphire wafer .","All experiments were made in ambient air at room temperature .","Matrix of 10 × 10 measurements ( step 500 nm ) was obtained for each papilla , with a 1µN force .","The Young’s Modulus was estimated using the Nanoscope Analysis ( Bruker ) software , using the Sneddon model with a < 200 nm indentation .","Flowers from stages 12 to 15 were emasculated and pollinated on plants with mature WT pollen .","One hour after pollination , pistils were cut in the middle of the ovary , deposited on a SEM platform and observed under Hirox SEM SH-3000 at −20°C , with an accelerating voltage of 15kV .","Images were processed with ImageJ software and pollen tube direction was quantified by counting the number of turns made by the tube , only on papillae that received one unique pollen grain .","Stage 13 flowers were emasculated and pollinated on plants with mature WT pollen .","One hour after pollination , pistils were immersed in fixative solution containing 2 . 5% glutaraldehyde and 2 . 5% paraformaldehyde in 0 . 1 M phosphate buffer ( pH 7 . 2 ) and after 4 rounds of 30 min vacuum , they were incubated in fixative for 12 hr at room temperature .","Pistils were then washed in phosphate buffer and further fixed in 1% osmium tetroxide in 0 . 1 M phosphate buffer ( pH 7 . 2 ) for 1 . 5 hr at room temperature .","After rinsing in phosphate buffer and distilled water , samples were dehydrated through an ethanol series , impregnated in increasing concentrations of SPURR resin over a period of 3 days before being polymerized at 70°C for 18 hr , sectioned ( 65 nm sections ) and imaged at 80 kV using an FEI TEM tecnaiSpirit with 4 k x 4 k eagle CCD .","Flowers from MAP65 lines were emasculated and stigmas were observed under confocal microscope .","Images were processed with ImageJ software and quantitative analyses of the average orientation and anisotropy of CMTs were performed using FibrilTool , an ImageJ plug-in ( Boudaoud et al . , 2014 ) .","Anisotropy values range from 0 to 1; 0 indicates pure isotropy , and one pure anisotropy .","A droplet of Adigor was applied at 2 . 5% ( v/v in water ) on the stigma during 4 hr to facilitate subsequent chemical treatments .","As described for hypocotyls ( Landrein and Hamant , 2013 ) , pistils were incubated in 12 . 5% glacial acetic acid for 1 hr and dehydrated in 100% and then 50% ethanol for 20 min each .","Pistils were then washed in water during 20 min and stored in 1M KOH during 2 days .","Pistils were then stained using 0 . 02% ( w/v ) Direct Red 23 dye ( Sigma-Aldrich ) during 4 hr and washed with distilled water .","Pistils were observed with a mRFP filter ( excited at 561 nm ) under a Zeiss LSM 880 confocal microscope using a 63x Plan-Apochromat objective ( numerical aperture 1 . 4 , oil immersion ) .","Stigmas were imaged by taking a z-stack of 0 . 2 μm sections in the papilla cells .","Image analysis was performed by doing a 3D-projection and measuring the fluorescence intensity longitudinally along the papilla axis using ImageJ software .","Flowers from LTI6B lines were emasculated and pollinated with mature pollen from the pACT11::RFP line .","20 min after pollination , stigmas were observed under confocal microscope .","Serial confocal images every 1 µm encompassing the entire volume of the stigma were recorded and processed with ImageJ software .","Plasma membrane deformation was estimated by choosing the slide from the stack that corresponded to the focus plan of the contact site with the RFP-labelled pollen tube .","On the bright field image corresponding to the selected slide , a line was drawn connecting the two ridges of the invagination of the papilla .","Two perpendicular lines , one toward the exterior ( ExtD ) of the papilla to the maximum point of deformation , and the other toward the interior ( IntD ) on the GFP image were measured , respectively .","To avoid contact of pollen grains with liquid , we performed local applications of oryzalin ( Chemical service , Supelco ) as described ( Sassi et al . , 2014 ) , using lanolin pasted around the style , just under the stigmatic cells , at 833 µg/mL ( DMSO ) , for 4 hr at 21°C .","Oryzalin-treated pistils were pollinated with mature WT pollen and 1 hr after pollination observed under SEM .","XyG composition of stigmas was performed following the oligosaccharide fingerprinting set up by Lerouxel et al . , 2002 .","10 stigmas per genotype were dissected and kept in 96% ethanol .","After ethanol removal , XyG oligosaccharides were generated by treating samples with endoglucanase in 50 mM sodium acetate buffer , pH 5 , overnight at 37°C .","Matrix-assisted laser-desorption ionization time of flight mass spectrometry of the XyG oligosaccharides was recorded with a MALDI/TOF Bruker Reflex III using super-DHB ( 9:1 mixture of 2 , 5-dihydroxy-benzoic acid and 2-hydroxy-5-methoxy-benzoic acid; Sigma-Aldrich , sigmaaldrich . com ) as matrix .","Alcohol insoluble residues were obtained from around 3 to 5 mg of fresh pistils .","They were collected for analysis and fixed in 96% ethanol before incubation for 30 min at 70°C .","The pellet was then washed twice with 96% ethanol and twice with acetone .","The remaining pellet is called alcohol insoluble residues ( AIR ) and was dried in a fume hood overnight at room temperature .","Cell wall monosaccharide content of the non‐cellulosic fraction was determined by hydrolysis of all the AIR obtained from the pistils with 2 M TFA for 1 hr at 120°C .","After cooling and centrifugation , the supernatant was dried under a vacuum , resuspended in 200 μl of water and retained for analysis .","To obtain the glucose content of the crystalline cellulose fraction , the TFA-insoluble pellet was further hydrolysed with 72% ( v/v ) sulfuric acid for 1 hr at room temperature .","The sulfuric acid was then diluted to 1 M with water and the samples incubated at 100°C for 3 hr .","All samples were filtered using a 20‐μm filter caps , and quantified by HPAEC‐PAD on a Dionex ICS‐5000 instrument ( ThermoFisher Scientific ) as described ( Fang et al . , 2016 ) .","Graph and statistics were obtained with R software or Excel .","Statistical tests performed are specified in figure legends ."]],"string":"[\n [\n \"Following deposition of dehydrated pollen grains on the receptive surface of the female organ , the stigma , pollen rehydrates , germinates and produces a pollen tube that carries the male gametes toward the ovules where the double fertilization takes place .\",\n \"This long itinerary through the different tissues of the pistil is finely controlled , avoiding misrouting of the pollen tube and hence assuring proper delivery of the sperm cells to the female gametes .\",\n \"How the pollen germinates a tube and how pollen tube growth is regulated have been the object of many investigations ( Palanivelu and Tsukamoto , 2012; Mizuta and Higashiyama , 2018 ) .\",\n \"The use of in vitro pollen germination as well as semi-in vivo fertilization assays , together with the analysis of mutants defective in pollen or ovule functions , have rapidly expanded our knowledge of the mechanisms that sustain pollen tube growth , its guidance toward the ovule and the final delivery of male gametes within the embryo sac ( Dresselhaus and Franklin-Tong , 2013; Higashiyama and Yang , 2017; Cameron and Geitmann , 2018 ) .\",\n \"At the cellular level , cytoskeleton has been extensively studied during pollen tube elongation ( Fu , 2015 ) , highlighting the critical role played by the actin microfilaments in pollen-tube tip growth through delivery of materials for the biosynthesis of the plasma membrane and cell wall .\",\n \"By contrast , CMTs seem to have a lower importance in the pollen tube elongation process , drugs affecting CMT polymerisation having no significant effects on pollen-tube growth rate , yet altering the capacity of pollen tubes to change their growth direction ( Gossot and Geitmann , 2007 ) .\",\n \"In Arabidopsis thaliana , pollen tubes grow within the cell wall of papillae of the stigmatic epidermis , and then through the transmitting tissue of the style and ovary ( Lennon and Lord , 2000 ) .\",\n \"The transmitting tissue has an essential function in pollen tube guidance , providing chemical attractants and nutrients ( Crawford and Yanofsky , 2008; Higashiyama and Hamamura , 2008 ) .\",\n \"In contrast to these accumulating data showing the existence of factors mediating pollen tube growth in the pistil , whether guidance cues exist at the very early stage of pollen tube emergence and growth in the papilla cell wall remains largely unknown .\",\n \"The cell wall constitutes a stiff substrate and hence a mechanical barrier to pollen tube progression .\",\n \"There are numerous examples in animal cells demonstrating that mechanical properties of the cellular environment , and in particular rigidity , mediate cell signalling , proliferation , differentiation and migration ( Discher et al . , 2005; Fu et al . , 2010; Ermis et al . , 2018 ) .\",\n \"In plant cells , cell wall rigidity depends mainly on its major component , cellulose , which is synthesized by plasma membrane-localized cellulose synthase complexes ( CSCs ) moving along cortical microtubule ( CMT ) tracks ( Paredez et al . , 2006 ) .\",\n \"While penetrating the cell wall , the pollen tube exerts a pressure onto the stigmatic cell ( Sanati Nezhad and Geitmann , 2013 ) .\",\n \"Physical forces are known to reorganise the cortical microtubules ( CMTs ) , which by directing CSCs to the plasma membrane , reinforce wall stiffness by novel cellulose microfibril ( CMF ) synthesis ( Paredez et al . , 2006; Sampathkumar et al . , 2014 ) .\",\n \"Hence , there is an intricate interconnection between CMT organisation , CMF deposition and cell wall rigidity ( Xiao and Anderson , 2016 ) .\",\n \"A major regulatory element of CMT dynamics is the KATANIN ( KTN1 ) microtubule-severing enzyme , which allows CMT reorientation following mechanical stimulation ( Uyttewaal et al . , 2012; Sampathkumar et al . , 2014; Louveaux et al . , 2016 ) .\",\n \"Here we investigated whether the CMT network of papilla cells might contribute to pollen tube growth and guidance in stigmatic cells by combining cell imaging techniques , genetic tools , chemical analysis of the cell wall and atomic force microscopy .\",\n \"We show that isotropic reorientation of CMTs occurs in aged Col-0 and ktn1-5 papilla cells , which is accompanied by a change in the growth direction of pollen tubes that tend to make coils on papillae .\",\n \"We show that CMT reorganisation is associated with isotropic rearrangement of CMFs , high content of crystalline cellulose and particular cell wall mechanics of ktn1-5 stigmas .\",\n \"Altogether , our results indicate that cytoskeleton dynamics and mechanical properties of the cell wall , which both depend on KTN1 activity , have a major role in guiding early pollen tube growth in stigma papillae .\"\n ],\n [\n \"To assess the functional role of stigmatic CMTs in pollen-papilla cell interaction , we first analysed their organisation in papillae at stages 12 to 15 of stigma development as described ( Smyth et al . , 1990; Figure 1A , B ) .\",\n \"We generated a transgenic line expressing the CMT marker MAP65 . 1-citrine under the control of the stigma-specific promoter SLR1 ( Fobis-Loisy et al . , 2007 ) .\",\n \"Before ( stage 12 ) and at anthesis ( stage 13 ) , where flowers are fully fertile ( Kandasamy et al . , 1994 ) , the CMTs were aligned perpendicularly to the longitudinal axis of papilla cells and were highly anisotropic ( median value of 0 . 40 ) ( Figure 1C , D ) .\",\n \"At stage 14 , when anthers extend above the stigma , the CMT pattern became less organised , with a higher variability in anisotropy values .\",\n \"Finally , at stage 15 when the stigma extends above anthers , CMT anisotropy had a median value of 0 . 09 indicative of an isotropic orientation of CMTs ( Figure 1C , D ) .\",\n \"These findings reveal that the papilla CMT cytoskeleton is dynamic during development , with a change of CMT array orientation from anisotropy to isotropy .\",\n \"We then wondered whether this change in CMT organisation could be correlated with pollen tube growth .\",\n \"To this end , we self-pollinated Col-0 papillae from stages 12 to 15 and examined pollen tube growth one hour after pollination by scanning electron microscopy ( SEM ) ( Figure 2A ) .\",\n \"At stage 12 and 13 , we found most ( ~60% ) pollen tubes grew straight in the papillae , whereas about 30% and 10% of tubes made half-turn or one turn around stigmatic cells , respectively ( Figure 2B ) .\",\n \"At later stages of development , the tendency to coil around the papillae increased , with 3 . 5-fold more pollen tubes at stage 14 and 5 . 5-fold more at stage 15 making one or more than one turn around papillae .\",\n \"These results suggest that CMT organisation in the papilla impacts the direction of growth of pollen tubes and that loss of CMT anisotropy is associated with coiled growth .\",\n \"We next asked whether the pressure exerted by the growing pollen tube in the cell wall may induce changes in the CMT organisation of the papilla cell .\",\n \"To this end , we set up a live imaging system where pollinated stigmas were maintained in air so as to prevent immediate hydration and burst of pollen grains .\",\n \"During the time course of the experiment , starting at the beginning of pollen tube emergence on the stigma papilla at stage 13 ( t0 ) up to 13 min after germination , we found no major alteration of CMT arrays from early to late penetration stages ( Figure 3A ) .\",\n \"This result was in contradiction with previous immunostaining analysis that showed fragmentation of CMTs following compatible pollination in Brassica napus ( Samuel et al . , 2011 ) .\",\n \"To confirm our result and gain better insight into CMT organisation , we then observed pollinated stigmas at a single time point , that is 30 min after pollination , using a mounting medium to get higher resolution images .\",\n \"Again , we did not detect any reorganisation or degradation of CMTs ( Figure 3B ) .\",\n \"However , we found the stability of CMTs to be very sensitive to strong pressure , as when the coverslip was applied not cautiously on the stigma , a break-down of CMT arrays occurred ( Figure 3—figure supplement 1 ) .\",\n \"These results indicate that the pressure exerted by the pollen tube while growing in the papilla cell wall does not alter the organisation of stigmatic CMTs .\",\n \"To test the direct impact of stigma CMTs on pollen tube growth direction , we examined whether the destabilization of CMTs in Col-0 papillae could affect pollen tube growth .\",\n \"To this end , we treated stigmas by local application of the depolymerizing microtubule drug oryzalin in lanolin pasted around the style .\",\n \"After 4 hr of drug treatment , no more CMT labelling was detected in papillae , while CMTs were clearly visible in mock-treated ( DMSO ) stigmas ( Figure 4A ) .\",\n \"Stigmas were then pollinated with Col-0 pollen and one hour later observed by SEM .\",\n \"Pollen tubes were found to make one turn more frequently on drug-treated than on control papillae , with more than a 3-fold increase of tubes making one turn ( Figure 4B , C ) .\",\n \"To confirm the relation between stigma CMTs and pollen behaviour , we examined pollen tube growth on stigmas of the katanin1-5 mutant , which is known to exhibit reduced CMT array anisotropy in root cells ( Bichet et al . , 2001; Burk et al . , 2001; Burk and Ye , 2002 ) .\",\n \"Because the CMT organisation in ktn1-5 papillae is unknown , we crossed ktn1-5 with the MAP65-1-citrine marker line and found that CMT arrays were more isotropic in ktn1-5 papillae when compared with those of the WT ( Figure 5A , C ) .\",\n \"We then analysed Col-0 pollen behaviour on ktn1-5 stigmatic cells at stage 13 ( Figure 5B ) .\",\n \"We observed that Col-0 pollen tubes acquired a strong tendency to coil around ktn1-5 papillae , with more than 60% of tubes making one or more than one turn around papillae , sometimes making up to six turns , before reaching the base of the cell ( Figure 5D; Figure 5—figure supplement 1A–C ) .\",\n \"In some rare cases , pollen tubes even grew upward in the ktn1-5 mutant and appeared blocked at the tip of the papilla ( Figure 5—figure supplement 1B , D ) .\",\n \"The number of coils was significantly higher than on oryzalin-treated papillae .\",\n \"Indeed , 25% of the pollen tubes made at least two coils in ktn1-5 papillae whereas this percentage represented only 4% on the oryzalin treated stigmas ( Figure 4C; Figure 5D ) .\",\n \"To check whether papilla receptivity to pollen might be impaired in ktn1-5 stigmas , we examined hydration and tube growth of Col-0 pollen deposited on stage-13 Col-0 or ktn1-5 stigmas .\",\n \"We found no significant differences in pollen hydration related to the stigma type ( Figure 5—figure supplement 2A ) and pollen tubes grew in the transmitting tract and reached the ovules in ktn1-5 pistils like in Col-0 ( Figure 5—figure supplement 2B ) .\",\n \"We then wondered whether alteration of the KATANIN function in pollen might affect pollen tube growth directionality on Col-0 or ktn1-5 stigmas .\",\n \"Cross-pollination revealed that ktn1-5 pollen tubes grew like Col-0 pollen tubes on stage-13 Col-0 papillae , while they coiled on ktn1-5 papillae ( Figure 5—figure supplement 2C ) .\",\n \"Seed counts showed that fertility slightly decreased when ktn1-5 pollen was used to pollinate Col-0 stigmas , and was strongly affected when ktn1-5 was used as female , irrespective of whether the pollen was Col-0 or ktn1-5 ( Figure 5—figure supplement 2D ) .\",\n \"This indicates that the fertilisation process is impaired in ktn1-5 ovules .\",\n \"Altogether , these results show that the coiled phenotype observed on ktn1-5 stigmas is only dependent on defects occurring at the papilla level , that the stigma receptivity of ktn1-5 mutant is not altered and confirm that stigmatic CMTs , somehow , contribute to the directional growth of pollen tubes in papilla cells .\",\n \"The katanin1/fra2 mutant was initially described as a mutant impaired in cell wall biosynthesis and CMT array organisation ( Burk et al . , 2001 ) .\",\n \"This prompted us to investigate whether other mutants affected in cell wall biogenesis might exhibit the coiled pollen tube phenotype .\",\n \"We selected mutants impaired in the cellulose synthase complex ( kor1 . 1 , prc1 and any1 ) , hemicellulose biosynthesis ( xxt1 xxt2 , xyl1 . 4 ) and pectin content ( qua2 . 1 ) , for which expression of the corresponding genes in stigma was confirmed ( Figure 5—source data 2 ) .\",\n \"Strikingly , none of the 6 cell wall mutants displayed the coiled pollen tube phenotype ( Figure 5—figure supplement 3A–F ) .\",\n \"To ascertain that the cell wall composition was altered in the stigma papillae of ktn1-5 and cell wall mutants , we undertook sugar analysis of ktn1-5 and xxt1 xxt2 stigmas and compared them with that of Col-0 .\",\n \"We chose the xxt1 xxt2 mutant because its cell wall composition had been already well described ( Cavalier et al . , 2008; Xiao et al . , 2016 ) .\",\n \"The relative abundance of monosaccharides in pistil cell walls varied between the three genotypes with major differences detected in the ktn1-5 cell wall composition that had an increased abundance of glucose , this latter deriving either from hemicellulose and amorphous cellulose or from crystalline cellulose ( Figure 5—figure supplement 4A ) .\",\n \"ktn1-5 cell wall was characterized by the highest relative abundance of crystalline cellulose , which was 25% higher than in Col-0 and xxt1 xxt2 .\",\n \"To explore whether side chains of hemicellulose might also be affected in the katanin mutant , we performed an enzymatic fingerprinting of xyloglucans from dissected stigmas of Col-0 and ktn1-5; xxt1xxt2 was not analysed as it lacks xyloglucan side chains .\",\n \"We found in ktn1-5 a decrease in relative abundance of short side chains with terminal xylose ( XXG and XXXG ) and an increase in the longer ones with terminal fucose ( e . g XXFG ) ( Figure 5—figure supplement 4B ) .\",\n \"The chemical analysis confirms that the cell wall composition is altered in ktn1-5 stigmas and presents some specific features compared with Col-0 and xxt1 xxt2 stigmas .\",\n \"However , combined with the results of pollination on the six cell wall mutants , we cannot establish a causal effect of chemical alterations on the pollen tube coiled phenotype .\",\n \"This suggests that the relation between CMT organisation , cell wall composition and pollen tube trajectory is more complex than anticipated .\",\n \"Because CMTs guide cellulose deposition , they also control the directional elongation of plant cells ( Baskin , 2005 ) .\",\n \"Indeed , katanin1/fra2 cells are wider and shorter than wild type ( Bichet et al . , 2001; Burk et al . , 2001 ) , and we may predict that the shape of papilla cells in the mutant might be similarly altered .\",\n \"The contribution of stigmatic CMTs to pollen tube growth may thus be mediated by papilla cell shape only; for instance , wider papilla cells in ktn1-5 would promote pollen tube coiling .\",\n \"To check that possibility , we measured the length and width of papilla cells in Col-0 ( at stage 13 and 15 ) , ktn1-5 , xxt1 xxt2 and any1 ( Figure 5—figure supplement 5 ) .\",\n \"We did not find any correlation between papilla length and the coiled phenotype .\",\n \"Indeed , coiled phenotype was observed in stage-15 WT papillae that were longer than those at stage-13 , as well as in ktn1-5 papillae that had a length similar to stage-13 WT papillae .\",\n \"The correlation between papilla width and coiled phenotype was also not clear-cut .\",\n \"As anticipated , ktn1-5 mutant exhibited wider papillae than Col-0 .\",\n \"However , xxt1 xxt2 and any1 papillae were also wider than the WT but did not display the coiled phenotype .\",\n \"Altogether , this suggests that papilla morphology is not sufficient to explain the coiled phenotype .\",\n \"Because the main role of CMTs in plant cells is to guide the trajectory of CSCs , thereby impacting the mechanical anisotropy of the cell wall , we analysed the cell walls of Col-0 and ktn1-5 papillae following pollination .\",\n \"First , using Transmission Electron Microscopy ( TEM ) , we found Col-0 pollen tubes penetrated the cuticle and grew between the two layers of the papilla cell wall , as previously described ( Kandasamy et al . , 1994 ) , in both Col-0 and ktn1-5 papilla cells ( Figure 6A ) .\",\n \"We did not detect any significant difference in the ultrastructure of cell walls ( Figure 6—figure supplement 1 ) .\",\n \"Interestingly , as the pollen tube progresses through the papilla cell wall , it generates a bump ( external deformation ) and an invagination ( internal deformation ) in the cell wall ( Figure 6A ) .\",\n \"As such deformations could reflect differences in wall properties , we quantified the external ( extD ) and internal ( intD ) deformation following pollination of Col-0 and ktn1-5 stigmas .\",\n \"To visualize more clearly this deformation , we pollinated stigmas expressing the plasma membrane protein LTI6B fused to GFP ( LTI6B-GFP ) with pollen whose tube was labelled with the red fluorescent protein RFP driven by the ACT11 promoter ( Rotman et al . , 2005; Figure 6B ) .\",\n \"We found that Col-0 pollen tubes grew with almost equal extD and intD values in Col-0 cell wall .\",\n \"However , the ratio between extD and intD was about three when Col-0 pollen tubes grew in ktn1-5 papilla cells ( Figure 6B–D ) .\",\n \"These quantitative data were consistent with the observation that pollen tubes appeared more prominent on ktn1-5 stigmatic cells using SEM ( Figure 5B; Figure 5—figure supplement 1B ) .\",\n \"Assuming that stigmatic cells are pressurized by their turgor pressure , this may reflect the presence of softer walls in ktn1-5 papillae .\",\n \"To test this hypothesis , we assessed the stiffness of Col-0 and ktn1-5 papilla cell walls using Atomic Force Microscopy ( AFM ) with a 200 nm indentation .\",\n \"We found that cell wall stiffness in ktn1-5 papilla cells was about 30% lower than that in stage 13 WT cells ( Figure 6E , F ) .\",\n \"To confirm this result , we investigated the stiffness of the papilla cell wall on WT stigmas at stage 15 , where increased coiled pollen tubes were detected ( Figure 2A , B ) .\",\n \"We found the cell wall to be softer than that of papillae at stage 13 but stiffer than that of the ktn1-5 ( Figure 6E , F ) .\",\n \"We then reasoned that the presence of softer walls should also affect the pollen tube growth rate , stiffer walls reducing growth .\",\n \"Thus , measuring the growth rate would provide an indication of the resistance force encountered by the tube while growing in the papilla cell wall .\",\n \"We thus monitored the growth rate of Col-0 pollen tubes in Col-0 and ktn1-5 papillae .\",\n \"We found that pollen tubes grew faster ( ~x 1 . 8 ) within ktn1-5 papillae ( Figure 6G ) .\",\n \"Similarly , we found that pollen tube growth on stage 15 papillae was faster ( ~x 1 . 6 ) than on stage 13 papillae ( Figure 6H ) .\",\n \"These results suggest that the coiled phenotype is associated with a higher pollen tube growth rate and hence , that papilla cell walls of ktn1-5 and stage-15 papillae exhibit less resistance to tube penetration .\",\n \"Altogether , these data suggest that KATANIN-dependent mechanical properties of papilla cell wall play a role in pollen tube guidance .\",\n \"Because CMT disorganisation in papillae affects both wall stiffness and mechanical anisotropy , we next investigated the relative contribution of these two parameters in pollen tube growth .\",\n \"Hypocotyl cells of the xxt1 xxt2 double mutant were reported to display CMT orientation defects and reduced stiffness of the cell wall ( Xiao et al . , 2016 ) , features that we found in ktn1-5 papilla cells .\",\n \"However , growing pollen tubes on xxt1 xxt2 stigmas did not coil and grew like on WT stigmas , questioning whether the cell wall stiffness of stigmatic cells was actually affected in the mutant .\",\n \"Using AFM , we found the cell wall of xxt1 xxt2 papillae to be about 30% softer than that of Col-0 papillae , i . e . very similar to that of ktn1-5 ( Figure 6F ) .\",\n \"Despite this similarity , the pollen tube growth rate in xxt1 xxt2 stigmas was identical to Col-0 stigmas at stage 13 ( Figure 6H ) .\",\n \"In addition , contrary to ktn1-5 , pollen tubes were not prominent while growing in xxt1 xxt2 papilla cell walls ( Figure 5—figure supplement 3G ) .\",\n \"These results suggested that although ktn1-5 and xxt1 xxt2 papillae had similar cell wall stiffness as inferred from AFM measurements , they exhibited different mechanical constraints to pollen tube growth .\",\n \"Because of the close relationship between CMT organisation , CMF deposition and cell wall rigidity ( Xiao et al . , 2016; Xiao and Anderson , 2016 ) , we suspected that CMT and CMF organisation might be different between the two mutants and be the possible causal agent of the mechanical differences and related tube coiled phenotype observed in ktn1-5 .\",\n \"To check this possibility , and to gain insight into the predominant cellulose pattern in the stigmatic cell walls , we stained CMFs by using Direct Red 23 .\",\n \"We found cellulose fibres to be highly aligned and slanted to the longitudinal axis of the papilla in stage 13 Col-0 stigmas , whereas ktn1-5 and stage 15 Col-0 papillae both displayed a clearly disordered CMF pattern ( Figure 7A ) with CMFs forming thicker and more spaced bundles ( Figure 7B ) .\",\n \"By contrast , CMFs in xxt1 xxt2 papillae at stage 13 organised in a dense and well oriented pattern of fibres , mostly perpendicular to the papilla long axis , which resembled Col-0 stage 13 CMF organisation ( Figure 7A , B ) .\",\n \"These results suggest that KTN1 loss-of-function alters mechanical properties of the papilla cell wall in a complex manner , lowering both its stiffness to pressure applied perpendicularly to its surface ( e . g . , by the AFM indenter ) and its resistance to pollen tube progression .\",\n \"This latter modification appears as the main cause of the pollen tube coiled phenotype .\",\n \"By contrast , loss of XXT1 and XXT2 functions , by perturbing cell wall stiffness to indenter pressure only , does not induce change in pollen tube growth .\",\n \"Altogether , our data suggest that the mechanical anisotropy of papilla cell walls plays a key role in pollen tube trajectory .\"\n ],\n [\n \"Previous work reported that rearrangements of actin microfilaments ( Iwano et al . , 2007; Rozier et al . , 2020 ) and destabilization of CMTs ( Samuel et al . , 2011 ) occur in stigmas following compatible pollination in Brassicaceae species .\",\n \"However , CMT pattern and dynamics during papilla development have never been described .\",\n \"Our data show that during the course of stigma maturation , which is associated with papilla cell elongation , CMT bundles progressively move from perpendicular ( anisotropic ) to the elongation axis at stage 12 to disorganised ( isotropic ) at stage 15 .\",\n \"This correlates with the known CMT dynamics during elongation in plant cells , where CMT arrays are highly anisotropic in young cells and become more isotropic as cells differentiate ( Baskin , 2005; Landrein and Hamant , 2013 ) .\",\n \"During invasion of plant cells by fungal or Oomycete pathogens or upon mechanical stimulation , the network of microtubules is rearranged , generally associated first with the depolymerization of CMTs at the contact site and later with the formation of an array of microtubules surrounding the invading organism or microneedle ( Hardham , 2013 ) .\",\n \"Interestingly , penetration of the pollen tube in the papilla cell wall does not lead to such microtubule reorganisation ( Figure 3 ) .\",\n \"This suggests that at the cellular level , the invading pollen tube does not elicit a defence-like response in the papilla , which accepts and supports pollen tube growth .\",\n \"In addition , we show that when the stigma is manually pressed between slide and coverslip , the mechanical stimulation applied perpendicularly to the wall leads to CMT fragmentation ( Figure 3—figure supplement 1 ) .\",\n \"This indicates that although the papilla microtubule network is sensitive to mechanical forces , the pressure exerted by the pollen tube is not strong enough to perturb CMT organisation .\",\n \"One possible explanation is that the penetration force developed by the growing pollen tube acts mainly in plane to the cell wall surface and hence would only have a little effect on the papilla cell , contrary to a pressure applied perpendicularly to the wall .\",\n \"Our data are in contradiction with the earlier observation that growing pollen tube provokes fragmentation of CMTs in the papilla ( Samuel et al . , 2011 ) .\",\n \"This discrepancy is likely to relate to the microscopic approaches used between the two studies , live imaging and a fluorescent microtubule probe in our case , chemically fixed material and immunostaining of microtubules in Samuel et al . , 2011 .\",\n \"The progressive randomisation of CMT orientation we observed in papilla cells during ageing is accompanied by an increased coiled growth of pollen tubes in papillae .\",\n \"Similarly , when CMTs are destabilized by the microtubule depolymerizing drug oryzalin , coiled pollen tubes are more frequently observed compared with untreated control stigmas .\",\n \"These results reveal a link between the stigmatic CMT cytoskeleton organisation and the trajectory that pollen tube takes while growing in the papilla cell wall , although the effects of oryzalin on cell wall properties remain unknown .\",\n \"The fact that the most striking effect on pollen tube growth was found on ktn1-5 mutant suggests that the coiled phenotype depends not only on the CMT organisation but implicates other factors .\",\n \"Among various phenotypic alterations described in loss-of-function mutants for KTN1 , are the impaired cell mechanical properties and cell elongation , defects in CMT organisation , cell wall composition and CMF orientation ( Burk et al . , 2001; Burk and Ye , 2002; Ryden et al . , 2003 ) .\",\n \"Interestingly , we found that the protuberance of pollen tubes at the surface of ktn1-5 papillae was associated with a faster growth rate and a lower rigidity of the cell wall compared with Col-0 ( Figure 6 ) .\",\n \"This places mechanics of the cell wall as a likely component involved in the coiled phenotype .\",\n \"Surprisingly , of the six cell wall mutants we analysed , including xxt1 xxt2 and prc1 known to exhibit both abnormal CMT organisation and softer cell walls , none induced the coiled phenotype displayed by ktn1-5 papillae .\",\n \"More remarkably , despite the similar stiffness of xxt1 xxt2 and ktn1-5 papilla cell walls as measured by AFM , pollen tubes behaved differently on these two stigma types , pollen tubes not only coiling but also growing faster on ktn1-5 stigmas .\",\n \"This observation is in accordance with the recent findings that changes in indentation properties of plant cell walls do not correlate with changes in tensile stiffness as described in cell-free strips of onion epidermal walls ( Zhang et al . , 2019 ) .\",\n \"In our experiments , indentations tell us about the elasticity of the wall to a pressure normal to the plane of the wall surface ( i . e . out-of-plane mechanics ) , while pollen tube growth rates inform about the resistance of the wall to a force in-plane to the papilla wall ( i . e . in-plane mechanics ) .\",\n \"Several lines of evidence lead us to suggest that mechanical properties of the cell wall are not identical in the two mutants .\",\n \"Previous work showed that cellulose crystallinity is one factor implicated in cell wall mechanics .\",\n \"Indeed , the epidermal cells of the inflorescence stem in the any1 mutant have the relative amount of crystalline cellulose significantly reduced , while the total content of cellulose in the cell wall is unaltered , and this is accompanied by reduced stiffness of the cell wall ( Fujita et al . , 2013; Altartouri et al . , 2019 ) .\",\n \"Our chemical analysis reveals that the cell wall composition is different between xxt1 xxt2 and ktn1-5 stigmas , with this latter having 25% more crystalline cellulose , and hence should exhibit stiffer cell walls ( Figure 5—figure supplement 4 ) .\",\n \"This data is at odds with AFM measurements but a possible explanation would be that the two layers detected by TEM in the papilla cell wall ( Figure 6—figure supplement 1 ) display different mechanical properties , the inner layer being stiffer than the outer one in the ktn1-5 mutant .\",\n \"This may explain the protruding growth of pollen tubes observed in ktn1-5 papillae , the tube pushing away the softer outer layer but not the inner ( Figure 6A–D ) .\",\n \"Interestingly , Zhang et al . , 2019 reported that cellulose networks largely determine in-plane mechanics whereas out-of-plane mechanics depends on both homogalacturonan ( pectin ) and cellulose networks .\",\n \"Cell walls of the qua2-1 mutant are specifically affected in pectins , with a 50% reduction in homogalacturonan content compared with Col-0 without observable changes in other polysaccharides ( Mouille et al . , 2007 ) .\",\n \"In light of these data , it is noteworthy that papilla cells of the qua2-1 mutant do not induce pollen tube turns ( Figure 5—figure supplement 3 ) , which suggests that pectins play no significant role in the pollen tube phenotype and that the main factor contributing to pollen tube guidance is in-plane mechanics , and hence relies on the cellulose network .\",\n \"The orientation of the rigid CMFs is another determinant of cell wall mechanics .\",\n \"The ktn1-5 mutant was described to have a severe reduction in cell length and an increase in cell width in all organs ( Burk et al . , 2001 ) .\",\n \"This cellular phenotype was attributed to the distorted deposition of CMFs correlated with the isotropic orientation of CMTs , whereas in WT cells , CMFs like CMTs are oriented perpendicularly to the elongation axis ( Burk and Ye , 2002 ) .\",\n \"Similarly , we found that orientation of CMFs is also altered in ktn1-5 papillae ( Figure 7 ) and this was associated with larger papilla cells ( Figure 5—figure supplement 5 ) .\",\n \"Hence , we may assume that mechanical anisotropy , described as the cell wall anisotropy made by the orientation of the rigid CMFs ( Sassi et al . , 2014 ) , is impaired in ktn1-5 papillae .\",\n \"Contrary to the ktn1-5 mutant , papillae of the xxt1 xxt2 double mutant display an anisotropic CMF pattern with parallel fibres approximatively oriented transversally to the papilla axis .\",\n \"These data are in agreement with those reported for etiolated hypocotyl cells , where CMFs are largely parallel to one another , straighter than in WT and oriented almost transversely to the long axis of the cell ( Xiao et al . , 2016 ) .\",\n \"In addition , we found the papilla cell shape of xxt1 xxt2 stigmas to be closer to that of Col-0 than that of ktn1-5 papillae , which is consistent with an anisotropic growth of the papillae in the xxt1 xxt2 mutant compared with ktn1-5 .\",\n \"Apart from the similar stiffness of papilla cell walls as deduced from AFM measurements , the main differences between ktn1-5 and xxt1 xxt2 cell walls are the cell wall composition , orientation of CMFs , and possible changes in molecular connections between cell wall components and plasma membrane and/or cytoskeleton proteins .\",\n \"Indeed , a recent proteomic study revealed that loss of KATANIN function is associated with the decrease in abundance of several cytoskeleton proteins , such as profilin 1 , actin-depolymerizing factor 3 and actin 7 ( Takáč et al . , 2017 ) , whereas targeted quantitative RT-PCR showed that several Microtubule-associated protein ( MAP ) and wall signal receptor genes showed lower expression levels in xxt1 xxt2 ( Xiao et al . , 2016 ) .\",\n \"In this latter study , KTN1 expression level was shown to be unchanged compared with Col-0 .\",\n \"Altogether , our study suggests that KTN1 , by maintaining the papilla mechanical anisotropy , has a key function in mediating early pollen tube guidance on stigma papillae .\",\n \"The coiled phenotype was not only observed in ktn1-5 but also in the WT Col-0 papillae at stage 15 .\",\n \"Remarkably , several lines of evidence reveal that papilla cells from stage 15 share common features with ktn1-5 papillae , such as CMT and CMF increased isotropy , decreased stiffness of the cell wall as deduced from AFM and less resistance of the wall to pollen tube growth .\",\n \"Isotropic orientation of the cytoskeleton at stage 15 is likely to relate to cell elongation , which is known to be accompanied by cytoskeleton reorganisation ( Crowell et al . , 2011; Zhang et al . , 2014 ) .\",\n \"At the organ level , it has been suggested that the mechanical anisotropy of the wall restrains organ emergence ( Sassi et al . , 2014 ) .\",\n \"The authors propose that for the same wall stiffness , a cell wall with isotropic properties would lead to larger outgrowth than a wall with anisotropic properties .\",\n \"Our data are consistent with this hypothesis , albeit at the subcellular scale , since large protuberance of papilla wall following pollen tube growth is observed in ktn1-5 papilla cells , exhibiting walls with isotropic properties .\",\n \"It remains unclear how mechanical anisotropy guides pollen tube growth .\",\n \"We can hypothesise that as pollen tube grows inside the wall , it encounters recently deposited CMFs on the inner side of the wall ( facing the cytoplasm ) and older CMFs on the outer side .\",\n \"It is likely that these layers have different mechanical properties related to CMF orientation ( Baskin , 2005 ) .\",\n \"KATANIN , by acting on CMT dynamics and CMF organisation , would fine-tune the mechanical properties of the matrix through which the pollen tube grows .\",\n \"It is worth noting that growth rate of pollen tubes germinated in vitro is slowed down when pollen tubes pass through a microgap of a microfluidic device , the tubes adapting their invasive force to the mechanical constraints ( Sanati Nezhad et al . , 2013 ) .\",\n \"Based on measurements of pollen tube growth rates , our data show that the tube tip , while progressing in the papilla wall , senses the mechanical features of its environment and reacts accordingly .\",\n \"Hence , it reveals some unanticipated internal and hidden properties of the cell wall .\",\n \"Our study shows that mechanics plays a key role in early pollen tube guidance in the papilla cell .\",\n \"This role is mediated by a specific CMT/CMF organisation and mechanical anisotropy of the papilla cell , which both are dependent on KTN1 .\",\n \"Importantly , these specific mechanical properties of the stigmatic cells prevent emerging pollen tubes to grow upward on papillae and straighten pollen tube direction , helping the tube to find its correct path to the stylar transmitting tract .\",\n \"Our findings also raise the question about the biological importance of the mechanical changes that occur during stigma development .\",\n \"Indeed , for aged stigmas , modifications of the mechanical properties of the cell wall , accompanied by an acceleration of pollen tube growth , could be seen as beneficial for the plant by favouring ultimate fertilization and seed set on old flowers , and hence supporting dissemination of the species .\",\n \"This assumption is particularly relevant given that the pistil length increases with ageing and similarly the journey the pollen tube has to travel to reach the ovules .\",\n \"A recent work showed that ageing is associated with decreased fertility due to stigma senescence , which is initiated at stage 16–17 ( Gao et al . , 2018 ) .\",\n \"The faster the pollen tube will grow in old stigmas , the more chance it will have to circumvent papilla cell death and hence to fertilize the ovule .\",\n \"We may suggest that KTN1 , by mediating mechanical anisotropy of stigmatic cells and promoting pollen tube growth in old stigmas , has played an evolutionary role in the success of fertilization .\",\n \"To conclude , we uncovered a yet unexpected function for KTN1 in early pollen tube guidance on the stigma .\",\n \"In addition , our study also clearly unveils that the mechanical properties of one single cell ( e . g . , the stigmatic papilla ) impact the behaviour of its neighbouring cell ( e . g . , the pollen tube ) .\"\n ],\n [\n \"Arabidopsis thaliana , ecotype Columbia ( Col-0 ) , Arabidopsis transgenic plants generated in this study and Arabidopsis mutants were grown in soil under long-day conditions ( 16 hr of light/8 hr of dark , 21 °C / 19°C ) with a relative humidity around 60% .\",\n \"ktn1-5 ( SAIL_343_D12 ) , xxt1xxt2 , prc1 . 1 , qua2 . 1 , xyl1 . 4 , kor1 . 1 and any1 mutant lines were described previously ( Cavalier et al . , 2008; Fagard et al . , 2000; Fujita et al . , 2013; Lin et al . , 2013; Mouille et al . , 2007; Nicol et al . , 1998; Sampedro et al . , 2010; Shoji et al . , 2004 ) .\",\n \"All mutants were in Col-0 background except kor1 . 1 which was in WS .\",\n \"All stigmas were analysed at stage 13 of flower development ( Smyth et al . , 1990 ) except when specified .\",\n \"We used the Gateway technology ( Life Technologies , USA ) and two sets of Gateway-compatible binary T-DNA destination vectors ( Hellens et al . , 2000; Karimi et al . , 2002 ) for expression of transgenes in A . thaliana .\",\n \"The DNA fragment containing the Brassica oleracea SLR1 promoter was inserted into the pDONP4-P1R vector .\",\n \"The 165 bp-LTI6B fragment was introduced into the pDONR207 vector .\",\n \"MAP65 gene spanning the coding region from start to stop codons was introduced into the pDONR221 vector .\",\n \"CDS from citrine or GFP were cloned into the pDONP2R-P3 vector .\",\n \"Final constructs , pSLR1::MAP65-citrine and pSLR1::LTI6B-GFP were obtained by a three-fragment recombination system ( Life Technologies ) using the pK7m34GW and the pB7m34GW destination vectors , respectively .\",\n \"We generated a pACT11::RFP construct by amplifying the promoter of the A . thaliana ACTIN-11 gene and cloning it into the pGreenII gateway vector in front of the RFP coding sequence .\",\n \"Transgenic lines were generated by Agrobacterium tumefaciens-mediated transformation of A . thaliana Col-0 as described ( Logemann et al . , 2006 ) .\",\n \"Unique insertion lines , homozygous for the transgene were selected .\",\n \"We introduced the pSLR1::LTI6B-GFP or pSLR1::MAP65-citrine construct in ktn1-5 background by crossing and further selecting the progeny on antibiotic containing medium .\",\n \"Flowers at stage 13 and 15 were emasculated and pollinated on plants with mature pollen from the pACT11::RFP line .\",\n \"Immediately after pollination , stigmas were mounted between two coverslips maintained separated by four grease plugs placed at each coverslip corner .\",\n \"To maintain a constant humidity without adding liquid directly on the stigma surface , we used a wet piece of tissue in contact with the base of the stigma .\",\n \"Pollinated stigmas were observed under a Zeiss microscope ( AxioObserver Z1 ) equipped with a spinning disk module ( CSU-W1-T3 , Yokogawa ) using a 40x Plan-Apochromat objective ( numerical aperture 1 . 1 , water immersion ) .\",\n \"Serial confocal images were acquired in the entire volume of the stigma every 1 µm and every minute .\",\n \"Images were processed with Image J software and pollen tube lengths were measured .\",\n \"Pistils were placed straight in a 2% agar MS medium and 0 . 8% low-melting agarose was added up to a certain level where the papilla cells were maintained well immobilised in agarose while leaving the top accessible to the indenter .\",\n \"This set up allowed accurate measurements of cell wall stiffness on the dome-shaped top of papilla cells .\",\n \"AFM indentation experiments were carried out with a Catalyst Bioscope ( Bruker Nano Surface , Santa Barbara , CA , USA ) that was mounted on an optical microscope ( MacroFluo , Leica , Germany ) equipped with a x10 objective .\",\n \"All quantitative measurements were performed using standard pyramidal tips ( RFESP-190 ( Bruker ) ) .\",\n \"The tip radius given by the manufacturer was 8–12 nm .\",\n \"The spring constant of the cantilever was measured using the thermal tune method and was 35 N/m .\",\n \"The deflection sensitivity of the cantilever was calibrated against a sapphire wafer .\",\n \"All experiments were made in ambient air at room temperature .\",\n \"Matrix of 10 × 10 measurements ( step 500 nm ) was obtained for each papilla , with a 1µN force .\",\n \"The Young’s Modulus was estimated using the Nanoscope Analysis ( Bruker ) software , using the Sneddon model with a < 200 nm indentation .\",\n \"Flowers from stages 12 to 15 were emasculated and pollinated on plants with mature WT pollen .\",\n \"One hour after pollination , pistils were cut in the middle of the ovary , deposited on a SEM platform and observed under Hirox SEM SH-3000 at −20°C , with an accelerating voltage of 15kV .\",\n \"Images were processed with ImageJ software and pollen tube direction was quantified by counting the number of turns made by the tube , only on papillae that received one unique pollen grain .\",\n \"Stage 13 flowers were emasculated and pollinated on plants with mature WT pollen .\",\n \"One hour after pollination , pistils were immersed in fixative solution containing 2 . 5% glutaraldehyde and 2 . 5% paraformaldehyde in 0 . 1 M phosphate buffer ( pH 7 . 2 ) and after 4 rounds of 30 min vacuum , they were incubated in fixative for 12 hr at room temperature .\",\n \"Pistils were then washed in phosphate buffer and further fixed in 1% osmium tetroxide in 0 . 1 M phosphate buffer ( pH 7 . 2 ) for 1 . 5 hr at room temperature .\",\n \"After rinsing in phosphate buffer and distilled water , samples were dehydrated through an ethanol series , impregnated in increasing concentrations of SPURR resin over a period of 3 days before being polymerized at 70°C for 18 hr , sectioned ( 65 nm sections ) and imaged at 80 kV using an FEI TEM tecnaiSpirit with 4 k x 4 k eagle CCD .\",\n \"Flowers from MAP65 lines were emasculated and stigmas were observed under confocal microscope .\",\n \"Images were processed with ImageJ software and quantitative analyses of the average orientation and anisotropy of CMTs were performed using FibrilTool , an ImageJ plug-in ( Boudaoud et al . , 2014 ) .\",\n \"Anisotropy values range from 0 to 1; 0 indicates pure isotropy , and one pure anisotropy .\",\n \"A droplet of Adigor was applied at 2 . 5% ( v/v in water ) on the stigma during 4 hr to facilitate subsequent chemical treatments .\",\n \"As described for hypocotyls ( Landrein and Hamant , 2013 ) , pistils were incubated in 12 . 5% glacial acetic acid for 1 hr and dehydrated in 100% and then 50% ethanol for 20 min each .\",\n \"Pistils were then washed in water during 20 min and stored in 1M KOH during 2 days .\",\n \"Pistils were then stained using 0 . 02% ( w/v ) Direct Red 23 dye ( Sigma-Aldrich ) during 4 hr and washed with distilled water .\",\n \"Pistils were observed with a mRFP filter ( excited at 561 nm ) under a Zeiss LSM 880 confocal microscope using a 63x Plan-Apochromat objective ( numerical aperture 1 . 4 , oil immersion ) .\",\n \"Stigmas were imaged by taking a z-stack of 0 . 2 μm sections in the papilla cells .\",\n \"Image analysis was performed by doing a 3D-projection and measuring the fluorescence intensity longitudinally along the papilla axis using ImageJ software .\",\n \"Flowers from LTI6B lines were emasculated and pollinated with mature pollen from the pACT11::RFP line .\",\n \"20 min after pollination , stigmas were observed under confocal microscope .\",\n \"Serial confocal images every 1 µm encompassing the entire volume of the stigma were recorded and processed with ImageJ software .\",\n \"Plasma membrane deformation was estimated by choosing the slide from the stack that corresponded to the focus plan of the contact site with the RFP-labelled pollen tube .\",\n \"On the bright field image corresponding to the selected slide , a line was drawn connecting the two ridges of the invagination of the papilla .\",\n \"Two perpendicular lines , one toward the exterior ( ExtD ) of the papilla to the maximum point of deformation , and the other toward the interior ( IntD ) on the GFP image were measured , respectively .\",\n \"To avoid contact of pollen grains with liquid , we performed local applications of oryzalin ( Chemical service , Supelco ) as described ( Sassi et al . , 2014 ) , using lanolin pasted around the style , just under the stigmatic cells , at 833 µg/mL ( DMSO ) , for 4 hr at 21°C .\",\n \"Oryzalin-treated pistils were pollinated with mature WT pollen and 1 hr after pollination observed under SEM .\",\n \"XyG composition of stigmas was performed following the oligosaccharide fingerprinting set up by Lerouxel et al . , 2002 .\",\n \"10 stigmas per genotype were dissected and kept in 96% ethanol .\",\n \"After ethanol removal , XyG oligosaccharides were generated by treating samples with endoglucanase in 50 mM sodium acetate buffer , pH 5 , overnight at 37°C .\",\n \"Matrix-assisted laser-desorption ionization time of flight mass spectrometry of the XyG oligosaccharides was recorded with a MALDI/TOF Bruker Reflex III using super-DHB ( 9:1 mixture of 2 , 5-dihydroxy-benzoic acid and 2-hydroxy-5-methoxy-benzoic acid; Sigma-Aldrich , sigmaaldrich . com ) as matrix .\",\n \"Alcohol insoluble residues were obtained from around 3 to 5 mg of fresh pistils .\",\n \"They were collected for analysis and fixed in 96% ethanol before incubation for 30 min at 70°C .\",\n \"The pellet was then washed twice with 96% ethanol and twice with acetone .\",\n \"The remaining pellet is called alcohol insoluble residues ( AIR ) and was dried in a fume hood overnight at room temperature .\",\n \"Cell wall monosaccharide content of the non‐cellulosic fraction was determined by hydrolysis of all the AIR obtained from the pistils with 2 M TFA for 1 hr at 120°C .\",\n \"After cooling and centrifugation , the supernatant was dried under a vacuum , resuspended in 200 μl of water and retained for analysis .\",\n \"To obtain the glucose content of the crystalline cellulose fraction , the TFA-insoluble pellet was further hydrolysed with 72% ( v/v ) sulfuric acid for 1 hr at room temperature .\",\n \"The sulfuric acid was then diluted to 1 M with water and the samples incubated at 100°C for 3 hr .\",\n \"All samples were filtered using a 20‐μm filter caps , and quantified by HPAEC‐PAD on a Dionex ICS‐5000 instrument ( ThermoFisher Scientific ) as described ( Fang et al . , 2016 ) .\",\n \"Graph and statistics were obtained with R software or Excel .\",\n \"Statistical tests performed are specified in figure legends .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules .","Pollen tubes first grow within the cell wall of the papilla cells , applying pressure to the cell .","Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules ( CMTs ) , which in turn mediate deposition of cellulose microfibrils ( CMFs ) .","Here , by combining imaging , genetic and chemical approaches , we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 ( ktn1-5 ) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae .","We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth .","Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall ."],"string":"[\n \"Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules .\",\n \"Pollen tubes first grow within the cell wall of the papilla cells , applying pressure to the cell .\",\n \"Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules ( CMTs ) , which in turn mediate deposition of cellulose microfibrils ( CMFs ) .\",\n \"Here , by combining imaging , genetic and chemical approaches , we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 ( ktn1-5 ) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae .\",\n \"We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth .\",\n \"Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Flowering plants produce small particles known as pollen that – with the help of the wind , bees and other animals – carry male sex cells ( sperm ) to female sex cells ( eggs ) contained within flowers .","When a grain of pollen lands on the female organ of a flower , called the pistil , it gives rise to a tube that grows through the pistil towards the egg cells at the base .","The surface of the pistil is covered in a layer of long cells named papillae .","Like most plant cells , the papillae are surrounded by a rigid structure known as the cell wall , which is mainly composed of strands known as microfibrils .","The pollen tube exerts pressure on a papilla to allow it to grow through the cell wall towards the base of the pistil .","Previous studies have shown that the pistil produces signals that guide pollen tubes to the eggs .","However , it remains unclear how pollen tubes orient themselves on the surface of papillae to grow in the right direction through the pistil .","Riglet et al . combined microscopy , genetic and chemical approaches to study how pollen tubes grow through the surface of the pistils of a small weed known as Arabidopsis thaliana .","The experiments showed that an enzyme called KATANIN conferred mechanical properties to the cell walls of papillae that allowed pollen tubes to grow towards the egg cells , and also altered the orientation of the microfibrils in these cell walls .","In A . thaliana plants that were genetically modified to lack KATANIN the pollen tubes coiled around the papillae and sometimes grew in the opposite direction to where the eggs were .","KATANIN is known to cut structural filaments inside the cells of plants , animals and most other living things .","By revealing an additional role for KATANIN in regulating the mechanical properties of the papilla cell wall , these findings indicate this enzyme may also regulate the mechanical properties of cells involved in other biological processes ."],"string":"[\n \"Flowering plants produce small particles known as pollen that – with the help of the wind , bees and other animals – carry male sex cells ( sperm ) to female sex cells ( eggs ) contained within flowers .\",\n \"When a grain of pollen lands on the female organ of a flower , called the pistil , it gives rise to a tube that grows through the pistil towards the egg cells at the base .\",\n \"The surface of the pistil is covered in a layer of long cells named papillae .\",\n \"Like most plant cells , the papillae are surrounded by a rigid structure known as the cell wall , which is mainly composed of strands known as microfibrils .\",\n \"The pollen tube exerts pressure on a papilla to allow it to grow through the cell wall towards the base of the pistil .\",\n \"Previous studies have shown that the pistil produces signals that guide pollen tubes to the eggs .\",\n \"However , it remains unclear how pollen tubes orient themselves on the surface of papillae to grow in the right direction through the pistil .\",\n \"Riglet et al . combined microscopy , genetic and chemical approaches to study how pollen tubes grow through the surface of the pistils of a small weed known as Arabidopsis thaliana .\",\n \"The experiments showed that an enzyme called KATANIN conferred mechanical properties to the cell walls of papillae that allowed pollen tubes to grow towards the egg cells , and also altered the orientation of the microfibrils in these cell walls .\",\n \"In A . thaliana plants that were genetically modified to lack KATANIN the pollen tubes coiled around the papillae and sometimes grew in the opposite direction to where the eggs were .\",\n \"KATANIN is known to cut structural filaments inside the cells of plants , animals and most other living things .\",\n \"By revealing an additional role for KATANIN in regulating the mechanical properties of the papilla cell wall , these findings indicate this enzyme may also regulate the mechanical properties of cells involved in other biological processes .\"\n]","isConversational":false},"year":{"kind":"string","value":"2020"}}},{"rowIdx":284,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"Decoding odor quality and intensity in the Drosophila brain"},"id":{"kind":"string","value":"elife-04147-v1"},"sections":{"kind":"list like","value":[["To navigate the environment in a way that optimizes their survival and reproduction , animals have evolved sensory systems .","These have three essential tasks: First , the external world has to be translated into an internal representation in the form of an accurate neural map .","Second , the neural map has to be readable and interpretable , that is , the generated neural code must allow common attributes to be extracted across stimuli to enable the animal to make the best decisions .","Third , the animal has to be able to adapt to environmental changes and to form a sensory memory of new stimuli .","Many studies have been dedicated to unraveling the primary transformation from a stimulus into an initial neural representation within various sensory systems ( Manni and Petrosini , 2004; Vosshall and Stocker , 2007; Sanes and Zipursky , 2010 ) and to elucidating neuronal plasticity and sensory memory formation in higher-level processing centers ( Heisenberg , 2003; Pasternak and Greenlee , 2005 ) .","The ability to extract features and integrate stimulus modalities have so far mainly been studied in the visual system ( Livingstone and Hubel , 1988; Bausenwein et al . , 1992; Nassi and Callaway , 2009 ) .","We addressed the question of how stimulus features such as odor valence and intensity are coded and integrated within the olfactory system using the model organism Drosophila melanogaster .","The olfactory system of the vinegar fly provides an excellent model system for deciphering olfactory processing mechanisms , since it displays remarkable similarities to the mammalian system but is less complex and highly genetically tractable .","Like other sensory systems , the olfactory system employs a spatio-temporal map to translate the variables in chemosensory space into neuronal activity patterns in the brain .","This map emerges when the olfactory sensory neurons ( OSNs ) with the same chemosensory receptors converge into one exclusive glomerulus in the antennal lobe ( AL ) which represents the equivalent to the mammalian olfactory bulb ( Hildebrand and Shepherd , 1997; Vosshall et al . , 2000; Vosshall and Stocker , 2007 ) .","Glomeruli , the functional and morphological units of the AL , are microcircuits comprising OSNs , multiglomerular local interneurons ( LNs ) and uniglomerular output neurons , so-called excitatory projection neurons ( ePNs ) ( Wilson and Mainen , 2006; Vosshall and Stocker , 2007 ) that convey the olfactory information to higher brain centers , as the mushroom body calyx ( MBc ) and the lateral horn ( LH ) ( Stocker et al . , 1997 ) .","The stringent spatial arrangement of OSNs and ePNs in the AL generates a spatial map containing characteristic combinatorial glomerular activity patterns for all odorants ( Fiala et al . , 2002; Wang et al . , 2003a; Couto et al . , 2005; Fishilevich and Vosshall , 2005 ) .","The MBc is involved in olfactory memory formation ( Heisenberg , 2003 ) and enables a contextualization of the odor space ( Caron et al . , 2013 ) .","By exclusion , the LH is believed to be involved in innate olfactory behavior ( de Belle and Heisenberg , 1994; Jefferis et al . , 2007 ) .","Excitatory PNs retain the sensory information encoded in the AL and form glomerulus-dependent , stereotypic axonal terminal fields in the LH ( Marin et al . , 2002; Wong et al . , 2002; Tanaka et al . , 2004 ) .","Compartmentalization in the LH has been observed in form of a spatial segregation of ePNs innervating specific glomerular subgroups ( Tanaka et al . , 2004 ) , fruit and pheromone odor information processing ePNs ( Jefferis et al . , 2007 ) as well as ammonia and amine vs carbon dioxide coding ePNs ( Min et al . , 2013 ) .","Like many other sensory networks , the olfactory circuit of the fly contains spatially distinct pathways to the higher brain , namely the inner , middle and outer antennocerebral tract ( iACT , mACT and oACT ) ( Stocker et al . , 1990 ) .","Notably , the mACT projects from the AL to the LH exclusively and consists of inhibitory PNs ( iPNs ) , which exhibit also uniglomerular but mainly multiglomerular AL innervations ( Ito et al . , 1997; Jefferis et al . , 2007; Lai et al . , 2008; Okada et al . , 2009; Liang et al . , 2013 ) .","Both PN populations have been attributed different coding properties: Although both PN populations exhibit odor responses to overlapping odor ligands , iPNs seems to be broader tuned than ePNs ( Wang et al . , 2014 ) .","Furthermore , while ePNs encode rather odor identity ( Wang et al . , 2003a; Wilson et al . , 2004; Silbering et al . , 2008 ) , iPNs have been shown to enhance innate discrimination of closely related odors ( Parnas et al . , 2013 ) .","Together , these PN populations process information on dual olfactory pathways ( Liang et al . , 2013; Wang et al . , 2014 ) , as do processing mechanisms in other sensory modalities ( Nassi and Callaway , 2009 ) , and most likely accomplish different olfactory behaviors .","The mainly multiglomerular AL pattern of iPNs suggests that these neurons extract characteristic stimulus features from the AL code and re-integrate this information into the LH to mediate innate odorant-guided behavior .","This assumption is further supported by two recent studies showing that the inhibitory input from the AL to the LH is module-specific , that is , selective for food odors and pheromones ( Liang et al . , 2013; Fisek and Wilson , 2014 ) , while the connectivity in the MBc is rather probabilistic ( Murthy et al . , 2008; Caron et al . , 2013 ) .","However , it still remains open if and how different odor features as hedonic valence or intensity are functionally coded and integrated in the LH .","In this study , we characterized and dissected the iPN olfactory processing pathway regarding the coding of odor quality and intensity at morphological , functional and behavioral levels .","By linking odor-evoked activity patterns in the LH to odor-guided behavior , we provide evidence that iPNs mediate odor attraction .","Furthermore , our data demonstrate a feature-based , spatially segregated activity map in the LH comprised of iPNs and third-order neurons and thus expand its role as a center for integrating behaviorally relevant olfactory information ."],["Cell bodies of iPNs are exclusively located in the ventral cell cluster which consists of ∼50 iPNs ( Lai et al . , 2008 ) that project via the mACT to the LH , thereby bypassing the MBc ( Ito et al . , 1997 ) ( Figure 1A , B ) .","In contrast , ePN somata are located anterodorsally and laterally of the AL , and their axons project through the iACT or oACT to the MBc and the LH ( Stocker et al . , 1997; Marin et al . , 2002; Wong et al . , 2002; Lai et al . , 2008 ) .","To analyze the innervation patterns of iPNs and ePNs , we labeled both PN populations simultaneously in vivo using the enhancer trap lines GH146-QF and MZ699-GAL4 that label the majority of ePNs ( 60% ) and iPNs ( 86% ) , respectively ( Lai et al . , 2008 ) .","Double-labeling shows that both PN types innervate overlapping regions in the AL and the LH , while a small posterior-lateral LH area is targeted only by ePNs ( Figure 1A , Figure 1—figure supplement 1 ) .","In GH146-positive ( GH146+ ) PNs , immunolabeling reveals GABA production in all ∼6 PNs of the ventral cell cluster ( Wilson and Laurent , 2005 ) , whereas ePNs of this line are exclusively cholinergic ( Shang et al . , 2007 ) .","For the ∼45 MZ699-positive ( MZ699+ ) iPNs ( Lai et al . , 2008 ) , GAD1 ( glutamic acid decarboxylase ) in situ hybridizations imply GABA synthesis ( Okada et al . , 2009 ) , which was recently verified via immunostaining ( Liang et al . , 2013; Parnas et al . , 2013 ) .","The polarity of both PN populations has been studied in detail , showing that both possess dendritic regions in the AL , indicating the AL as their cholinergic input site , while the LH represents their major output site ( Jefferis et al . , 2001; Okada et al . , 2009; Liang et al . , 2013; Parnas et al . , 2013 ) . 10 . 7554/eLife . 04147 . 003Figure 1 . Detailed glomerular innervations of excitatory and inhibitory projection neurons in the AL .","( A ) Simultaneous labeling of inhibitory projections neurons ( iPNs , labeled by MZ699-GAL4;G-CaMP ) and excitatory projection neurons ( ePNs , labeled by GH146-QF;tdTomato ) in vivo reveals distinct projections to the lateral horn ( LH ) .","All iPNs bypass the mushroom body calyx ( MBc ) and innervate the LH exclusively .","The MZ699 line labels a few ventrolateral protocerebral neurons ( vlPr neurons ) projecting via the posterior lateral fascicle ( plF ) from the ventrolateral protocerebrum ( vlPr ) to the LH .","( B ) Schematic of the PN connectivity relay from the antennal lobe ( AL ) to higher brain centers ( ePNs in magenta , iPNs in green , and vlPr neurons in orange ) .","( C ) Above , complete glomerular assignment of the AL neuropil ( right AL ) , labeled with elav-n-synaptobrevin:DsRed ( END1-2 ) .","Below , glomerular innervations of both PN populations related to in vivo images in Figure 1—figure supplement 2 .","Depicted are the ventral level ( ∼−40 µm ) , the medial level ( ∼−20 µm ) and the dorsal view onto the AL .","Color annotation: blue glomeruli are not innervated by any of the used GAL4-lines; green glomeruli are innervated by MZ699+ iPNs and magenta by GH146+ ePNs; white glomeruli are innervated by both enhancer trap lines .","Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00310 . 7554/eLife . 04147 . 004Figure 1—figure supplement 1 . Characterization of excitatory and inhibitory projection neurons . Overlap of ePNs ( QUAS-tdTomato ) and iPNs ( UAS-GCaMP3 . 0 ) in the LH area .","The circle indicates the posterior lateral region , which is sparsely innervated by iPNs and dominated by ePN axonal terminal fields .","Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00410 . 7554/eLife . 04147 . 005Figure 1—figure supplement 2 . Glomerular innervations of ePNs and iPNs .","( A ) Representative in vivo images of glomerular innervations .","MZ699- and GH146-GAL4 lines have been reconstructed with the END1-2 background staining ( two upper planes ) and dual labeling via the Q-system and the GAL4-UAS expression system ( lowest plane ) .","Scale bar , 20 µm .","( B ) Detailed glomerular AL innervation .","Green filled cells indicate innervation by MZ699-GAL4 , magenta GH146-GAL4 innervation , respectively and grey , no innervation by the indicated line .","Bottom rows , total number of innervated glomeruli with percentage share indicated below .","Merge column: white filled with ‘x’ indicates glomeruli innervated by both lines , grey only one line .","Blue filled rows are glomeruli labeled by none of the enhancer trap lines . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 005 To further characterize PNs labeled by MZ699-GAL4 and GH146-GAL4 , we analyzed their precise glomerular innervation to unravel how selectively they acquire information in the AL .","To allow glomerulus identification in vivo , we employed a transgenic fly carrying elav-n-synaptobrevin:DsRed ( END1-2 ) to express the presynaptically targeted fusion protein under the control of the neuron-specific elav promotor ( Figure 1—figure supplement 2A ) ( Grabe et al . , 2014 ) .","The reconstruction and identification of all AL glomeruli provided 53 glomeruli , of which 75% were innervated by MZ699+ iPNs ( 40 ) while 70% ( 37 ) were covered by GH146+ ePNs ( Figure 1C , Figure 1—figure supplement 2B ) .","55% of all glomeruli were innervated by both lines .","Notably , dendritic MZ699-GAL4 innervation density was not homogeneous .","Certain glomeruli were densely innervated ( e . g . , DM2 , DM4 and DM5 ) , while others did not reveal any postsynaptic sites ( e . g . , DL1 , DL4 and DL5 ) .","Hence MZ699+ iPNs target specific glomerular subsets selectively , which suggests that these neurons have a particular function within the olfactory network .","Probabilistic synaptic density maps of GH146+ PNs predicted a regionalized neuronal activity in the LH ( Jefferis et al . , 2007 ) .","Do iPNs functionally segregate in a comparable way ?","To address this question , we expressed the Ca2+-sensitive reporter G-CaMP3 . 0 ( Nakai et al . , 2001; Tian et al . , 2009 ) in iPNs using MZ699-GAL4 and performed functional imaging in the LH ( Figure 2A–C ) .","We initially tested three odors with potential relevance for Drosophila at different concentrations: acetoin acetate , an attractive byproduct of the yeast fermentation process , balsamic vinegar , an attractive natural odor mixture , and benzaldehyde , a well-known fly repellant ( Magee and Kosaric , 1987; Keene et al . , 2004; Semmelhack and Wang , 2009 ) .","We observed that odor evoked Ca2+ responses separate in certain regions of the LH in an odor-specific and concentration-dependent manner ( Figure 2C ) .","Acetoin acetate and balsamic vinegar evoked Ca2+ activity in spatially similar regions .","At higher concentrations , an additional region was recruited .","Benzaldehyde elicited no response at very low concentrations , but induced clear activity at median and high concentrations in a third region , which was completely separate from the regions activated by the other two odors .","Observed patterns were highly reproducible within one animal and stereotypic among different individuals , as shown for the stimulation with 1-octen-3-ol ( Figure 2D ) as well as other odors ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 04147 . 006Figure 2 . Odors evoke specific and stereotypic calcium responses in the LH subdivided into three distinct odor response domains .","( A ) Schematic of the olfactory circuit with the investigated area highlighted .","( B ) RAW image of the LH ( top picture ) depicting the recorded area of figures ( C–E ) and the false color image ( bottom picture ) during the solvent application .","The ΔF/F scale bar applies for all false color-coded pictures; the alpha-bar for the pixel participation xk of the indicated colors applies for ( E–F ) .","( C ) Representative LH Ca2+ responses ( ΔF/F% ) of acetoin acetate , balsamic vinegar and benzaldehyde at three concentrations .","Numbers in the lower right corner indicate individual maxima .","( D ) Odor-evoked Ca2+ responses ( ΔF/F% ) are exemplarily depicted for 1-octen-3ol- at three concentrations in four animals .","( E ) NNMF-extracted LH odor response domains ( ORD ) of four representative animals: three LH ORDs were fully reproducible after being extracted from all measured animals .","Domains classified as identical are similarly color-coded: the green ORD is located in the posterior-medial region of the LH ( LH-PM ) ; blue , in the anterior-medial ( LH-AM ) , and red in the anterior-lateral LH area ( LH-AL ) .","The alpha-bar for green , blue and red shades is placed in ( B ) .","( F ) Left , schematic outlines of the LH with indicated ORDs .","Right , median activity traces of all odors at three concentrations are depicted for each colored ORD .","Shadows represent lower and upper quartiles ( n = 6–7 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00610 . 7554/eLife . 04147 . 007Figure 2—figure supplement 1 . Odor-evoked activity patterns in the LH are reproducible and stereotypic . Odor-evoked Ca2+ responses ( ΔF/F% ) in the LH for the three odors acetoin acetate , balsamic vinegar and benzaldehyde in four animals are shown as false-color coded images .","Two measurements in each animal are given to reveal that the activity patterns are highly reproducible within one animal .","Comparison between the patterns among individuals shows that the activity regions are stereotypic .","Numbers in the lower right corner indicate individual maxima .","The ΔF/F scale bar is shown at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00710 . 7554/eLife . 04147 . 008Figure 2—figure supplement 2 . Odor-evoked activity patterns in the LH can be reconstructed with five components . Above , Odor-evoked Ca2+ responses ( ΔF/F% ) in the LH for the three odors acetic acid , balsamic vinegar and 1-octen-3-ol in three animals are shown as false-color coded images .","The ΔF/F scale bar is shown at the bottom .","Middle , activity patterns were reconstructed using NNMF with five components .","Below , residue of the pattern reconstructions with five components ( as shown in the middle panel ) revealing that no stimulus related activity remained . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00810 . 7554/eLife . 04147 . 009Figure 2—figure supplement 3 . Odor-evoked activity patterns in the LH cluster into three components . Hierachichal clustering ( UMPGA ) of the odor response spectra of the NNMF components with a reliable stimulus response ( trial-to-trial correlation >0 . 7 , that is , 28 out of 35 components in seven animals ) .","The response spectra segregate into three distinct clusters according to their stimulus response spectra .","The corresponding response areas ( left pictures ) are located in similar regions of the LH . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 009 Due to the lack of morphological landmarks in the LH , functional data were analyzed using the pattern recognition algorithm Non-Negative Matrix Factorization ( NNMF ) ( Lee and Seung , 1999 ) , which automatically extracts spatial areas possessing a common distinct time-course , further termed LH odor response domains ( ORDs ) .","The NNMF analysis extracted three clearly reproducible and spatially robust ORDs ( Figure 2E , see NNMF part in the ‘Materials and methods’ section ) .","Notably , ORDs occupying common temporal kinetics exhibited highly stereotypic spatial patterns .","We termed the ORDs LH-PM ( LH-posterior-medial ) , LH-AM ( LH-anterior-medial ) and LH-AL ( LH-anterior-lateral ) according to their anatomical positions .","To validate our observations , we extended our stimulus array to 11 additional odorants and applied each at three concentrations .","Odorants were chosen according to chemical classes , hedonic valence and biological value .","Hence , the odor set included acids , lactones , terpenes , aromatics , alcohols , esters , ketones and the natural blend , balsamic vinegar .","Remarkably , analysis of the additional odorants revealed neuronal activity exclusively within the three described ORDs ( Figure 2F , Figure 2—figure supplements 2 , 3 ) .","Furthermore , median NNMF-extracted Ca2+ response traces with indicated statistical quartiles illustrate very low variability and highly reproducible LH signals .","The LH-PM area chiefly revealed robust odor-evoked responses across concentrations , while the LH-AM and LH-AL were mainly activated at very high odor concentrations by distinct odorants .","The global responsiveness within separate ORDs in the LH substantiates our finding of a relatively broad AL input to MZ699+ iPNs which converges into three spatially regionalized and stereotypic LH activity domains .","We next investigated if the spatially regionalized odor-evoked response patterns are reflected in the axonal terminal fields of MZ699+ iPNs in the LH .","To analyze these neurons at the single neuron level , we performed neural tracing by employing a genetically encoded photoactivatable GFP ( PA-GFP ) ( Patterson and Lippincott-Schwartz , 2002; Datta et al . , 2008; Ruta et al . , 2010 ) .","The photoconversion of all MZ699+ neurons leaving the AL confirmed the homogeneous distribution of iPN neurites in the LH and the sparse innervation of the posterior-lateral region as mentioned above ( Figure 3A ) .","Next we illuminated PA-GFP in single somata to selectively label individual MZ699+ iPNs from the soma up to the farthest axonal terminals in the LH .","Individual iPNs were reconstructed and transformed into a reference brain using the END1-2 background ( Grabe et al . , 2014 ) to align neurons of different individuals .","Based on their innervation pattern in the LH , MZ699+ iPNs could be assigned to two major morphological classes ( Figure 3B , C ) .","As expected from the extracted ORDs , one iPN group diverged to the LH-PM region ( 8/25 of iPNs ) , while a second group extended their axonal terminations within the LH-AM area ( 10/25 of iPNs ) .","In order to statistically substantiate our observation , we performed a cluster analysis based on a similarity score ( Kohl et al . , 2013 ) of the target areas of all terminals of each iPN in the LH ( for details see ‘Materials and methods’ ) .","The dendrogram of morphological similarity between each individual iPN shows that all , except one iPN , could be clustered according to their target region in either the LH-AM or LH-PM area ( Figure 3D ) which confirms the classification into two major categories .","Additionally , we performed a principal component analysis based on the distances of the similarity scores showing that both neuronal classes possess significantly different target areas in the LH ( Figure 3—figure supplement 1A; p < 0 . 001 , one-way ANOSIM ) . 10 . 7554/eLife . 04147 . 010Figure 3 . iPNs can be classified according to their projection pattern in three distinct LH zones .","( A ) Complete population of MZ699+ iPNs labeled using PA-GFP ( left image ) , the posterior-lateral LH region is encircled , arrowhead indicates the final common projection point of iPN axons .","Middle image: photoactivation of all vlPr neurons of the MZ699-GAL4 line that project from the LH to the vlPr via the plF .","Right image: exemplary single iPN , labeled by photoconverting PA-GFP in a single soma ( arrow ) .","Scale bar , 20 µm .","( B ) Framed images: neuronal reconstructions of all iPNs projecting to the LH-PM zone ( n = 8 ) with outlined olfactory neuropils .","View from dorsal ( left ) and lateral ( right ) .","Right part represents two exemplary registered individual iPNs .","( C ) Neuronal reconstructions of all iPNs projecting into the LH-AM zone ( n = 10 ) , images are arranged as in ( B ) .","( D and E )","Cluster analyses based on the target areas of all terminals of each iPN in the LH ( D ) or based on the innervated glomeruli in the AL ( E ) .","The dendrograms are split into colored subclusters .","Below each dendrogram , each individual iPN is specified according to the labels in Figure 3—figure supplement 2 .","Note , that iPNs can be morphologically clustered according to their target or input regions .","( F ) Neuronal reconstruction of vlPr neurons projecting through the plF to the LH-AL zone .","( G ) Combination of all registered neurons .","( H ) Dual combinations of all registered neurons with their projections in the LH . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01010 . 7554/eLife . 04147 . 011Figure 3—figure supplement 1 . iPNs can be morphologically segregated according to their target and input region .","( A ) Principal component analysis based on the distances of the similarity scores of all terminal points of each individual iPN in the LH ( for details see ‘Materials and methods’ ) .","LH-AM iPNs ( blue ) and LH-PM iPNs ( green ) form significantly distinct clusters ( ***p < 0 . 001 , One-Way ANOSIM , Bray–Curtis ) .","( B ) Principal component analysis based on the glomerular innervations of each individual iPN in the AL .","Again , LH-AM iPNs ( blue ) and LH-PM iPNs ( green ) form significantly distinct clusters ( ***p < 0 . 001 , One-Way ANOSIM , Bray–Curtis ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01110 . 7554/eLife . 04147 . 012Figure 3—figure supplement 2 . Glomerular innervations of individual iPNs . Binary innervation patterns of 25 individually labeled MZ699+ iPNs using PA-GFP .","Columns represent innervation patterns of individual neurons which have been grouped according to their innervation properties; rows represent 51 glomeruli in the AL along with the innervation in the specific odor response domains in the LH ( LH-PM , LH-AM ) and/or the mushroom body calyx ( MBc ) .","Glomeruli have been sorted according to their iPN innervation .","Grey , innervated; white , not innervated . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 012 We did not observe any clear panglomerular innervations of individual MZ699+ iPNs that spanned the entire AL , consistent with Liang et al . ( 2013 ) .","Instead , MZ699+ iPNs develop mainly oligoglomerular patterns innervating on average 5 . 4 ± 3 . 9 glomeruli ( mean ± SD ) , which are not necessarily in close proximity .","It is important to note here , that the glomerular innervations of iPNs are rather sparse in comparison to the innervation of ePNs which complicates the identification of truly innervated glomeruli .","After classifying all registered neurons according to their LH zones along with their glomerular innervations , we noted a spatial subdivision of MZ699+ iPN dendritic fields in the AL ( Figure 3—figure supplement 2 ) .","Whereas LH-PM iPNs extended dendrites mainly into glomeruli from the ventro- or dorsomedial area of the AL ( e . g . , DM4 , DM2 , VM7 , VM5d ) , iPNs targeting the LH-AM zone innervated glomeruli ranging from the ventro- and dorsoanterior to the dorsocentral region ( e . g . , DC3 , VC1 , VA6 , VL1 ) .","We observed that a glomerulus is typically innervated by only LH-PM iPNs or LH-AM iPNs .","However , we also found a few cases where a glomerulus can be innervated by both iPN types ( e . g . , glomeruli D and DC2 ) .","In order to analyze whether the two categories of iPNs can also be statistically separated according to their glomerular innervations in the AL , we performed a cluster analysis based on the glomeruli innervated by each individual iPN ( Figure 3E ) .","Notably , the two iPN classes could be clearly clustered into two groups due to their specific AL innervations .","This finding is further supported by a principal component analysis showing that iPNs targeting the LH-PM region innervate a significant different glomerular subset than iPNs that send their axonal terminals to the LH-AM area ( Figure 3—figure supplement 1B; p < 0 . 001 , one-way ANOSIM ) .","In accordance with our finding of two major iPN categories is the study by Lai et al . ( 2008 ) who observed several different stereotyped projection patterns of multiglomerular MZ699+ single-cell clones that could be broadly categorized into two groups based on the dendritic and axonal projection patterns .","While we observed corresponding innervated areas in the AL , the described target areas in the LH seem to differ .","However , due to the lack of 3D reconstruction of the single-cell clone data , the innervation patterns cannot be compared in detail .","In addition to the oligoglomerular iPNs , we observed a few uniglomerular MZ699+ iPNs innervating either glomerulus DA1 or VL1 ( 4/25 of iPNs ) , consistent with Lai et al . ( 2008 ) , which target the LH-AM region ( Figure 3—figure supplement 2 ) .","Moreover , we identified three other MZ699+ neurons that did not innervate the AL and sent their axons through the mACT to the LH and/or the MBc .","Since the MZ699-GAL4 line labels also neurons connecting the LH and the ventrolateral protocerebrum ( vlPr ) ( Ito et al . , 1997; Liang et al . , 2013; Parnas et al . , 2013 ) , we illuminated a small fraction of the posterior lateral fascicle ( plF ) to target these putative third-order neurons ( Figure 3A ) .","The plF comprised axons of ventrolateral protocerebral neurons ( vlPr neurons ) , which bifurcated within the LH-AL ( Figure 3F ) .","Combinations of all registered neuron types within the assigned zones revealed that iPNs of the LH-AM area and vlPr neurons of the LH-AL region intermingle ( Figure 3G , H ) .","To illustrate higher-order connectivity , we labeled the three major neuron types , that is , MZ699+ iPNs , GH146+ ePNs and vlPr neurons , targeting the LH within the olfactory circuitry using PA-GFP ( Figure 4A ) .","Since our observed Ca2+ responses in the LH-AL region might reflect activity from vlPr neurons rather than iPNs , we dissected the neuronal contributions within each extracted ORD by conducting transection experiments using two-photon laser-mediated microdissection ( Figure 4B ) .","By transecting the mACT , we aimed at abolishing LH-responses deriving from MZ699+ iPNs , while cutting the plF connection should eliminate potential odor-evoked vlPr neuron activity .","To achieve unambiguous and comparable results , functional imaging was performed in both brain hemispheres simultaneously .","Immediately after the intact brain areas were imaged , the tracts were selectively transected on one brain side each ( Figure 4C ) and the imaging procedure was repeated .","We applied a reduced odor set that elicited activity in all ORDs and performed NNMF for pre- and post-lesion recordings .","Transecting the mACT significantly reduced responses in the LH-PM and LH-AM region , whereas LH-AL responses were significantly abolished by plF-ablation ( Figure 4D ) .","Notably , we observed that LH-AL responses to some odors were significantly increased after mACT transection as a consequence of the suppression of iPN inhibition of vlPr neurons confirming the study by Liang et al . ( 2013 ) .","Hence , activity in the LH-PM and LH-AM domain can be assigned to MZ699+ iPNs , while LH-AL activity is mainly evoked by vlPr neurons ( Figure 4E ) . 10 . 7554/eLife . 04147 . 013Figure 4 . Distinct odor response domains in the LH constitute neuronal activity of iPNs and vlPr neurons .","( A ) Representation of all ePNs ( magenta ) and iPNs ( green ) labeled by GH146-GAL4 and MZ699-GAL4 using PA-GFP , respectively .","Photoactivation of vlPr neurons ( orange , MZ699-GAL4 ) connecting the LH and the vlPr via the plF .","The overlay image depicts a pseudo-merge image of the different GAL4-driver lines .","( B ) Schematic of the olfactory circuit with integrated layout of the transection experiment .","After simultaneous Ca2+ imaging of bilateral LHs , the ipsilateral plF and contralateral mACT was transected ( red zigzag line ) with an infrared laser ( dashed red arrow ) .","( C ) Projection images of a 7 µm stack of the LH area prior and post transection .","Left images , mACT transected; right image , plF transected .","The ablated region is indicated by the dashed red arrow .","Scale bar , 20 µm .","( D ) Median time traces displaying percental change of ΔF/F values for indicated ORDs prior to post transection of the mACT ( green , left ) and the plF ( orange , right ) for different odorants .","Significant changes of odor-evoked Ca2+ signals due to transection are shown in the column SIG difference .","Differences were tested with a two-tailed paired Student's t test ( p < 0 . 05 ) .","Color codes are indicated by the corresponding scale bar below , n = 4–5 .","Transecting the mACT eliminates Ca2+ signals in the LH-PM and LH-AM domain , while lesioning the plF significantly abolishes LH-AL responses .","Notably , the LH-AL domain is significantly stronger activated after mACT transection following application of 1-octen-3-ol and γ-butyrolactone .","( E ) Summarized cartoon of the neuron populations contributing to ORD activity prior and post transection of axons of iPNs or vlPr neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 013 We next addressed the behavioral relevance of MZ699+ iPN activity in the LH for innate odor-guided behavior .","To precisely target iPN function , we expressed an RNAi construct against glutamic acid decarboxylase 1 ( GADi ) to selectively knock-down the GABA synthesis in MZ699+ iPNs ( Figure 5A ) .","We confirmed the reduction in GABA production via immunostaining ( Figure 5B ) .","Since vlPr neurons are not GABAergic , they were not affected by the RNAi expression ( Liang et al . , 2013; Parnas et al . , 2013 ) .","Using wild-type flies and parental controls , we conducted T-maze assays ( Tully and Quinn , 1985; Chakraborty et al . , 2009 ) with nine of the odorants applied in functional imaging experiments at medium and high concentrations .","Notably , flies with silenced MZ699+ iPN GABA production revealed a neutral or aversive behavioral response to attractive odors , while repellent odors evoked an even stronger aversion ( Figure 5C ) .","To compare the T-maze data more accurately , we calculated the average change of behavioral response indices ( RIs ) between GADi flies and parental controls ( Figure 5D ) .","Indeed , all responses changed in a negative direction , indicating MZ699+ iPNs play a crucial role in mediating attraction behavior .","The sole exception involved high concentrations of the most repulsive odor , acetophenone , since this odor had already induced maximum aversion .","Overall , these experiments reveal a crucial function of MZ699+ iPNs in mediating attraction behavior by releasing GABA in the LH . 10 . 7554/eLife . 04147 . 014Figure 5 . iPN GABA release in the LH mediates odor attraction behavior .","( A ) Experimental layout: iPN GABA production was selectively silenced via GADi expression in MZ699+ iPNs; ePN and vlPr neuron activity remained unaffected .","( B ) Immunostaining against GABA and GFP within AL somata ( left ) and LH neurites ( right ) of iPNs with intact ( top ) and silenced GABA production ( bottom ) .","GADi flies show GABA signals in somata of iPNs labeled by GH146-GAL4 only ( arrowhead ) .","The arrow head points to an exemplary GABA-positive bouton in the LH .","Scale bar , 20 µm .","( C ) Averaged behavioral response indices ( RIs ) determined with a T-maze assay for wild-type flies ( dark blue ) , parental controls ( light blue ) and experimental animals ( magenta ) for nine odorants at two concentrations .","Empty boxes display no response ( Wilcoxon signed-rank test ) .","Dunn's Multiple Comparison Test was used for global differences in the dataset followed by a posthoc test for selected pairs ( p* < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .","Error bars represent SEM .","( D ) RI differences between GADi flies and averaged parental controls .","RI differences are negative for all but one odor indicating that GADi expression shifts odor-guided behavior towards aversion .","Error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 014 The behavioral effect of the iPN knock-down suggests that MZ699+ iPNs encode positive hedonic valences .","To correlate the complete ORD pattern array with innate behavioral preferences , we assigned behavioral RIs for all odors at median and high odor concentrations using the T-maze assay as in our previous experiment ( Figure 6A ) .","Since extremely low concentrations rarely evoked any behavioral response , we excluded the 10−6 concentration in this analysis .","It is important to note here , that different behavioral assays for testing olfactory preferences in flies might lead to contradictory results .","However , the majority of odors used here was also tested in two other behavioral paradigms , the trap assay ( Stökl et al . , 2010; Knaden et al . , 2012 ) and the FlyWalk ( Steck et al . , 2012 ) ( pers . comm . M Knaden ) and yielded similar results ( see Figure 6—figure supplement 1 ) .","When we plotted median odor-evoked activity in a three-dimensional space defined by the three ORDs , we saw a clear clustering of responses evoked by aversive and attractive odorants ( Figure 6B ) .","The LH-AL domain , constituted mainly by vlPr neurons , is coding aversive odors , while attractive odors activated only the LH-PM and LH-AM domains that derive from MZ699+ iPNs .","This result is in accordance with our finding that iPNs mediate odor attraction . 10 . 7554/eLife . 04147 . 015Figure 6 . Integration of hedonic valence and odor concentration into ORDs .","( A ) Response indices of wild type flies for all odors at median and high concentrations .","Odors are sorted from highly aversive ( −1 , red ) to highly attractive ( +1 , green ) .","( B ) 3D-scatter plot of median Ca2+ responses of all odors based on the three ORDs .","Odor-dots are labeled due to their RI shown in ( A ) .","Same odors at different concentrations are connected with a line: the dot at the end depicts 10−2 , the centered dot 10−4 , and the end of the line 10−6 .","Attractive and aversive odor representations form separate clusters .","( C and D )","Left , schematic LH outlines with colored ORDs corresponding to data on the right .","Correlation score r ( upper right corner ) between median activity and measured RI in T-maze experiments or odor concentration , respectively , with significance denoted below .","Student's t test , *p < 0 . 05 , ***p < 0 . 001 .","( E ) Complete correlation matrices for Ca2+ response patterns of OSNs in the AL ( left ) and iPNs in the LH ( right ) .","The odors are arranged according to single linkage clustering of the LH activity patterns .","Heatmap color-code refers to the correlation distance scale bar on the right .","Correlation distance is defined as 1 − r , where r is the Pearson correlation coefficient between the response patterns of two odorants .","Odor letters are color-coded according to hedonic valence; 10−6 RI values are labeled in grey ( complete list right hand ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01510 . 7554/eLife . 04147 . 016Figure 6—figure supplement 1 . Odor valences determined with three different behavioral assays . Odor-evoked behavioral responses of wild type flies for the 14 odors used in this study determined by T-maze assay , trap assay and the FlyWalk .","The color denotes an attractive ( green ) , aversive ( red ) or a neutral ( light yellow ) behavioral response .","N/T , not tested .","The majority of odors yielded similar results independent of the behavioral assay used .","In a few cases an attractive odor evoked a neutral response ( i . e . , no response ) , but never induced an aversive response in another assay . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01610 . 7554/eLife . 04147 . 017Figure 6—figure supplement 2 . Calcium responses of OSNs .","( A ) Representative glomerular Ca2+-responses of OSNs in the AL for a subset of odorants at three concentrations .","Scale bar to the right .","Control ( mineral oil ) recordings are shown additionally as full false-color coded images .","( B ) Glomerular AL atlas used for glomerular identification .","( C ) Median Ca2+-activity traces of all glomeruli for all odorants at the three indicated concentrations .","Scale bar and control measurement in the center .","Odor application is indicated by the grey bar below the heatmaps ( n = 6–7 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01710 . 7554/eLife . 04147 . 018Figure 6—figure supplement 3 . Correlation matrices for odor-evoked responses in the AL and LH . Complete correlation matrices for calcium activity patterns of OSNs in the AL ( left ) and iPNs in the LH ( right ) .","The odors are arranged according to single linkage clustering of the AL activity patterns .","Heatmap color-code refers to the correlation distance scale bar below each matrix .","Odor letters are color-coded according to hedonic valence; 10−6 RI values are labeled in grey ( complete list right hand ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 018 We next correlated ORD activity to odor valence separately for all ORDs .","This evaluation enabled us to analyze iPN and vlPr neuron coding properties apart from each other ( Figure 6C ) .","As expected , the analysis revealed a significant correlation between positive valence and the LH-PM domain , whereas Ca2+ responses in the LH-AL were strongly negatively correlated to hedonic valence .","The LH-AM domain exhibited a positive but not significant correlation for odor valence .","Remarkably , activity within the LH-PM was totally independent of concentration , whereas activity in both anterior domains was significantly correlated to odor intensity ( Figure 6D ) .","Hence , MZ699+ iPNs integrate odor attraction information into the LH-PM domain independent of odor intensity , confirming behavioral experiments .","Intensity coding is in turn conducted separately by distinct iPNs within the LH-AM domain .","In contrast , putative third-order vlPr neurons projecting into the LH-AL area code both negative valence and odor intensity .","Finally , we wondered if this valence-specific LH representation is already reflected at the primary level of olfactory processing .","The odor-evoked responses in iPNs are generally similar to those in OSNs ( Wang et al . , 2014 ) , indicating a straight forward transduction of cholinergic OSN responses .","We therefore performed functional imaging of odor-evoked Ca2+ dynamics at the AL input level by expressing G-CaMP3 . 0 in OSNs using Orco-GAL4 ( Larsson et al . , 2004 ) ( Figure 6—figure supplement 2 ) .","In order to compare the activity patterns at both processing levels , we calculated correlation distances for all pair-wise combinations of odor-evoked response patterns and plotted these with respect to maximal ORD pattern similarity in the LH ( Figure 6E ) .","As expected , odor representations in the LH clearly clustered within three separated parts of the matrix , reflecting our observed ORDs .","However , this coding similarity could not predict AL activity patterns , even if the correlation matrix was sorted with respect to pattern similarity in the AL ( Figure 6—figure supplement 3 ) ."],["We augment our present understanding of the Drosophila olfactory circuitry by elucidating the coding properties of a parallel and behaviorally relevant higher-order processing pathway to the LH .","Morphological , functional and behavioral approaches provide strong evidence for a functional subdivision of iPNs into neurons coding either odor attraction or odor intensity .","Our behavioral experiments reveal that inhibitory properties of iPNs are necessary for innate odor-guided attraction .","In addition , we characterize a third neural pathway coding odor repellence .","Do MZ699+ iPNs fulfill anatomical requirements to constitute a distinct processing channel in addition to ePNs ?","A remarkable anatomical feature of MZ699+ iPNs is their glomerular innervation pattern in the AL .","Whereas GH146+ ePNs are uniglomerular and retain the topographic code in their axonal arrangement ( Marin et al . , 2002; Wong et al . , 2002; Jefferis et al . , 2007 ) , most MZ699+ iPNs possess oligoglomerular innervations suggesting that these neurons might not convey precise odor-identity information .","In addition , MZ699+ iPNs in the AL diverge only into specific glomerular subsets , and so might be pre-determined to selectively extract common features of distinct odors .","We have previously shown that the AL map at the PN level exhibits a spatial segregation of valence representation ( Knaden et al . , 2012 ) .","Certain glomeruli , which have been classified as aversion coding at the GH146+ ePN level , are omitted by MZ699+ iPNs , whereas most glomeruli classified as attraction coding are particularly densely innervated .","These results suggest that within the MZ699+ iPN population , mainly positive odor traits are extracted , whereas the odor information of negative valence is neglected .","This conclusion is consistent with the recent finding that one type of LH neurons is receiving input from PNs that mainly innervate glomeruli coding fruity-smelling acetates ( Fisek and Wilson , 2014 ) which represent attractive odor cues ( Knaden et al . , 2012 ) .","We furthermore demonstrate that the MZ699+ iPN population is split into two major morphological classes possessing a clear spatial segregation in the AL which is strictly maintained within the LH .","It has to be kept in mind that we do not cover all iPNs by using MZ699-GAL4 .","Further experiments characterizing the ∼6 missing MZ699– iPNs , which are labeled by GH146-GAL4 ( Wilson and Laurent , 2005; Lai et al . , 2008 ) , will elucidate if our assumptions apply for the whole iPN population .","So far only a handful neuroanatomical studies targeting GH146+ ePNs have dealt with the question of how olfactory information is integrated and read out by higher brain structures , in particular the LH ( Marin et al . , 2002; Wong et al . , 2002; Tanaka et al . , 2004; Jefferis et al . , 2007 ) .","A recent study that traced the projection pattern of PNs coding ammonia and amines as attractive stimuli and carbon dioxide and acids as repulsive signals suggests that sensory stimuli of opposing valence are represented in spatially distinct areas within the LH ( Min et al . , 2013 ) .","In addition the study by Liang et al . ( 2013 ) showed that MZ699+ iPNs selectively suppress the activity of vlPr neurons to food odors , while pheromone responses were not affected verifying the assumption that different odor features are processed separately .","However , functional evidence for a feature-based , spatially segregated activity map in the LH was so far missing .","To unravel the coding properties of MZ699+ iPNs within the LH , we conducted Ca2+ imaging experiments of MZ699+ iPNs in the LH to odorants having different hedonic valences and intensities , and could classify the LH into three functional ORDs .","Our neuronal tracing and transection experiments validated the LH segmentation into two medial domains that derive from MZ699+ iPNs , and the LH-AL domain formed by vlPr neurons .","In line with our observations are morphological studies on ePNs and third-order LH neurons revealing a similarly tight constriction into three zones within the LH ( Tanaka et al . , 2004 ) , while single-cell labeling combined with image registration resulted in five ePN target zones ( Jefferis et al . , 2007 ) .","However , the ePN terminal zones do not necessarily correspond to the target domains of iPNs , since it has recently been shown that MZ699+ iPNs do not inhibit odor responses of GH146+ ePNs ( Liang et al . , 2013 ) and that the presynaptic sites of iPNs are spatially separated from those of ePNs ( Wang et al . , 2014 ) .","Hence both PN populations represent parallel processing pathways that most likely accomplish distinct processing tasks analogous to the honeybee olfactory system which possesses dual olfactory pathways to the higher brain that accomplish parallel processing of similar odors ( Brill et al . , 2013 ) .","Silencing MB function revealed that the LH alone is sufficient for basic olfactory behavior ( de Belle and Heisenberg , 1994; Connolly et al . , 1996; Heimbeck et al . , 2001 ) .","Our behavioral results demonstrate that selectively silencing MZ699+ iPNs severely reduced the flies' odor attraction behavior .","Hence our results suggest that MZ699+ iPNs are capable of extracting specific features from the combinatorial code emerging in the AL .","A behavioral study revealed that silencing MBc neurons impairs odor attraction but not repulsion ( Wang et al . , 2003b ) .","The authors drew the conclusion that the LH is involved in mediating innate repulsion rather than attraction .","These results are not necessarily contradictory to ours since some ePNs might activate the LH-AL domain exclusively ( i . e . , vlPr neurons ) .","On the other hand , Wang et al . ( 2003b ) did not include highly concentrated attractive odors .","Therefore it is possible that in their experiments , the odor detection threshold was simply reduced , so that only highly concentrated odors , which induced odor aversion , could be distinguished .","Our behavioral results , in contrast , revealed the constant influence of the MZ699+ iPNs in mediating attraction for odorants over a range of concentrations .","Our data suggests that odors with opposing hedonic valences are encoded by an interplay of distinct processing pathways .","The study by Liang et al . ( 2013 ) showed that GABA release from MZ699+ iPNs directly inhibits responses of vlPr neurons to food odors as mentioned above .","This finding fits well to our observations that iPNs are activated mainly by attractive odors while vlPr neurons are not , likely due to the inhibitory input from iPNs .","VlPr neurons are , on the other hand , almost solely activated by repellent odors , which do hardly activate iPNs and therefore do not induce a strong inhibition to vlPr neurons .","Repellent odors most likely activate vlPr neurons via ACh release of ePNs which is supported by immunostainings with pre- and postsynaptic markers indicating that vlPr neurons receive input in the LH , while the vlPr represents their major output region ( Parnas et al . , 2013 ) .","The vlPr is supposedly also a target of visual neurons from the optic lobe ( Tanaka et al . , 2004 ) implying that a certain integration of different sensory modalities takes place at this central processing relay .","Given that iPNs are inhibitory neurons , the underlying mechanism of odor attraction behavior might therefore be an inhibition of aversive neuronal circuits from the LH to the vlPr that are mainly composed of vlPr neurons .","However , this assumption needs to be verified with further experiments elucidating if vlPr neurons are sufficient and necessary to mediate odor aversion .","What is known about odor coding in the LH in other insect species ?","Notably , in locusts it has been shown that LH neurons receiving convergent PN input appeared to encode stimulus intensity in their net firing rates and in the phases of their spikes ( Gupta and Stopfer , 2012 ) .","Hence these results support the idea that within the LH , general stimulus features such as odor intensity are extracted , which is well in line with our observation of the anterior LH domains whose activity is also significantly correlated to odor intensity .","Also in line with our results is a study from honeybees which shows that the representation of different pheromone types is spatially segregated in the LH ( Roussel et al . , 2014 ) , indicating that odors eliciting specific behaviors are coded according to their biological values .","In conclusion , our study provides an important step in unraveling higher olfactory processing mechanisms that are crucial for mediating innate behaviors in Drosophila .","We provide functional evidence for a feature-based spatial arrangement of the LH decoding opposing hedonic valences and odor intensity .","The role of the LH as a center for integrating biological values towards innate decisions by computing conveyed information of two processing pathways is thus expanded ."],["All fly stocks were maintained on conventional cornmeal-agar-molasses medium under L:D 12:12 , RH = 70% and 25°C .","For wild-type controls D . melanogaster of the Canton-S strain was used .","Transgenic lines were obtained from Bloomington Stock Center ( http://flystocks . bio . indiana . edu/ ) and Vienna RNAi stock center ( http://www . vdrc . at ) .","Other fly stocks were kindly provided by Kei Ito ( MZ699-GAL4 ) and Maria Luisa Vasconcelos ( UAS-C3PA ) .","The END1-2 fly line is published in Grabe et al . ( 2014 ) .","Whole-mount immunofluorescence staining was carried out as described ( Laissue et al . , 1999; Vosshall et al . , 2000 ) .","Initially brains were dissected in Ringer's solution ( 130 mM NaCl , 5 mM KCl , 2 mM MgCl2 , 2 mM CaCl2 , 36 mM saccharose , 5 mM HEPES , [pH 7 . 3] ) ( Estes et al . , 1996 ) and fixed in 4% PFA in PBS-T ( PBS , 0 . 2–1% Triton-X ) .","After washing with PBS-T brains were blocked with PBS-T , 5% normal goat serum ( NGS ) .","Primary antibodies were diluted in blocking solution or PBS-T and incubated at 4°C for 2–3 days .","Secondary antibody incubation lasted 1–2 days .","Brains were mounted in VectaShield ( Vector Labs , Burlingame , CA ) .","The following primary antibodies were used: rabbit α-GABA ( 1:500 ) ( Sigma ) , mouse α-GFP ( 1:500 ) ( Invitrogen ) .","The following secondary antibodies were used: Alexa Fluor 488 , goat anti-mouse ( 1:500 ) ; Alexa Fluor 546 , goat anti-rabbit ( 1:500 ) ; ( all IgG Invitrogen ) .","Fly preparation and functional imaging of the AL was conducted as previously described ( Stökl et al . , 2010; Strutz et al . , 2012 ) .","LH imaging was conducted similarly , except for the higher resolution achieved with a 60× water immersion objective ( LUMPlanFl 60×/0 . 90 W Olympus ) .","The optical plane was ∼30 µm below the most dorsal entrance point of the iPN tract into the LH .","Binning on the CCD-camera chip resulted in a resolution of 1 pixel = 0 . 4 × 0 . 4 µm .","For bilateral LH imaging during transection a 20× water immersion objective ( NA 0 . 95 , XLUM Plan FI , Olympus ) was employed .","All recordings lasted 10 s with a frame rate of 4 Hz .","Odors included acids ( propionic acid , acetic acid ) , lactones ( γ-butyrolactone ) , terpenes ( linalool ) , aromatics ( acetophenone , methyl salicylate , benzaldehyde , phenylacetic acid ) , alcohols ( 1-octen-3-ol ) , esters ( acetoin acetate , cis-vaccenyl acetate , 2-phenethyl acetate ) , ketones ( 2 , 3 butanedione ) and balsamic vinegar diluted in mineral oil ( all from Sigma Aldrich ) .","Odors were applied during frame 8–14 ( i . e . , after 2 s , lasting for 2 s ) .","Flies were imaged for up to 1 hr , with a minimum inter-stimulus interval of one minute .","We selected conventional widefield Ca2+ imaging as the method of choice , since we were able to obtain single bouton resolution with this technique .","Calcium imaging data of AL were analyzed with custom-written IDL software ( ITT Visual Information Solutions ) provided by Mathias Ditzen as previously described ( Stökl et al . , 2010; Strutz et al . , 2012 ) .","Regarding the Ca2+ imaging data in the LH , we repeated recordings of each odor at each concentration two to three times to ensure the reliability of the extracted domain information .","To execute NNMF analysis ( see below ) , at least 6–7 valid measurements , that is , animals with repeated identical recordings , were collected for each odor and employed for the analysis .","Individual odor measurements were aligned using ImageJ ( Fiji ) to correct movement artifacts .","Fluorescence changes ( ∆F/F ) for each odor were calculated in relation to background fluorescence using frames 0–6 ( i . e . , 2–0 . 5 s before odor application ) .","A Gaussian low-pass filter ( ó = 1px ) was applied to compensate for remaining movement artifacts and pixel noise .","To reduce the computational load , the frame rate was averaged by two consecutive frames , and recordings were spatially down-sampled by a factor of two .","The resulting concatenated time-series of the recordings is denoted as measurement matrix Y with element Yt , p being the tth observed value of pixel","p . In contrast to the AL , which consists of highly ordered glomerular subunits , the LH comprises a mainly homogenous neuropil which does not provide spatial or functional landmarks .","Therefore , we used the automatic method NNMF to extract Ca2+ signals that exhibit common spatial or temporal features .","NNMF , like other matrix factorization techniques ( e . g . , Principal Component Analysis ( PCA ) and Independent Component Analysis ( ICA ) ) , decompose the measurement matrix Y into k components , Y=∑kxk∗akT+R .","The time-course ak of each component contains a common underlying time-courses of all pixels and each pixel participation xk declares how strongly each pixel is involved in this time-course .","The residual matrix R contains the unexplained data .","In order to perform NNMF , we implemented the HALS algorithm in Python including a spatial smoothness constraint ( asm = 0 . 1 ) ( Cichocki and Phan , 2009 ) and an additional spatial decorrelation constraint ( ade = 0 . 1 ) ( Chen and Cichocki , 2005 ) .","In PCA decomposition is performed such that either timecourses ak or pixel participation xk are uncorrelated , whereas ICA aims for timecourses ( temporal ICA ) or pixel participation ( spatial ICA ) to be independent .","Although spatial ICA is able to segregate signals into functional similar neuropils ( Reidl et al . , 2007 ) , we chose the NNMF approach , because it is known to achieve even a better parts-based representation compared to the more holistic results of PCA or ICA ( Lee and Seung , 1999 ) .","In contrast to PCA and ICA , NNMF constrains both the extracted time-courses and pixel participations to be positive .","Positive pixel participation enabled us to make a straightforward physiological interpretation , reading the participation values as the contribution strength of an underlying physiological domain .","The restriction to positive time-courses reflects the fact that we did not observe any significant decrease of fluorescence in response to an odor in the original measurement data .","For each animal we performed decomposition into k = 5 components .","This was sufficient to explain most of the data's variance ( 88% + 8% , error is standard deviation across individuals ) .","The remaining variance in the residual matrix R contained no additional domains but rather reflected remaining movement artifacts of the measurements ( Figure 2—figure supplement 2 ) .","Of the five components extracted by NNMF , three stood out prominently ( Figure 2—figure supplement 3 ) : First , they were extracted in all animals at very clearly defined anatomical positions .","Second , their responses to stimuli repetitions were highly reproducible in contrast to the other two components , that is , they exhibited a significant ( p < 2*10−8 , t test ) higher trial-to-trial correlation of 0 . 72 ± 0 . 20 in contrast to 0 . 52 ± 0 . 26 for the remaining components; third , the odorant spectra of their responses were characteristic across animals .","Though we cannot completely rule out that the remaining components of the factorization are ORDs of their own , there are several indications that they are not .","On the one hand , they exhibit a lower trial-to-trial correlation than the three selected components .","Second , those components did not consistently appear at similar anatomical position .","Third , they were spatially overlapping with the selected three components .","Instead of independent ORDs , these regions might convey fluorescence changes independent of odor stimulation or an overlapping region of two of the reliable ORDs .","A validation of our NNMF-based results with spatial ICA yielded very similar , but slightly worse results .","Whereas the three reliable ORDs from NNMF were also extracted in spatial ICA , the two remaining components exhibited much higher variability than when obtained with NNMF .","Hence , we conclude that the LH area comprising MZ699+ neurons consists of three ORDs .","We labeled those three components according to the anatomical position of their pixel participation within the LH .","To determine the coding properties of extracted odor response domains ( ORDs ) , we calculated the mean response of each animal within a time window of 1–4 s after stimulus onset .","Hence , median responses over all animals defined the standard stimulated response rORDo of an ORD to an odor","o . Initially , regions were evaluated individually , and correlations were calculated between standard response spectra and the behavioral response index ( RI ) , or odor concentration , respectively , using the ‘linregress’ function of the Python scipy . stat module .","To analyze the combined ORD representations of odor patterns po= ( rPMo , rAMo , rALo ) we calculated for all odor pairs the pattern similarity as correlation distance do1 , o2=1−corr ( po1 , po2 ) .","In order to visualize the correlation matrix in a comprehensible way , we then arranged odors according to the single linkage clustering of the Python scipy . cluster . hierarchy module .","To compare the representation in the LH to those of the AL , we applied the same procedure to the dorsal glomerular odor activation pattern .","For in vivo photoactivation experiments , 1–6 day old flies ( genotype: END1-2 , UAS-C3PA;MZ699-GAL4 ) were dissected as in the imaging experiments except that tracts of the salivary glands were cut to prevent movement .","Photoactivation was accomplished via continuous illumination with 760 nm for 15–25 min .","After a 5-min break to permit full diffusion of the photoconverted molecules , 925 nm z-stacks of the whole brain were acquired and subsequently used for neuronal 3D-reconstruction .","For all 3D reconstructions , the segmentation software AMIRA 4 . 1 . 1 & 5 . 3 . 3 ( FEI Visualization Sciences Group , Burlington , MA ) was used .","Neurons of different individuals were embedded into the reference brain using a labelfield registration as previously described ( Rybak et al . , 2010 ) .","Briefly , segmented labels of brain neuropils ( AL , MBc , LH ) were registered onto a reference brain image using affine registration followed by elastic warping .","In a second step , the calculated transformation matrix was applied to the respective neuron morphology that was then aligned to the reference brain image .","For morphological analysis of reconstructed iPNs , we first determined all terminal points of each iPN in the LH area .","For each combination of terminals we calculated a similarity score ( s ) in analogy to ( Kohl et al . , 2013 ) as follows:s ( t1 , t2 ) =√e−Δ ( t1 , t2 ) 2/2σ2 , where t is the terminal position , Δ ( t1 , t2 ) is the Euclidean distance and σ is a free parameter that determines how close in space terminal points must be to be considered similar; analogue to Kohl et al . ( 2013 ) we set this parameter to 3 µm .","Finally we calculated the pairwise similarity score between two neurons as their average all-to-all terminal similarity scores , normalized to their self-scores as follows:S ( n1 , n2 ) =∑t1 , t2s ( t1 , t2 ) /∑t1s ( t1 , t1 ) ∗∑t2s ( t2 , t2 ) Effectively this quantifies the relative overlap of the target area of all pairs of iPNs .","For clustering , the similarity scores were converted to distances ( i . e . , 1-S ) and a hierarchical clustering was performed using UPGMA method .","Principal component analysis and one-way ANOSIM was performed using the statistical software PAST 3 . x ( Paleontological statistics software package for education and data analysis ) .","Transections of either the plF tract or the mACT were conducted in one brain hemisphere , each of the same fly .","The target area was monitored with 925 nm and chosen to be close to the LH but distant enough not to affect neurites ramifying in the LH neuropil .","For both tracts , lesioned areas had an average size of 34 µm and were illuminated with short pulses of 710 nm every 40 ms for 250 ms in 60 ( plF ) to 80 ( mACT ) cycles in a single focal plane .","After a fast z-stack with 925 nm to confirm complete lesion , a 5-min neuronal recovery interval followed before continuing the imaging procedure .","Data were analyzed using NNMF .","Photoactivation and transection procedures as well as image acquisition following immunohistochemistry were accomplished with a 2-photon confocal laser scanning microscope ( 2PCLSM , Zeiss LSM 710 NLO ) equipped with a 40× ( W Plan-Apochromat 40×/1 . 0 DIC M27 , Zeiss ) or 20× ( W N-Achroplan 20×/0 . 5 M27 , Zeiss ) .","The 2PCLSM was placed on a smart table UT2 ( Newport Corporation , Irvine , CA , USA ) and equipped with an infrared Chameleon Ultra diode-pumped laser ( Coherent , Santa Clara , CA , USA ) .","Z-stacks were performed with argon 488 nm and helium-neon 543 nm laser or the Chameleon Laser 925 nm ( BP500-550 for G-CaMP and LP555 for DsRed/tdTomato ) and had a resolution of 1024 or 512 square pixels .","The maximum step size for immuno-preparations or single neuron projections was 1 µm and for AL reconstructions 2 µm .","Flies carrying P[GAD1-RNAi];P[MZ699-GAL4] were crossed just before the experiment to prevent dosage compensation effects .","T-maze experiments were performed as described ( Stensmyr et al . , 2012 ) .","WT , parental controls ( P[GAD1-RNAi] or P[MZ699-GAL4] ) and test flies carrying both insertions were tested separately under identical conditions .","The response index ( RI ) was calculated as ( O-C ) /T , where O is the number of flies in the odor arm , C is the number of flies in the control arm , and T is the total number of flies used in the trial .","Hence , the RI ranges from −1 ( complete avoidance ) to 1 ( complete attraction ) .","Each experiment was carried out on 30 flies and was repeated 12 times .","Dunn's Multiple Comparison Test was used for global differences in the dataset .","Whenever the Multiple Comparison Test was significant ( i . e . , p < 0 . 05 ) , a posthoc test for selected pairs was performed , that is , between the GADi-flies and the other three control lines as we were not interested in differences among the different control lines .","All RI were tested against 0 ( no response ) by using the Wilcoxon-rank-sum test ."]],"string":"[\n [\n \"To navigate the environment in a way that optimizes their survival and reproduction , animals have evolved sensory systems .\",\n \"These have three essential tasks: First , the external world has to be translated into an internal representation in the form of an accurate neural map .\",\n \"Second , the neural map has to be readable and interpretable , that is , the generated neural code must allow common attributes to be extracted across stimuli to enable the animal to make the best decisions .\",\n \"Third , the animal has to be able to adapt to environmental changes and to form a sensory memory of new stimuli .\",\n \"Many studies have been dedicated to unraveling the primary transformation from a stimulus into an initial neural representation within various sensory systems ( Manni and Petrosini , 2004; Vosshall and Stocker , 2007; Sanes and Zipursky , 2010 ) and to elucidating neuronal plasticity and sensory memory formation in higher-level processing centers ( Heisenberg , 2003; Pasternak and Greenlee , 2005 ) .\",\n \"The ability to extract features and integrate stimulus modalities have so far mainly been studied in the visual system ( Livingstone and Hubel , 1988; Bausenwein et al . , 1992; Nassi and Callaway , 2009 ) .\",\n \"We addressed the question of how stimulus features such as odor valence and intensity are coded and integrated within the olfactory system using the model organism Drosophila melanogaster .\",\n \"The olfactory system of the vinegar fly provides an excellent model system for deciphering olfactory processing mechanisms , since it displays remarkable similarities to the mammalian system but is less complex and highly genetically tractable .\",\n \"Like other sensory systems , the olfactory system employs a spatio-temporal map to translate the variables in chemosensory space into neuronal activity patterns in the brain .\",\n \"This map emerges when the olfactory sensory neurons ( OSNs ) with the same chemosensory receptors converge into one exclusive glomerulus in the antennal lobe ( AL ) which represents the equivalent to the mammalian olfactory bulb ( Hildebrand and Shepherd , 1997; Vosshall et al . , 2000; Vosshall and Stocker , 2007 ) .\",\n \"Glomeruli , the functional and morphological units of the AL , are microcircuits comprising OSNs , multiglomerular local interneurons ( LNs ) and uniglomerular output neurons , so-called excitatory projection neurons ( ePNs ) ( Wilson and Mainen , 2006; Vosshall and Stocker , 2007 ) that convey the olfactory information to higher brain centers , as the mushroom body calyx ( MBc ) and the lateral horn ( LH ) ( Stocker et al . , 1997 ) .\",\n \"The stringent spatial arrangement of OSNs and ePNs in the AL generates a spatial map containing characteristic combinatorial glomerular activity patterns for all odorants ( Fiala et al . , 2002; Wang et al . , 2003a; Couto et al . , 2005; Fishilevich and Vosshall , 2005 ) .\",\n \"The MBc is involved in olfactory memory formation ( Heisenberg , 2003 ) and enables a contextualization of the odor space ( Caron et al . , 2013 ) .\",\n \"By exclusion , the LH is believed to be involved in innate olfactory behavior ( de Belle and Heisenberg , 1994; Jefferis et al . , 2007 ) .\",\n \"Excitatory PNs retain the sensory information encoded in the AL and form glomerulus-dependent , stereotypic axonal terminal fields in the LH ( Marin et al . , 2002; Wong et al . , 2002; Tanaka et al . , 2004 ) .\",\n \"Compartmentalization in the LH has been observed in form of a spatial segregation of ePNs innervating specific glomerular subgroups ( Tanaka et al . , 2004 ) , fruit and pheromone odor information processing ePNs ( Jefferis et al . , 2007 ) as well as ammonia and amine vs carbon dioxide coding ePNs ( Min et al . , 2013 ) .\",\n \"Like many other sensory networks , the olfactory circuit of the fly contains spatially distinct pathways to the higher brain , namely the inner , middle and outer antennocerebral tract ( iACT , mACT and oACT ) ( Stocker et al . , 1990 ) .\",\n \"Notably , the mACT projects from the AL to the LH exclusively and consists of inhibitory PNs ( iPNs ) , which exhibit also uniglomerular but mainly multiglomerular AL innervations ( Ito et al . , 1997; Jefferis et al . , 2007; Lai et al . , 2008; Okada et al . , 2009; Liang et al . , 2013 ) .\",\n \"Both PN populations have been attributed different coding properties: Although both PN populations exhibit odor responses to overlapping odor ligands , iPNs seems to be broader tuned than ePNs ( Wang et al . , 2014 ) .\",\n \"Furthermore , while ePNs encode rather odor identity ( Wang et al . , 2003a; Wilson et al . , 2004; Silbering et al . , 2008 ) , iPNs have been shown to enhance innate discrimination of closely related odors ( Parnas et al . , 2013 ) .\",\n \"Together , these PN populations process information on dual olfactory pathways ( Liang et al . , 2013; Wang et al . , 2014 ) , as do processing mechanisms in other sensory modalities ( Nassi and Callaway , 2009 ) , and most likely accomplish different olfactory behaviors .\",\n \"The mainly multiglomerular AL pattern of iPNs suggests that these neurons extract characteristic stimulus features from the AL code and re-integrate this information into the LH to mediate innate odorant-guided behavior .\",\n \"This assumption is further supported by two recent studies showing that the inhibitory input from the AL to the LH is module-specific , that is , selective for food odors and pheromones ( Liang et al . , 2013; Fisek and Wilson , 2014 ) , while the connectivity in the MBc is rather probabilistic ( Murthy et al . , 2008; Caron et al . , 2013 ) .\",\n \"However , it still remains open if and how different odor features as hedonic valence or intensity are functionally coded and integrated in the LH .\",\n \"In this study , we characterized and dissected the iPN olfactory processing pathway regarding the coding of odor quality and intensity at morphological , functional and behavioral levels .\",\n \"By linking odor-evoked activity patterns in the LH to odor-guided behavior , we provide evidence that iPNs mediate odor attraction .\",\n \"Furthermore , our data demonstrate a feature-based , spatially segregated activity map in the LH comprised of iPNs and third-order neurons and thus expand its role as a center for integrating behaviorally relevant olfactory information .\"\n ],\n [\n \"Cell bodies of iPNs are exclusively located in the ventral cell cluster which consists of ∼50 iPNs ( Lai et al . , 2008 ) that project via the mACT to the LH , thereby bypassing the MBc ( Ito et al . , 1997 ) ( Figure 1A , B ) .\",\n \"In contrast , ePN somata are located anterodorsally and laterally of the AL , and their axons project through the iACT or oACT to the MBc and the LH ( Stocker et al . , 1997; Marin et al . , 2002; Wong et al . , 2002; Lai et al . , 2008 ) .\",\n \"To analyze the innervation patterns of iPNs and ePNs , we labeled both PN populations simultaneously in vivo using the enhancer trap lines GH146-QF and MZ699-GAL4 that label the majority of ePNs ( 60% ) and iPNs ( 86% ) , respectively ( Lai et al . , 2008 ) .\",\n \"Double-labeling shows that both PN types innervate overlapping regions in the AL and the LH , while a small posterior-lateral LH area is targeted only by ePNs ( Figure 1A , Figure 1—figure supplement 1 ) .\",\n \"In GH146-positive ( GH146+ ) PNs , immunolabeling reveals GABA production in all ∼6 PNs of the ventral cell cluster ( Wilson and Laurent , 2005 ) , whereas ePNs of this line are exclusively cholinergic ( Shang et al . , 2007 ) .\",\n \"For the ∼45 MZ699-positive ( MZ699+ ) iPNs ( Lai et al . , 2008 ) , GAD1 ( glutamic acid decarboxylase ) in situ hybridizations imply GABA synthesis ( Okada et al . , 2009 ) , which was recently verified via immunostaining ( Liang et al . , 2013; Parnas et al . , 2013 ) .\",\n \"The polarity of both PN populations has been studied in detail , showing that both possess dendritic regions in the AL , indicating the AL as their cholinergic input site , while the LH represents their major output site ( Jefferis et al . , 2001; Okada et al . , 2009; Liang et al . , 2013; Parnas et al . , 2013 ) . 10 . 7554/eLife . 04147 . 003Figure 1 . Detailed glomerular innervations of excitatory and inhibitory projection neurons in the AL .\",\n \"( A ) Simultaneous labeling of inhibitory projections neurons ( iPNs , labeled by MZ699-GAL4;G-CaMP ) and excitatory projection neurons ( ePNs , labeled by GH146-QF;tdTomato ) in vivo reveals distinct projections to the lateral horn ( LH ) .\",\n \"All iPNs bypass the mushroom body calyx ( MBc ) and innervate the LH exclusively .\",\n \"The MZ699 line labels a few ventrolateral protocerebral neurons ( vlPr neurons ) projecting via the posterior lateral fascicle ( plF ) from the ventrolateral protocerebrum ( vlPr ) to the LH .\",\n \"( B ) Schematic of the PN connectivity relay from the antennal lobe ( AL ) to higher brain centers ( ePNs in magenta , iPNs in green , and vlPr neurons in orange ) .\",\n \"( C ) Above , complete glomerular assignment of the AL neuropil ( right AL ) , labeled with elav-n-synaptobrevin:DsRed ( END1-2 ) .\",\n \"Below , glomerular innervations of both PN populations related to in vivo images in Figure 1—figure supplement 2 .\",\n \"Depicted are the ventral level ( ∼−40 µm ) , the medial level ( ∼−20 µm ) and the dorsal view onto the AL .\",\n \"Color annotation: blue glomeruli are not innervated by any of the used GAL4-lines; green glomeruli are innervated by MZ699+ iPNs and magenta by GH146+ ePNs; white glomeruli are innervated by both enhancer trap lines .\",\n \"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00310 . 7554/eLife . 04147 . 004Figure 1—figure supplement 1 . Characterization of excitatory and inhibitory projection neurons . Overlap of ePNs ( QUAS-tdTomato ) and iPNs ( UAS-GCaMP3 . 0 ) in the LH area .\",\n \"The circle indicates the posterior lateral region , which is sparsely innervated by iPNs and dominated by ePN axonal terminal fields .\",\n \"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00410 . 7554/eLife . 04147 . 005Figure 1—figure supplement 2 . Glomerular innervations of ePNs and iPNs .\",\n \"( A ) Representative in vivo images of glomerular innervations .\",\n \"MZ699- and GH146-GAL4 lines have been reconstructed with the END1-2 background staining ( two upper planes ) and dual labeling via the Q-system and the GAL4-UAS expression system ( lowest plane ) .\",\n \"Scale bar , 20 µm .\",\n \"( B ) Detailed glomerular AL innervation .\",\n \"Green filled cells indicate innervation by MZ699-GAL4 , magenta GH146-GAL4 innervation , respectively and grey , no innervation by the indicated line .\",\n \"Bottom rows , total number of innervated glomeruli with percentage share indicated below .\",\n \"Merge column: white filled with ‘x’ indicates glomeruli innervated by both lines , grey only one line .\",\n \"Blue filled rows are glomeruli labeled by none of the enhancer trap lines . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 005 To further characterize PNs labeled by MZ699-GAL4 and GH146-GAL4 , we analyzed their precise glomerular innervation to unravel how selectively they acquire information in the AL .\",\n \"To allow glomerulus identification in vivo , we employed a transgenic fly carrying elav-n-synaptobrevin:DsRed ( END1-2 ) to express the presynaptically targeted fusion protein under the control of the neuron-specific elav promotor ( Figure 1—figure supplement 2A ) ( Grabe et al . , 2014 ) .\",\n \"The reconstruction and identification of all AL glomeruli provided 53 glomeruli , of which 75% were innervated by MZ699+ iPNs ( 40 ) while 70% ( 37 ) were covered by GH146+ ePNs ( Figure 1C , Figure 1—figure supplement 2B ) .\",\n \"55% of all glomeruli were innervated by both lines .\",\n \"Notably , dendritic MZ699-GAL4 innervation density was not homogeneous .\",\n \"Certain glomeruli were densely innervated ( e . g . , DM2 , DM4 and DM5 ) , while others did not reveal any postsynaptic sites ( e . g . , DL1 , DL4 and DL5 ) .\",\n \"Hence MZ699+ iPNs target specific glomerular subsets selectively , which suggests that these neurons have a particular function within the olfactory network .\",\n \"Probabilistic synaptic density maps of GH146+ PNs predicted a regionalized neuronal activity in the LH ( Jefferis et al . , 2007 ) .\",\n \"Do iPNs functionally segregate in a comparable way ?\",\n \"To address this question , we expressed the Ca2+-sensitive reporter G-CaMP3 . 0 ( Nakai et al . , 2001; Tian et al . , 2009 ) in iPNs using MZ699-GAL4 and performed functional imaging in the LH ( Figure 2A–C ) .\",\n \"We initially tested three odors with potential relevance for Drosophila at different concentrations: acetoin acetate , an attractive byproduct of the yeast fermentation process , balsamic vinegar , an attractive natural odor mixture , and benzaldehyde , a well-known fly repellant ( Magee and Kosaric , 1987; Keene et al . , 2004; Semmelhack and Wang , 2009 ) .\",\n \"We observed that odor evoked Ca2+ responses separate in certain regions of the LH in an odor-specific and concentration-dependent manner ( Figure 2C ) .\",\n \"Acetoin acetate and balsamic vinegar evoked Ca2+ activity in spatially similar regions .\",\n \"At higher concentrations , an additional region was recruited .\",\n \"Benzaldehyde elicited no response at very low concentrations , but induced clear activity at median and high concentrations in a third region , which was completely separate from the regions activated by the other two odors .\",\n \"Observed patterns were highly reproducible within one animal and stereotypic among different individuals , as shown for the stimulation with 1-octen-3-ol ( Figure 2D ) as well as other odors ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 04147 . 006Figure 2 . Odors evoke specific and stereotypic calcium responses in the LH subdivided into three distinct odor response domains .\",\n \"( A ) Schematic of the olfactory circuit with the investigated area highlighted .\",\n \"( B ) RAW image of the LH ( top picture ) depicting the recorded area of figures ( C–E ) and the false color image ( bottom picture ) during the solvent application .\",\n \"The ΔF/F scale bar applies for all false color-coded pictures; the alpha-bar for the pixel participation xk of the indicated colors applies for ( E–F ) .\",\n \"( C ) Representative LH Ca2+ responses ( ΔF/F% ) of acetoin acetate , balsamic vinegar and benzaldehyde at three concentrations .\",\n \"Numbers in the lower right corner indicate individual maxima .\",\n \"( D ) Odor-evoked Ca2+ responses ( ΔF/F% ) are exemplarily depicted for 1-octen-3ol- at three concentrations in four animals .\",\n \"( E ) NNMF-extracted LH odor response domains ( ORD ) of four representative animals: three LH ORDs were fully reproducible after being extracted from all measured animals .\",\n \"Domains classified as identical are similarly color-coded: the green ORD is located in the posterior-medial region of the LH ( LH-PM ) ; blue , in the anterior-medial ( LH-AM ) , and red in the anterior-lateral LH area ( LH-AL ) .\",\n \"The alpha-bar for green , blue and red shades is placed in ( B ) .\",\n \"( F ) Left , schematic outlines of the LH with indicated ORDs .\",\n \"Right , median activity traces of all odors at three concentrations are depicted for each colored ORD .\",\n \"Shadows represent lower and upper quartiles ( n = 6–7 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00610 . 7554/eLife . 04147 . 007Figure 2—figure supplement 1 . Odor-evoked activity patterns in the LH are reproducible and stereotypic . Odor-evoked Ca2+ responses ( ΔF/F% ) in the LH for the three odors acetoin acetate , balsamic vinegar and benzaldehyde in four animals are shown as false-color coded images .\",\n \"Two measurements in each animal are given to reveal that the activity patterns are highly reproducible within one animal .\",\n \"Comparison between the patterns among individuals shows that the activity regions are stereotypic .\",\n \"Numbers in the lower right corner indicate individual maxima .\",\n \"The ΔF/F scale bar is shown at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00710 . 7554/eLife . 04147 . 008Figure 2—figure supplement 2 . Odor-evoked activity patterns in the LH can be reconstructed with five components . Above , Odor-evoked Ca2+ responses ( ΔF/F% ) in the LH for the three odors acetic acid , balsamic vinegar and 1-octen-3-ol in three animals are shown as false-color coded images .\",\n \"The ΔF/F scale bar is shown at the bottom .\",\n \"Middle , activity patterns were reconstructed using NNMF with five components .\",\n \"Below , residue of the pattern reconstructions with five components ( as shown in the middle panel ) revealing that no stimulus related activity remained . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 00810 . 7554/eLife . 04147 . 009Figure 2—figure supplement 3 . Odor-evoked activity patterns in the LH cluster into three components . Hierachichal clustering ( UMPGA ) of the odor response spectra of the NNMF components with a reliable stimulus response ( trial-to-trial correlation >0 . 7 , that is , 28 out of 35 components in seven animals ) .\",\n \"The response spectra segregate into three distinct clusters according to their stimulus response spectra .\",\n \"The corresponding response areas ( left pictures ) are located in similar regions of the LH . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 009 Due to the lack of morphological landmarks in the LH , functional data were analyzed using the pattern recognition algorithm Non-Negative Matrix Factorization ( NNMF ) ( Lee and Seung , 1999 ) , which automatically extracts spatial areas possessing a common distinct time-course , further termed LH odor response domains ( ORDs ) .\",\n \"The NNMF analysis extracted three clearly reproducible and spatially robust ORDs ( Figure 2E , see NNMF part in the ‘Materials and methods’ section ) .\",\n \"Notably , ORDs occupying common temporal kinetics exhibited highly stereotypic spatial patterns .\",\n \"We termed the ORDs LH-PM ( LH-posterior-medial ) , LH-AM ( LH-anterior-medial ) and LH-AL ( LH-anterior-lateral ) according to their anatomical positions .\",\n \"To validate our observations , we extended our stimulus array to 11 additional odorants and applied each at three concentrations .\",\n \"Odorants were chosen according to chemical classes , hedonic valence and biological value .\",\n \"Hence , the odor set included acids , lactones , terpenes , aromatics , alcohols , esters , ketones and the natural blend , balsamic vinegar .\",\n \"Remarkably , analysis of the additional odorants revealed neuronal activity exclusively within the three described ORDs ( Figure 2F , Figure 2—figure supplements 2 , 3 ) .\",\n \"Furthermore , median NNMF-extracted Ca2+ response traces with indicated statistical quartiles illustrate very low variability and highly reproducible LH signals .\",\n \"The LH-PM area chiefly revealed robust odor-evoked responses across concentrations , while the LH-AM and LH-AL were mainly activated at very high odor concentrations by distinct odorants .\",\n \"The global responsiveness within separate ORDs in the LH substantiates our finding of a relatively broad AL input to MZ699+ iPNs which converges into three spatially regionalized and stereotypic LH activity domains .\",\n \"We next investigated if the spatially regionalized odor-evoked response patterns are reflected in the axonal terminal fields of MZ699+ iPNs in the LH .\",\n \"To analyze these neurons at the single neuron level , we performed neural tracing by employing a genetically encoded photoactivatable GFP ( PA-GFP ) ( Patterson and Lippincott-Schwartz , 2002; Datta et al . , 2008; Ruta et al . , 2010 ) .\",\n \"The photoconversion of all MZ699+ neurons leaving the AL confirmed the homogeneous distribution of iPN neurites in the LH and the sparse innervation of the posterior-lateral region as mentioned above ( Figure 3A ) .\",\n \"Next we illuminated PA-GFP in single somata to selectively label individual MZ699+ iPNs from the soma up to the farthest axonal terminals in the LH .\",\n \"Individual iPNs were reconstructed and transformed into a reference brain using the END1-2 background ( Grabe et al . , 2014 ) to align neurons of different individuals .\",\n \"Based on their innervation pattern in the LH , MZ699+ iPNs could be assigned to two major morphological classes ( Figure 3B , C ) .\",\n \"As expected from the extracted ORDs , one iPN group diverged to the LH-PM region ( 8/25 of iPNs ) , while a second group extended their axonal terminations within the LH-AM area ( 10/25 of iPNs ) .\",\n \"In order to statistically substantiate our observation , we performed a cluster analysis based on a similarity score ( Kohl et al . , 2013 ) of the target areas of all terminals of each iPN in the LH ( for details see ‘Materials and methods’ ) .\",\n \"The dendrogram of morphological similarity between each individual iPN shows that all , except one iPN , could be clustered according to their target region in either the LH-AM or LH-PM area ( Figure 3D ) which confirms the classification into two major categories .\",\n \"Additionally , we performed a principal component analysis based on the distances of the similarity scores showing that both neuronal classes possess significantly different target areas in the LH ( Figure 3—figure supplement 1A; p < 0 . 001 , one-way ANOSIM ) . 10 . 7554/eLife . 04147 . 010Figure 3 . iPNs can be classified according to their projection pattern in three distinct LH zones .\",\n \"( A ) Complete population of MZ699+ iPNs labeled using PA-GFP ( left image ) , the posterior-lateral LH region is encircled , arrowhead indicates the final common projection point of iPN axons .\",\n \"Middle image: photoactivation of all vlPr neurons of the MZ699-GAL4 line that project from the LH to the vlPr via the plF .\",\n \"Right image: exemplary single iPN , labeled by photoconverting PA-GFP in a single soma ( arrow ) .\",\n \"Scale bar , 20 µm .\",\n \"( B ) Framed images: neuronal reconstructions of all iPNs projecting to the LH-PM zone ( n = 8 ) with outlined olfactory neuropils .\",\n \"View from dorsal ( left ) and lateral ( right ) .\",\n \"Right part represents two exemplary registered individual iPNs .\",\n \"( C ) Neuronal reconstructions of all iPNs projecting into the LH-AM zone ( n = 10 ) , images are arranged as in ( B ) .\",\n \"( D and E )\",\n \"Cluster analyses based on the target areas of all terminals of each iPN in the LH ( D ) or based on the innervated glomeruli in the AL ( E ) .\",\n \"The dendrograms are split into colored subclusters .\",\n \"Below each dendrogram , each individual iPN is specified according to the labels in Figure 3—figure supplement 2 .\",\n \"Note , that iPNs can be morphologically clustered according to their target or input regions .\",\n \"( F ) Neuronal reconstruction of vlPr neurons projecting through the plF to the LH-AL zone .\",\n \"( G ) Combination of all registered neurons .\",\n \"( H ) Dual combinations of all registered neurons with their projections in the LH . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01010 . 7554/eLife . 04147 . 011Figure 3—figure supplement 1 . iPNs can be morphologically segregated according to their target and input region .\",\n \"( A ) Principal component analysis based on the distances of the similarity scores of all terminal points of each individual iPN in the LH ( for details see ‘Materials and methods’ ) .\",\n \"LH-AM iPNs ( blue ) and LH-PM iPNs ( green ) form significantly distinct clusters ( ***p < 0 . 001 , One-Way ANOSIM , Bray–Curtis ) .\",\n \"( B ) Principal component analysis based on the glomerular innervations of each individual iPN in the AL .\",\n \"Again , LH-AM iPNs ( blue ) and LH-PM iPNs ( green ) form significantly distinct clusters ( ***p < 0 . 001 , One-Way ANOSIM , Bray–Curtis ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01110 . 7554/eLife . 04147 . 012Figure 3—figure supplement 2 . Glomerular innervations of individual iPNs . Binary innervation patterns of 25 individually labeled MZ699+ iPNs using PA-GFP .\",\n \"Columns represent innervation patterns of individual neurons which have been grouped according to their innervation properties; rows represent 51 glomeruli in the AL along with the innervation in the specific odor response domains in the LH ( LH-PM , LH-AM ) and/or the mushroom body calyx ( MBc ) .\",\n \"Glomeruli have been sorted according to their iPN innervation .\",\n \"Grey , innervated; white , not innervated . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 012 We did not observe any clear panglomerular innervations of individual MZ699+ iPNs that spanned the entire AL , consistent with Liang et al . ( 2013 ) .\",\n \"Instead , MZ699+ iPNs develop mainly oligoglomerular patterns innervating on average 5 . 4 ± 3 . 9 glomeruli ( mean ± SD ) , which are not necessarily in close proximity .\",\n \"It is important to note here , that the glomerular innervations of iPNs are rather sparse in comparison to the innervation of ePNs which complicates the identification of truly innervated glomeruli .\",\n \"After classifying all registered neurons according to their LH zones along with their glomerular innervations , we noted a spatial subdivision of MZ699+ iPN dendritic fields in the AL ( Figure 3—figure supplement 2 ) .\",\n \"Whereas LH-PM iPNs extended dendrites mainly into glomeruli from the ventro- or dorsomedial area of the AL ( e . g . , DM4 , DM2 , VM7 , VM5d ) , iPNs targeting the LH-AM zone innervated glomeruli ranging from the ventro- and dorsoanterior to the dorsocentral region ( e . g . , DC3 , VC1 , VA6 , VL1 ) .\",\n \"We observed that a glomerulus is typically innervated by only LH-PM iPNs or LH-AM iPNs .\",\n \"However , we also found a few cases where a glomerulus can be innervated by both iPN types ( e . g . , glomeruli D and DC2 ) .\",\n \"In order to analyze whether the two categories of iPNs can also be statistically separated according to their glomerular innervations in the AL , we performed a cluster analysis based on the glomeruli innervated by each individual iPN ( Figure 3E ) .\",\n \"Notably , the two iPN classes could be clearly clustered into two groups due to their specific AL innervations .\",\n \"This finding is further supported by a principal component analysis showing that iPNs targeting the LH-PM region innervate a significant different glomerular subset than iPNs that send their axonal terminals to the LH-AM area ( Figure 3—figure supplement 1B; p < 0 . 001 , one-way ANOSIM ) .\",\n \"In accordance with our finding of two major iPN categories is the study by Lai et al . ( 2008 ) who observed several different stereotyped projection patterns of multiglomerular MZ699+ single-cell clones that could be broadly categorized into two groups based on the dendritic and axonal projection patterns .\",\n \"While we observed corresponding innervated areas in the AL , the described target areas in the LH seem to differ .\",\n \"However , due to the lack of 3D reconstruction of the single-cell clone data , the innervation patterns cannot be compared in detail .\",\n \"In addition to the oligoglomerular iPNs , we observed a few uniglomerular MZ699+ iPNs innervating either glomerulus DA1 or VL1 ( 4/25 of iPNs ) , consistent with Lai et al . ( 2008 ) , which target the LH-AM region ( Figure 3—figure supplement 2 ) .\",\n \"Moreover , we identified three other MZ699+ neurons that did not innervate the AL and sent their axons through the mACT to the LH and/or the MBc .\",\n \"Since the MZ699-GAL4 line labels also neurons connecting the LH and the ventrolateral protocerebrum ( vlPr ) ( Ito et al . , 1997; Liang et al . , 2013; Parnas et al . , 2013 ) , we illuminated a small fraction of the posterior lateral fascicle ( plF ) to target these putative third-order neurons ( Figure 3A ) .\",\n \"The plF comprised axons of ventrolateral protocerebral neurons ( vlPr neurons ) , which bifurcated within the LH-AL ( Figure 3F ) .\",\n \"Combinations of all registered neuron types within the assigned zones revealed that iPNs of the LH-AM area and vlPr neurons of the LH-AL region intermingle ( Figure 3G , H ) .\",\n \"To illustrate higher-order connectivity , we labeled the three major neuron types , that is , MZ699+ iPNs , GH146+ ePNs and vlPr neurons , targeting the LH within the olfactory circuitry using PA-GFP ( Figure 4A ) .\",\n \"Since our observed Ca2+ responses in the LH-AL region might reflect activity from vlPr neurons rather than iPNs , we dissected the neuronal contributions within each extracted ORD by conducting transection experiments using two-photon laser-mediated microdissection ( Figure 4B ) .\",\n \"By transecting the mACT , we aimed at abolishing LH-responses deriving from MZ699+ iPNs , while cutting the plF connection should eliminate potential odor-evoked vlPr neuron activity .\",\n \"To achieve unambiguous and comparable results , functional imaging was performed in both brain hemispheres simultaneously .\",\n \"Immediately after the intact brain areas were imaged , the tracts were selectively transected on one brain side each ( Figure 4C ) and the imaging procedure was repeated .\",\n \"We applied a reduced odor set that elicited activity in all ORDs and performed NNMF for pre- and post-lesion recordings .\",\n \"Transecting the mACT significantly reduced responses in the LH-PM and LH-AM region , whereas LH-AL responses were significantly abolished by plF-ablation ( Figure 4D ) .\",\n \"Notably , we observed that LH-AL responses to some odors were significantly increased after mACT transection as a consequence of the suppression of iPN inhibition of vlPr neurons confirming the study by Liang et al . ( 2013 ) .\",\n \"Hence , activity in the LH-PM and LH-AM domain can be assigned to MZ699+ iPNs , while LH-AL activity is mainly evoked by vlPr neurons ( Figure 4E ) . 10 . 7554/eLife . 04147 . 013Figure 4 . Distinct odor response domains in the LH constitute neuronal activity of iPNs and vlPr neurons .\",\n \"( A ) Representation of all ePNs ( magenta ) and iPNs ( green ) labeled by GH146-GAL4 and MZ699-GAL4 using PA-GFP , respectively .\",\n \"Photoactivation of vlPr neurons ( orange , MZ699-GAL4 ) connecting the LH and the vlPr via the plF .\",\n \"The overlay image depicts a pseudo-merge image of the different GAL4-driver lines .\",\n \"( B ) Schematic of the olfactory circuit with integrated layout of the transection experiment .\",\n \"After simultaneous Ca2+ imaging of bilateral LHs , the ipsilateral plF and contralateral mACT was transected ( red zigzag line ) with an infrared laser ( dashed red arrow ) .\",\n \"( C ) Projection images of a 7 µm stack of the LH area prior and post transection .\",\n \"Left images , mACT transected; right image , plF transected .\",\n \"The ablated region is indicated by the dashed red arrow .\",\n \"Scale bar , 20 µm .\",\n \"( D ) Median time traces displaying percental change of ΔF/F values for indicated ORDs prior to post transection of the mACT ( green , left ) and the plF ( orange , right ) for different odorants .\",\n \"Significant changes of odor-evoked Ca2+ signals due to transection are shown in the column SIG difference .\",\n \"Differences were tested with a two-tailed paired Student's t test ( p < 0 . 05 ) .\",\n \"Color codes are indicated by the corresponding scale bar below , n = 4–5 .\",\n \"Transecting the mACT eliminates Ca2+ signals in the LH-PM and LH-AM domain , while lesioning the plF significantly abolishes LH-AL responses .\",\n \"Notably , the LH-AL domain is significantly stronger activated after mACT transection following application of 1-octen-3-ol and γ-butyrolactone .\",\n \"( E ) Summarized cartoon of the neuron populations contributing to ORD activity prior and post transection of axons of iPNs or vlPr neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 013 We next addressed the behavioral relevance of MZ699+ iPN activity in the LH for innate odor-guided behavior .\",\n \"To precisely target iPN function , we expressed an RNAi construct against glutamic acid decarboxylase 1 ( GADi ) to selectively knock-down the GABA synthesis in MZ699+ iPNs ( Figure 5A ) .\",\n \"We confirmed the reduction in GABA production via immunostaining ( Figure 5B ) .\",\n \"Since vlPr neurons are not GABAergic , they were not affected by the RNAi expression ( Liang et al . , 2013; Parnas et al . , 2013 ) .\",\n \"Using wild-type flies and parental controls , we conducted T-maze assays ( Tully and Quinn , 1985; Chakraborty et al . , 2009 ) with nine of the odorants applied in functional imaging experiments at medium and high concentrations .\",\n \"Notably , flies with silenced MZ699+ iPN GABA production revealed a neutral or aversive behavioral response to attractive odors , while repellent odors evoked an even stronger aversion ( Figure 5C ) .\",\n \"To compare the T-maze data more accurately , we calculated the average change of behavioral response indices ( RIs ) between GADi flies and parental controls ( Figure 5D ) .\",\n \"Indeed , all responses changed in a negative direction , indicating MZ699+ iPNs play a crucial role in mediating attraction behavior .\",\n \"The sole exception involved high concentrations of the most repulsive odor , acetophenone , since this odor had already induced maximum aversion .\",\n \"Overall , these experiments reveal a crucial function of MZ699+ iPNs in mediating attraction behavior by releasing GABA in the LH . 10 . 7554/eLife . 04147 . 014Figure 5 . iPN GABA release in the LH mediates odor attraction behavior .\",\n \"( A ) Experimental layout: iPN GABA production was selectively silenced via GADi expression in MZ699+ iPNs; ePN and vlPr neuron activity remained unaffected .\",\n \"( B ) Immunostaining against GABA and GFP within AL somata ( left ) and LH neurites ( right ) of iPNs with intact ( top ) and silenced GABA production ( bottom ) .\",\n \"GADi flies show GABA signals in somata of iPNs labeled by GH146-GAL4 only ( arrowhead ) .\",\n \"The arrow head points to an exemplary GABA-positive bouton in the LH .\",\n \"Scale bar , 20 µm .\",\n \"( C ) Averaged behavioral response indices ( RIs ) determined with a T-maze assay for wild-type flies ( dark blue ) , parental controls ( light blue ) and experimental animals ( magenta ) for nine odorants at two concentrations .\",\n \"Empty boxes display no response ( Wilcoxon signed-rank test ) .\",\n \"Dunn's Multiple Comparison Test was used for global differences in the dataset followed by a posthoc test for selected pairs ( p* < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .\",\n \"Error bars represent SEM .\",\n \"( D ) RI differences between GADi flies and averaged parental controls .\",\n \"RI differences are negative for all but one odor indicating that GADi expression shifts odor-guided behavior towards aversion .\",\n \"Error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 014 The behavioral effect of the iPN knock-down suggests that MZ699+ iPNs encode positive hedonic valences .\",\n \"To correlate the complete ORD pattern array with innate behavioral preferences , we assigned behavioral RIs for all odors at median and high odor concentrations using the T-maze assay as in our previous experiment ( Figure 6A ) .\",\n \"Since extremely low concentrations rarely evoked any behavioral response , we excluded the 10−6 concentration in this analysis .\",\n \"It is important to note here , that different behavioral assays for testing olfactory preferences in flies might lead to contradictory results .\",\n \"However , the majority of odors used here was also tested in two other behavioral paradigms , the trap assay ( Stökl et al . , 2010; Knaden et al . , 2012 ) and the FlyWalk ( Steck et al . , 2012 ) ( pers . comm . M Knaden ) and yielded similar results ( see Figure 6—figure supplement 1 ) .\",\n \"When we plotted median odor-evoked activity in a three-dimensional space defined by the three ORDs , we saw a clear clustering of responses evoked by aversive and attractive odorants ( Figure 6B ) .\",\n \"The LH-AL domain , constituted mainly by vlPr neurons , is coding aversive odors , while attractive odors activated only the LH-PM and LH-AM domains that derive from MZ699+ iPNs .\",\n \"This result is in accordance with our finding that iPNs mediate odor attraction . 10 . 7554/eLife . 04147 . 015Figure 6 . Integration of hedonic valence and odor concentration into ORDs .\",\n \"( A ) Response indices of wild type flies for all odors at median and high concentrations .\",\n \"Odors are sorted from highly aversive ( −1 , red ) to highly attractive ( +1 , green ) .\",\n \"( B ) 3D-scatter plot of median Ca2+ responses of all odors based on the three ORDs .\",\n \"Odor-dots are labeled due to their RI shown in ( A ) .\",\n \"Same odors at different concentrations are connected with a line: the dot at the end depicts 10−2 , the centered dot 10−4 , and the end of the line 10−6 .\",\n \"Attractive and aversive odor representations form separate clusters .\",\n \"( C and D )\",\n \"Left , schematic LH outlines with colored ORDs corresponding to data on the right .\",\n \"Correlation score r ( upper right corner ) between median activity and measured RI in T-maze experiments or odor concentration , respectively , with significance denoted below .\",\n \"Student's t test , *p < 0 . 05 , ***p < 0 . 001 .\",\n \"( E ) Complete correlation matrices for Ca2+ response patterns of OSNs in the AL ( left ) and iPNs in the LH ( right ) .\",\n \"The odors are arranged according to single linkage clustering of the LH activity patterns .\",\n \"Heatmap color-code refers to the correlation distance scale bar on the right .\",\n \"Correlation distance is defined as 1 − r , where r is the Pearson correlation coefficient between the response patterns of two odorants .\",\n \"Odor letters are color-coded according to hedonic valence; 10−6 RI values are labeled in grey ( complete list right hand ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01510 . 7554/eLife . 04147 . 016Figure 6—figure supplement 1 . Odor valences determined with three different behavioral assays . Odor-evoked behavioral responses of wild type flies for the 14 odors used in this study determined by T-maze assay , trap assay and the FlyWalk .\",\n \"The color denotes an attractive ( green ) , aversive ( red ) or a neutral ( light yellow ) behavioral response .\",\n \"N/T , not tested .\",\n \"The majority of odors yielded similar results independent of the behavioral assay used .\",\n \"In a few cases an attractive odor evoked a neutral response ( i . e . , no response ) , but never induced an aversive response in another assay . DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01610 . 7554/eLife . 04147 . 017Figure 6—figure supplement 2 . Calcium responses of OSNs .\",\n \"( A ) Representative glomerular Ca2+-responses of OSNs in the AL for a subset of odorants at three concentrations .\",\n \"Scale bar to the right .\",\n \"Control ( mineral oil ) recordings are shown additionally as full false-color coded images .\",\n \"( B ) Glomerular AL atlas used for glomerular identification .\",\n \"( C ) Median Ca2+-activity traces of all glomeruli for all odorants at the three indicated concentrations .\",\n \"Scale bar and control measurement in the center .\",\n \"Odor application is indicated by the grey bar below the heatmaps ( n = 6–7 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 01710 . 7554/eLife . 04147 . 018Figure 6—figure supplement 3 . Correlation matrices for odor-evoked responses in the AL and LH . Complete correlation matrices for calcium activity patterns of OSNs in the AL ( left ) and iPNs in the LH ( right ) .\",\n \"The odors are arranged according to single linkage clustering of the AL activity patterns .\",\n \"Heatmap color-code refers to the correlation distance scale bar below each matrix .\",\n \"Odor letters are color-coded according to hedonic valence; 10−6 RI values are labeled in grey ( complete list right hand ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04147 . 018 We next correlated ORD activity to odor valence separately for all ORDs .\",\n \"This evaluation enabled us to analyze iPN and vlPr neuron coding properties apart from each other ( Figure 6C ) .\",\n \"As expected , the analysis revealed a significant correlation between positive valence and the LH-PM domain , whereas Ca2+ responses in the LH-AL were strongly negatively correlated to hedonic valence .\",\n \"The LH-AM domain exhibited a positive but not significant correlation for odor valence .\",\n \"Remarkably , activity within the LH-PM was totally independent of concentration , whereas activity in both anterior domains was significantly correlated to odor intensity ( Figure 6D ) .\",\n \"Hence , MZ699+ iPNs integrate odor attraction information into the LH-PM domain independent of odor intensity , confirming behavioral experiments .\",\n \"Intensity coding is in turn conducted separately by distinct iPNs within the LH-AM domain .\",\n \"In contrast , putative third-order vlPr neurons projecting into the LH-AL area code both negative valence and odor intensity .\",\n \"Finally , we wondered if this valence-specific LH representation is already reflected at the primary level of olfactory processing .\",\n \"The odor-evoked responses in iPNs are generally similar to those in OSNs ( Wang et al . , 2014 ) , indicating a straight forward transduction of cholinergic OSN responses .\",\n \"We therefore performed functional imaging of odor-evoked Ca2+ dynamics at the AL input level by expressing G-CaMP3 . 0 in OSNs using Orco-GAL4 ( Larsson et al . , 2004 ) ( Figure 6—figure supplement 2 ) .\",\n \"In order to compare the activity patterns at both processing levels , we calculated correlation distances for all pair-wise combinations of odor-evoked response patterns and plotted these with respect to maximal ORD pattern similarity in the LH ( Figure 6E ) .\",\n \"As expected , odor representations in the LH clearly clustered within three separated parts of the matrix , reflecting our observed ORDs .\",\n \"However , this coding similarity could not predict AL activity patterns , even if the correlation matrix was sorted with respect to pattern similarity in the AL ( Figure 6—figure supplement 3 ) .\"\n ],\n [\n \"We augment our present understanding of the Drosophila olfactory circuitry by elucidating the coding properties of a parallel and behaviorally relevant higher-order processing pathway to the LH .\",\n \"Morphological , functional and behavioral approaches provide strong evidence for a functional subdivision of iPNs into neurons coding either odor attraction or odor intensity .\",\n \"Our behavioral experiments reveal that inhibitory properties of iPNs are necessary for innate odor-guided attraction .\",\n \"In addition , we characterize a third neural pathway coding odor repellence .\",\n \"Do MZ699+ iPNs fulfill anatomical requirements to constitute a distinct processing channel in addition to ePNs ?\",\n \"A remarkable anatomical feature of MZ699+ iPNs is their glomerular innervation pattern in the AL .\",\n \"Whereas GH146+ ePNs are uniglomerular and retain the topographic code in their axonal arrangement ( Marin et al . , 2002; Wong et al . , 2002; Jefferis et al . , 2007 ) , most MZ699+ iPNs possess oligoglomerular innervations suggesting that these neurons might not convey precise odor-identity information .\",\n \"In addition , MZ699+ iPNs in the AL diverge only into specific glomerular subsets , and so might be pre-determined to selectively extract common features of distinct odors .\",\n \"We have previously shown that the AL map at the PN level exhibits a spatial segregation of valence representation ( Knaden et al . , 2012 ) .\",\n \"Certain glomeruli , which have been classified as aversion coding at the GH146+ ePN level , are omitted by MZ699+ iPNs , whereas most glomeruli classified as attraction coding are particularly densely innervated .\",\n \"These results suggest that within the MZ699+ iPN population , mainly positive odor traits are extracted , whereas the odor information of negative valence is neglected .\",\n \"This conclusion is consistent with the recent finding that one type of LH neurons is receiving input from PNs that mainly innervate glomeruli coding fruity-smelling acetates ( Fisek and Wilson , 2014 ) which represent attractive odor cues ( Knaden et al . , 2012 ) .\",\n \"We furthermore demonstrate that the MZ699+ iPN population is split into two major morphological classes possessing a clear spatial segregation in the AL which is strictly maintained within the LH .\",\n \"It has to be kept in mind that we do not cover all iPNs by using MZ699-GAL4 .\",\n \"Further experiments characterizing the ∼6 missing MZ699– iPNs , which are labeled by GH146-GAL4 ( Wilson and Laurent , 2005; Lai et al . , 2008 ) , will elucidate if our assumptions apply for the whole iPN population .\",\n \"So far only a handful neuroanatomical studies targeting GH146+ ePNs have dealt with the question of how olfactory information is integrated and read out by higher brain structures , in particular the LH ( Marin et al . , 2002; Wong et al . , 2002; Tanaka et al . , 2004; Jefferis et al . , 2007 ) .\",\n \"A recent study that traced the projection pattern of PNs coding ammonia and amines as attractive stimuli and carbon dioxide and acids as repulsive signals suggests that sensory stimuli of opposing valence are represented in spatially distinct areas within the LH ( Min et al . , 2013 ) .\",\n \"In addition the study by Liang et al . ( 2013 ) showed that MZ699+ iPNs selectively suppress the activity of vlPr neurons to food odors , while pheromone responses were not affected verifying the assumption that different odor features are processed separately .\",\n \"However , functional evidence for a feature-based , spatially segregated activity map in the LH was so far missing .\",\n \"To unravel the coding properties of MZ699+ iPNs within the LH , we conducted Ca2+ imaging experiments of MZ699+ iPNs in the LH to odorants having different hedonic valences and intensities , and could classify the LH into three functional ORDs .\",\n \"Our neuronal tracing and transection experiments validated the LH segmentation into two medial domains that derive from MZ699+ iPNs , and the LH-AL domain formed by vlPr neurons .\",\n \"In line with our observations are morphological studies on ePNs and third-order LH neurons revealing a similarly tight constriction into three zones within the LH ( Tanaka et al . , 2004 ) , while single-cell labeling combined with image registration resulted in five ePN target zones ( Jefferis et al . , 2007 ) .\",\n \"However , the ePN terminal zones do not necessarily correspond to the target domains of iPNs , since it has recently been shown that MZ699+ iPNs do not inhibit odor responses of GH146+ ePNs ( Liang et al . , 2013 ) and that the presynaptic sites of iPNs are spatially separated from those of ePNs ( Wang et al . , 2014 ) .\",\n \"Hence both PN populations represent parallel processing pathways that most likely accomplish distinct processing tasks analogous to the honeybee olfactory system which possesses dual olfactory pathways to the higher brain that accomplish parallel processing of similar odors ( Brill et al . , 2013 ) .\",\n \"Silencing MB function revealed that the LH alone is sufficient for basic olfactory behavior ( de Belle and Heisenberg , 1994; Connolly et al . , 1996; Heimbeck et al . , 2001 ) .\",\n \"Our behavioral results demonstrate that selectively silencing MZ699+ iPNs severely reduced the flies' odor attraction behavior .\",\n \"Hence our results suggest that MZ699+ iPNs are capable of extracting specific features from the combinatorial code emerging in the AL .\",\n \"A behavioral study revealed that silencing MBc neurons impairs odor attraction but not repulsion ( Wang et al . , 2003b ) .\",\n \"The authors drew the conclusion that the LH is involved in mediating innate repulsion rather than attraction .\",\n \"These results are not necessarily contradictory to ours since some ePNs might activate the LH-AL domain exclusively ( i . e . , vlPr neurons ) .\",\n \"On the other hand , Wang et al . ( 2003b ) did not include highly concentrated attractive odors .\",\n \"Therefore it is possible that in their experiments , the odor detection threshold was simply reduced , so that only highly concentrated odors , which induced odor aversion , could be distinguished .\",\n \"Our behavioral results , in contrast , revealed the constant influence of the MZ699+ iPNs in mediating attraction for odorants over a range of concentrations .\",\n \"Our data suggests that odors with opposing hedonic valences are encoded by an interplay of distinct processing pathways .\",\n \"The study by Liang et al . ( 2013 ) showed that GABA release from MZ699+ iPNs directly inhibits responses of vlPr neurons to food odors as mentioned above .\",\n \"This finding fits well to our observations that iPNs are activated mainly by attractive odors while vlPr neurons are not , likely due to the inhibitory input from iPNs .\",\n \"VlPr neurons are , on the other hand , almost solely activated by repellent odors , which do hardly activate iPNs and therefore do not induce a strong inhibition to vlPr neurons .\",\n \"Repellent odors most likely activate vlPr neurons via ACh release of ePNs which is supported by immunostainings with pre- and postsynaptic markers indicating that vlPr neurons receive input in the LH , while the vlPr represents their major output region ( Parnas et al . , 2013 ) .\",\n \"The vlPr is supposedly also a target of visual neurons from the optic lobe ( Tanaka et al . , 2004 ) implying that a certain integration of different sensory modalities takes place at this central processing relay .\",\n \"Given that iPNs are inhibitory neurons , the underlying mechanism of odor attraction behavior might therefore be an inhibition of aversive neuronal circuits from the LH to the vlPr that are mainly composed of vlPr neurons .\",\n \"However , this assumption needs to be verified with further experiments elucidating if vlPr neurons are sufficient and necessary to mediate odor aversion .\",\n \"What is known about odor coding in the LH in other insect species ?\",\n \"Notably , in locusts it has been shown that LH neurons receiving convergent PN input appeared to encode stimulus intensity in their net firing rates and in the phases of their spikes ( Gupta and Stopfer , 2012 ) .\",\n \"Hence these results support the idea that within the LH , general stimulus features such as odor intensity are extracted , which is well in line with our observation of the anterior LH domains whose activity is also significantly correlated to odor intensity .\",\n \"Also in line with our results is a study from honeybees which shows that the representation of different pheromone types is spatially segregated in the LH ( Roussel et al . , 2014 ) , indicating that odors eliciting specific behaviors are coded according to their biological values .\",\n \"In conclusion , our study provides an important step in unraveling higher olfactory processing mechanisms that are crucial for mediating innate behaviors in Drosophila .\",\n \"We provide functional evidence for a feature-based spatial arrangement of the LH decoding opposing hedonic valences and odor intensity .\",\n \"The role of the LH as a center for integrating biological values towards innate decisions by computing conveyed information of two processing pathways is thus expanded .\"\n ],\n [\n \"All fly stocks were maintained on conventional cornmeal-agar-molasses medium under L:D 12:12 , RH = 70% and 25°C .\",\n \"For wild-type controls D . melanogaster of the Canton-S strain was used .\",\n \"Transgenic lines were obtained from Bloomington Stock Center ( http://flystocks . bio . indiana . edu/ ) and Vienna RNAi stock center ( http://www . vdrc . at ) .\",\n \"Other fly stocks were kindly provided by Kei Ito ( MZ699-GAL4 ) and Maria Luisa Vasconcelos ( UAS-C3PA ) .\",\n \"The END1-2 fly line is published in Grabe et al . ( 2014 ) .\",\n \"Whole-mount immunofluorescence staining was carried out as described ( Laissue et al . , 1999; Vosshall et al . , 2000 ) .\",\n \"Initially brains were dissected in Ringer's solution ( 130 mM NaCl , 5 mM KCl , 2 mM MgCl2 , 2 mM CaCl2 , 36 mM saccharose , 5 mM HEPES , [pH 7 . 3] ) ( Estes et al . , 1996 ) and fixed in 4% PFA in PBS-T ( PBS , 0 . 2–1% Triton-X ) .\",\n \"After washing with PBS-T brains were blocked with PBS-T , 5% normal goat serum ( NGS ) .\",\n \"Primary antibodies were diluted in blocking solution or PBS-T and incubated at 4°C for 2–3 days .\",\n \"Secondary antibody incubation lasted 1–2 days .\",\n \"Brains were mounted in VectaShield ( Vector Labs , Burlingame , CA ) .\",\n \"The following primary antibodies were used: rabbit α-GABA ( 1:500 ) ( Sigma ) , mouse α-GFP ( 1:500 ) ( Invitrogen ) .\",\n \"The following secondary antibodies were used: Alexa Fluor 488 , goat anti-mouse ( 1:500 ) ; Alexa Fluor 546 , goat anti-rabbit ( 1:500 ) ; ( all IgG Invitrogen ) .\",\n \"Fly preparation and functional imaging of the AL was conducted as previously described ( Stökl et al . , 2010; Strutz et al . , 2012 ) .\",\n \"LH imaging was conducted similarly , except for the higher resolution achieved with a 60× water immersion objective ( LUMPlanFl 60×/0 . 90 W Olympus ) .\",\n \"The optical plane was ∼30 µm below the most dorsal entrance point of the iPN tract into the LH .\",\n \"Binning on the CCD-camera chip resulted in a resolution of 1 pixel = 0 . 4 × 0 . 4 µm .\",\n \"For bilateral LH imaging during transection a 20× water immersion objective ( NA 0 . 95 , XLUM Plan FI , Olympus ) was employed .\",\n \"All recordings lasted 10 s with a frame rate of 4 Hz .\",\n \"Odors included acids ( propionic acid , acetic acid ) , lactones ( γ-butyrolactone ) , terpenes ( linalool ) , aromatics ( acetophenone , methyl salicylate , benzaldehyde , phenylacetic acid ) , alcohols ( 1-octen-3-ol ) , esters ( acetoin acetate , cis-vaccenyl acetate , 2-phenethyl acetate ) , ketones ( 2 , 3 butanedione ) and balsamic vinegar diluted in mineral oil ( all from Sigma Aldrich ) .\",\n \"Odors were applied during frame 8–14 ( i . e . , after 2 s , lasting for 2 s ) .\",\n \"Flies were imaged for up to 1 hr , with a minimum inter-stimulus interval of one minute .\",\n \"We selected conventional widefield Ca2+ imaging as the method of choice , since we were able to obtain single bouton resolution with this technique .\",\n \"Calcium imaging data of AL were analyzed with custom-written IDL software ( ITT Visual Information Solutions ) provided by Mathias Ditzen as previously described ( Stökl et al . , 2010; Strutz et al . , 2012 ) .\",\n \"Regarding the Ca2+ imaging data in the LH , we repeated recordings of each odor at each concentration two to three times to ensure the reliability of the extracted domain information .\",\n \"To execute NNMF analysis ( see below ) , at least 6–7 valid measurements , that is , animals with repeated identical recordings , were collected for each odor and employed for the analysis .\",\n \"Individual odor measurements were aligned using ImageJ ( Fiji ) to correct movement artifacts .\",\n \"Fluorescence changes ( ∆F/F ) for each odor were calculated in relation to background fluorescence using frames 0–6 ( i . e . , 2–0 . 5 s before odor application ) .\",\n \"A Gaussian low-pass filter ( ó = 1px ) was applied to compensate for remaining movement artifacts and pixel noise .\",\n \"To reduce the computational load , the frame rate was averaged by two consecutive frames , and recordings were spatially down-sampled by a factor of two .\",\n \"The resulting concatenated time-series of the recordings is denoted as measurement matrix Y with element Yt , p being the tth observed value of pixel\",\n \"p . In contrast to the AL , which consists of highly ordered glomerular subunits , the LH comprises a mainly homogenous neuropil which does not provide spatial or functional landmarks .\",\n \"Therefore , we used the automatic method NNMF to extract Ca2+ signals that exhibit common spatial or temporal features .\",\n \"NNMF , like other matrix factorization techniques ( e . g . , Principal Component Analysis ( PCA ) and Independent Component Analysis ( ICA ) ) , decompose the measurement matrix Y into k components , Y=∑kxk∗akT+R .\",\n \"The time-course ak of each component contains a common underlying time-courses of all pixels and each pixel participation xk declares how strongly each pixel is involved in this time-course .\",\n \"The residual matrix R contains the unexplained data .\",\n \"In order to perform NNMF , we implemented the HALS algorithm in Python including a spatial smoothness constraint ( asm = 0 . 1 ) ( Cichocki and Phan , 2009 ) and an additional spatial decorrelation constraint ( ade = 0 . 1 ) ( Chen and Cichocki , 2005 ) .\",\n \"In PCA decomposition is performed such that either timecourses ak or pixel participation xk are uncorrelated , whereas ICA aims for timecourses ( temporal ICA ) or pixel participation ( spatial ICA ) to be independent .\",\n \"Although spatial ICA is able to segregate signals into functional similar neuropils ( Reidl et al . , 2007 ) , we chose the NNMF approach , because it is known to achieve even a better parts-based representation compared to the more holistic results of PCA or ICA ( Lee and Seung , 1999 ) .\",\n \"In contrast to PCA and ICA , NNMF constrains both the extracted time-courses and pixel participations to be positive .\",\n \"Positive pixel participation enabled us to make a straightforward physiological interpretation , reading the participation values as the contribution strength of an underlying physiological domain .\",\n \"The restriction to positive time-courses reflects the fact that we did not observe any significant decrease of fluorescence in response to an odor in the original measurement data .\",\n \"For each animal we performed decomposition into k = 5 components .\",\n \"This was sufficient to explain most of the data's variance ( 88% + 8% , error is standard deviation across individuals ) .\",\n \"The remaining variance in the residual matrix R contained no additional domains but rather reflected remaining movement artifacts of the measurements ( Figure 2—figure supplement 2 ) .\",\n \"Of the five components extracted by NNMF , three stood out prominently ( Figure 2—figure supplement 3 ) : First , they were extracted in all animals at very clearly defined anatomical positions .\",\n \"Second , their responses to stimuli repetitions were highly reproducible in contrast to the other two components , that is , they exhibited a significant ( p < 2*10−8 , t test ) higher trial-to-trial correlation of 0 . 72 ± 0 . 20 in contrast to 0 . 52 ± 0 . 26 for the remaining components; third , the odorant spectra of their responses were characteristic across animals .\",\n \"Though we cannot completely rule out that the remaining components of the factorization are ORDs of their own , there are several indications that they are not .\",\n \"On the one hand , they exhibit a lower trial-to-trial correlation than the three selected components .\",\n \"Second , those components did not consistently appear at similar anatomical position .\",\n \"Third , they were spatially overlapping with the selected three components .\",\n \"Instead of independent ORDs , these regions might convey fluorescence changes independent of odor stimulation or an overlapping region of two of the reliable ORDs .\",\n \"A validation of our NNMF-based results with spatial ICA yielded very similar , but slightly worse results .\",\n \"Whereas the three reliable ORDs from NNMF were also extracted in spatial ICA , the two remaining components exhibited much higher variability than when obtained with NNMF .\",\n \"Hence , we conclude that the LH area comprising MZ699+ neurons consists of three ORDs .\",\n \"We labeled those three components according to the anatomical position of their pixel participation within the LH .\",\n \"To determine the coding properties of extracted odor response domains ( ORDs ) , we calculated the mean response of each animal within a time window of 1–4 s after stimulus onset .\",\n \"Hence , median responses over all animals defined the standard stimulated response rORDo of an ORD to an odor\",\n \"o . Initially , regions were evaluated individually , and correlations were calculated between standard response spectra and the behavioral response index ( RI ) , or odor concentration , respectively , using the ‘linregress’ function of the Python scipy . stat module .\",\n \"To analyze the combined ORD representations of odor patterns po= ( rPMo , rAMo , rALo ) we calculated for all odor pairs the pattern similarity as correlation distance do1 , o2=1−corr ( po1 , po2 ) .\",\n \"In order to visualize the correlation matrix in a comprehensible way , we then arranged odors according to the single linkage clustering of the Python scipy . cluster . hierarchy module .\",\n \"To compare the representation in the LH to those of the AL , we applied the same procedure to the dorsal glomerular odor activation pattern .\",\n \"For in vivo photoactivation experiments , 1–6 day old flies ( genotype: END1-2 , UAS-C3PA;MZ699-GAL4 ) were dissected as in the imaging experiments except that tracts of the salivary glands were cut to prevent movement .\",\n \"Photoactivation was accomplished via continuous illumination with 760 nm for 15–25 min .\",\n \"After a 5-min break to permit full diffusion of the photoconverted molecules , 925 nm z-stacks of the whole brain were acquired and subsequently used for neuronal 3D-reconstruction .\",\n \"For all 3D reconstructions , the segmentation software AMIRA 4 . 1 . 1 & 5 . 3 . 3 ( FEI Visualization Sciences Group , Burlington , MA ) was used .\",\n \"Neurons of different individuals were embedded into the reference brain using a labelfield registration as previously described ( Rybak et al . , 2010 ) .\",\n \"Briefly , segmented labels of brain neuropils ( AL , MBc , LH ) were registered onto a reference brain image using affine registration followed by elastic warping .\",\n \"In a second step , the calculated transformation matrix was applied to the respective neuron morphology that was then aligned to the reference brain image .\",\n \"For morphological analysis of reconstructed iPNs , we first determined all terminal points of each iPN in the LH area .\",\n \"For each combination of terminals we calculated a similarity score ( s ) in analogy to ( Kohl et al . , 2013 ) as follows:s ( t1 , t2 ) =√e−Δ ( t1 , t2 ) 2/2σ2 , where t is the terminal position , Δ ( t1 , t2 ) is the Euclidean distance and σ is a free parameter that determines how close in space terminal points must be to be considered similar; analogue to Kohl et al . ( 2013 ) we set this parameter to 3 µm .\",\n \"Finally we calculated the pairwise similarity score between two neurons as their average all-to-all terminal similarity scores , normalized to their self-scores as follows:S ( n1 , n2 ) =∑t1 , t2s ( t1 , t2 ) /∑t1s ( t1 , t1 ) ∗∑t2s ( t2 , t2 ) Effectively this quantifies the relative overlap of the target area of all pairs of iPNs .\",\n \"For clustering , the similarity scores were converted to distances ( i . e . , 1-S ) and a hierarchical clustering was performed using UPGMA method .\",\n \"Principal component analysis and one-way ANOSIM was performed using the statistical software PAST 3 . x ( Paleontological statistics software package for education and data analysis ) .\",\n \"Transections of either the plF tract or the mACT were conducted in one brain hemisphere , each of the same fly .\",\n \"The target area was monitored with 925 nm and chosen to be close to the LH but distant enough not to affect neurites ramifying in the LH neuropil .\",\n \"For both tracts , lesioned areas had an average size of 34 µm and were illuminated with short pulses of 710 nm every 40 ms for 250 ms in 60 ( plF ) to 80 ( mACT ) cycles in a single focal plane .\",\n \"After a fast z-stack with 925 nm to confirm complete lesion , a 5-min neuronal recovery interval followed before continuing the imaging procedure .\",\n \"Data were analyzed using NNMF .\",\n \"Photoactivation and transection procedures as well as image acquisition following immunohistochemistry were accomplished with a 2-photon confocal laser scanning microscope ( 2PCLSM , Zeiss LSM 710 NLO ) equipped with a 40× ( W Plan-Apochromat 40×/1 . 0 DIC M27 , Zeiss ) or 20× ( W N-Achroplan 20×/0 . 5 M27 , Zeiss ) .\",\n \"The 2PCLSM was placed on a smart table UT2 ( Newport Corporation , Irvine , CA , USA ) and equipped with an infrared Chameleon Ultra diode-pumped laser ( Coherent , Santa Clara , CA , USA ) .\",\n \"Z-stacks were performed with argon 488 nm and helium-neon 543 nm laser or the Chameleon Laser 925 nm ( BP500-550 for G-CaMP and LP555 for DsRed/tdTomato ) and had a resolution of 1024 or 512 square pixels .\",\n \"The maximum step size for immuno-preparations or single neuron projections was 1 µm and for AL reconstructions 2 µm .\",\n \"Flies carrying P[GAD1-RNAi];P[MZ699-GAL4] were crossed just before the experiment to prevent dosage compensation effects .\",\n \"T-maze experiments were performed as described ( Stensmyr et al . , 2012 ) .\",\n \"WT , parental controls ( P[GAD1-RNAi] or P[MZ699-GAL4] ) and test flies carrying both insertions were tested separately under identical conditions .\",\n \"The response index ( RI ) was calculated as ( O-C ) /T , where O is the number of flies in the odor arm , C is the number of flies in the control arm , and T is the total number of flies used in the trial .\",\n \"Hence , the RI ranges from −1 ( complete avoidance ) to 1 ( complete attraction ) .\",\n \"Each experiment was carried out on 30 flies and was repeated 12 times .\",\n \"Dunn's Multiple Comparison Test was used for global differences in the dataset .\",\n \"Whenever the Multiple Comparison Test was significant ( i . e . , p < 0 . 05 ) , a posthoc test for selected pairs was performed , that is , between the GADi-flies and the other three control lines as we were not interested in differences among the different control lines .\",\n \"All RI were tested against 0 ( no response ) by using the Wilcoxon-rank-sum test .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["To internally reflect the sensory environment , animals create neural maps encoding the external stimulus space .","From that primary neural code relevant information has to be extracted for accurate navigation .","We analyzed how different odor features such as hedonic valence and intensity are functionally integrated in the lateral horn ( LH ) of the vinegar fly , Drosophila melanogaster .","We characterized an olfactory-processing pathway , comprised of inhibitory projection neurons ( iPNs ) that target the LH exclusively , at morphological , functional and behavioral levels .","We demonstrate that iPNs are subdivided into two morphological groups encoding positive hedonic valence or intensity information and conveying these features into separate domains in the LH .","Silencing iPNs severely diminished flies' attraction behavior .","Moreover , functional imaging disclosed a LH region tuned to repulsive odors comprised exclusively of third-order neurons .","We provide evidence for a feature-based map in the LH , and elucidate its role as the center for integrating behaviorally relevant olfactory information ."],"string":"[\n \"To internally reflect the sensory environment , animals create neural maps encoding the external stimulus space .\",\n \"From that primary neural code relevant information has to be extracted for accurate navigation .\",\n \"We analyzed how different odor features such as hedonic valence and intensity are functionally integrated in the lateral horn ( LH ) of the vinegar fly , Drosophila melanogaster .\",\n \"We characterized an olfactory-processing pathway , comprised of inhibitory projection neurons ( iPNs ) that target the LH exclusively , at morphological , functional and behavioral levels .\",\n \"We demonstrate that iPNs are subdivided into two morphological groups encoding positive hedonic valence or intensity information and conveying these features into separate domains in the LH .\",\n \"Silencing iPNs severely diminished flies' attraction behavior .\",\n \"Moreover , functional imaging disclosed a LH region tuned to repulsive odors comprised exclusively of third-order neurons .\",\n \"We provide evidence for a feature-based map in the LH , and elucidate its role as the center for integrating behaviorally relevant olfactory information .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Organisms need to sense and adapt to their environment in order to survive .","Senses such as vision and smell allow an organism to absorb information about the external environment and translate it into a meaningful internal image .","This internal image helps the organism to remember incidents and act accordingly when they encounter similar situations again .","A typical example is when organisms are repeatedly attracted to odors that are essential for survival , such as food and pheromones , and are repulsed by odors that threaten survival .","Strutz et al . addressed how attractiveness or repulsiveness of a smell , and also the strength of a smell , are processed by a part of the olfactory system called the lateral horn in fruit flies .","This involved mapping the neuronal patterns that were generated in the lateral horn when a fly was exposed to particular odors .","Strutz et al . found that a subset of neurons called inhibitory projection neurons processes information about whether the odor is attractive or repulsive , and that a second subset of these neurons process information about the intensity of the odor .","Other insects , such as honey bees and hawk moths , have olfactory systems with a similar architecture and might also employ a similar spatial approach to encode information regarding the intensity and identity of odors .","Locusts , on the other hand , employ a temporal approach to encoding information about odors .","The work of Strutz et al . shows that certain qualities of odors are contained in a spatial map in a specific brain region of the fly .","This opens up the question of how the information in this spatial map influences decisions made by the fly ."],"string":"[\n \"Organisms need to sense and adapt to their environment in order to survive .\",\n \"Senses such as vision and smell allow an organism to absorb information about the external environment and translate it into a meaningful internal image .\",\n \"This internal image helps the organism to remember incidents and act accordingly when they encounter similar situations again .\",\n \"A typical example is when organisms are repeatedly attracted to odors that are essential for survival , such as food and pheromones , and are repulsed by odors that threaten survival .\",\n \"Strutz et al . addressed how attractiveness or repulsiveness of a smell , and also the strength of a smell , are processed by a part of the olfactory system called the lateral horn in fruit flies .\",\n \"This involved mapping the neuronal patterns that were generated in the lateral horn when a fly was exposed to particular odors .\",\n \"Strutz et al . found that a subset of neurons called inhibitory projection neurons processes information about whether the odor is attractive or repulsive , and that a second subset of these neurons process information about the intensity of the odor .\",\n \"Other insects , such as honey bees and hawk moths , have olfactory systems with a similar architecture and might also employ a similar spatial approach to encode information regarding the intensity and identity of odors .\",\n \"Locusts , on the other hand , employ a temporal approach to encoding information about odors .\",\n \"The work of Strutz et al . shows that certain qualities of odors are contained in a spatial map in a specific brain region of the fly .\",\n \"This opens up the question of how the information in this spatial map influences decisions made by the fly .\"\n]","isConversational":false},"year":{"kind":"string","value":"2014"}}},{"rowIdx":285,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["biochemistry and chemical biology","cell biology"],"string":"[\n \"biochemistry and chemical biology\",\n \"cell biology\"\n]","isConversational":false},"title":{"kind":"string","value":"The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase"},"id":{"kind":"string","value":"elife-17578-v1"},"sections":{"kind":"list like","value":[["Alzheimer’s disease ( AD ) is the most common form of dementia and currently the sixth leading cause of death in the United States , with no disease-modifying therapeutics available .","A central and pathological hallmark of AD is the deposition of amyloid-beta ( Aβ ) plaques in the brain ( Hardy and Selkoe , 2002 ) .","These plaques are comprised of aggregates of Aβ peptides , which are formed by the sequential cleavage of the membrane embedded amyloid precursor protein ( APP ) by two proteases—β-secretase first removes the ectodomain of APP , then γ-secretase cleaves the remaining C-terminal fragment within its transmembrane domain ( TMD ) to liberate Aβ from cellular membranes .","Via its proteolytic component presenilin ( Li et al . , 2000; Wolfe et al . , 1999 ) , γ-secretase processes the TMD of APP into Aβ peptides of differing lengths , mostly producing a more benign Aβ species 40 amino acids in length , termed Aβ40 , as well as lesser amounts of a longer , more aggregation prone and pathogenic species , Aβ42 .","The total amount of Aβ42 , as well as the ratio of Aβ42/40 , are thought to be key mediators of Aβ pathogenesis , a hypothesis strongly supported by the fact that nearly all the 200-plus autosomal-dominant familial Alzheimer’s disease ( FAD ) mutations in presenilin-1 , -2 and APP increase the Aβ42/40 ratio ( see www . alzforum . org/mutations ) .","Due to the strong link between γ-secretase catalyzed Aβ formation and AD pathogenesis , the development of γ-secretase targeting therapeutics has been of high interest over the past two decades ( Golde et al . , 2013; De Strooper , 2014 ) .","Both γ-secretase inhibitors ( GSIs ) and a γ-secretase modulator ( GSM ) , the latter working by an unknown mechanism to influence γ-secretase to produce shorter , presumably less pathogenic Aβ species , have failed in recent clinical trials .","The failure of GSIs is due at least in part to toxicities from cleavage inhibition of other γ-secretase substrates such as Notch ( Doody et al . , 2013; Golde et al . , 2013 ) .","Little is known about how γ-secretase recognizes the transmembrane domain of substrates , given that no consensus amino acid cleavage motif has been identified for the more than one hundred γ-secretase substrates discovered to date ( Haapasalo and Kovacs , 2011 ) .","Unfortunately , the further development of safe and effective γ-secretase targeting therapeutics has been held back by a fundamental lack of understanding of how γ-secretase recognizes and cleaves the TMDs of its many substrates , especially APP .","Elucidation of this basic mechanism should at the very least add to our understanding of how Aβ is produced and may also aid in the development of safe and effective disease-modifying therapeutics .","Mass spectrometry studies have identified a complex mixture of products generated from γ-secretase’s cleavage of the transmembrane domain of APP ( Matsumura et al . , 2014 ) .","Looking at the formation of these products over time , it is apparent that the TMD of APP is mostly processed via two major pathways .","γ-secretase predominantly initiates endoproteolysis at a so-called epsilon ( ε ) cleavage site—after Leu49 or Thr48 , generating Aβ49 or Aβ48 and two different APP intracellular domains ( AICD ) , AICD 50–99 or AICD 49–99 , respectively ( Kakuda et al . , 2006; Sato et al . , 2003 ) .","Aβ49 and Aβ48 are then sequentially cleaved in increments of three amino acids to produce mostly Aβ40 and Aβ42 , respectively ( Takami et al . , 2009 ) .","The two major pathways are therefore Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 ( Figure 1A ) ( Fernandez et al . , 2014; Takami et al . , 2009 ) .","There are , however , other Aβ species generated by γ-secretase through usually minor and sometimes overlapping , alternative pathways ( Matsumura et al . , 2014; Olsson et al . , 2014 ) .","Importantly a shorter peptide , Aβ38 , can be formed from both major pathways , originating from Aβ42 or Aβ43 ( Okochi et al . , 2013 ) .","Additionally , a third , sparingly used site of ε cleavage can lead to the production of Aβ47 , which rather than being processed to Aβ44 is instead mostly cleaved to Aβ43 , subsequently generating Aβ40 ( Aβ47 → Aβ43 → Aβ40 ) ( Matsumura et al . , 2014 ) . 10 . 7554/eLife . 17578 . 003Figure 1 . Tripeptide fragments of APP inhibit γ-secretase .","( A ) Schematic diagram of the major sequential cleavage pathways of the transmembrane domain of APP ( Aβ49 → Aβ46 → Aβ43 → Aβ40 in red and Aβ48 → Aβ45 → Aβ42 in blue ) .","Mutations causing Familial Alzheimer’s disease are below the APP TMD in blue .","( B ) IC50 curves from the inhibition of γ-secretase activity by APP product tripeptide fragments .","Mean ± SD , n = 2 .","( C ) Noncompetitive inhibition of γ-secretase with VIV tripeptide , R2 = 0 . 98 .","( D ) Yonetani-Theorell plot for the mutually exclusive binding of VIV and the noncompetitive transition-state analog inhibitor III-31-C , R2 = 0 . 98 .","( E ) Cartoon representation of the three S’ pockets of presenilin ( PSEN ) along with three P’ amino acids of substrate and the transition-state analog L685 , 458 .","( F ) IC50 curves from the inhibition of γ-secretase activity with FAF and AFA synthetic tripeptides .","Mean ± SD , n = 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 003 Normally , γ-secretase uses the Aβ49 → Aβ40 and the Aβ48 → Aβ42 pathways to produce mostly Aβ40 and Aβ42 via a stepwise , tripeptide cleavage process .","The mechanism that dictates this preferred tripeptide cleavage ( and thus the driving force behind γ-secretase catalysis and Aβ formation ) is completely unknown .","In this study , we report that γ-secretase tripeptide cleavage is driven by three S’ pockets within the active site of the enzyme .","We identify specific substrate cleavage preferences dictated by the three S’ pockets and exploit these preferences to determine the predominant mechanism of each FAD mutation within the transmembrane domain of APP , including a novel mechanism in which final cleavage products are uncoupled from initial ε pathway preference ."],["When studying enzyme catalysis much focus is appropriately placed on determining how an enzyme interacts with its substrate .","However , oftentimes the manner in which an enzyme interacts with product ( in the form of product inhibition ) can be equally informative with regard to its catalytic mechanism .","To this end , we asked whether the naturally produced tripeptide fragments of APP are inhibitors of γ-secretase .","We found that all five tripeptides produced from the TMD of APP are indeed capable of inhibiting γ-secretase activity , albeit rather weakly with IC50 values ranging from ~150 μM to several mM ( Figure 1B ) .","Although these binding affinities are too low for the tripeptides to be involved in any form of biologically relevant feedback inhibition , we imagined the manner in which they inhibit γ-secretase could be instructive in elucidating the basic cleavage mechanism of the protease .","We characterized the mode of inhibition of the most potent of the tripeptides , VIV , finding that these data fit well to a noncompetitive inhibition model , with a global R2 of 0 . 98 ( Figure 1C ) .","Given that the tripeptide segments of the TMD of APP must occupy the active site of γ-secretase during catalysis , we hypothesized that VIV may compete for the same binding site on the enzyme as transition-state analogs .","An inhibitor cross-competition analysis reveals this is likely true , with a series of parallel lines resulting from a Yonetani-Theorell plot demonstrating mutually exclusive binding of VIV and the transition-state analog III-31-C ( Figure 1D ) .","We noticed that nearly all of the γ-secretase targeting transition-state analog inhibitors developed to date ( e . g . L685 , 458 , III-31-C ) contain essentially a tripeptide fragment C-terminal to the transition-state-mimicking hydroxyl isostere ( Figure 1E ) .","Structure-activity relationship ( SAR ) studies have demonstrated that transition-state analog inhibitors containing only two amino acids here are relatively weak inhibitors compared to those comprised of three amino acids , while adding a fourth amino acid does not achieve additional potency ( Esler et al . , 2004 ) .","This suggests that there are three , and only three , putative S’ pockets in the presenilin active site that contribute to inhibitor binding .","Of note , the SAR studies also suggest that while the putative S1’ and S3’ pockets are large and can accommodate amino acids of varying size , the S2’ pocket is small and inhibitors with an aromatic amino acid ( phenylalanine ) at this position have decreased potency by two orders of magnitude compared to those with less bulky aliphatic amino acids ( Esler et al . , 2004 ) .","We imagined that the tripeptide APP TMD products may be binding these three putative S’ pockets to achieve inhibition .","In agreement with this hypothesis , a synthetic tripeptide of the predicted optimal binding sequence ( FAF , to fit in the large-small-large S1’-S2’-S3’ pockets ) was a more potent inhibitor of γ-secretase activity than any of the naturally occurring APP-derived tripeptides , while a peptide predicted to clash with this binding site ( AFA ) is a very weak inhibitor , even at mM concentrations ( Figure 1F ) .","Based on these results , we reasoned that the three putative S’ pockets within the γ-secretase active site are likely occupied by substrate prior to hydrolysis of the scissile bond as a means for γ-secretase to stabilize a transition-state-like scission complex with substrate .","This would provide a simple mechanism for the preferred cleavage of APP in three amino acid increments , as well as provide an explanation for why γ-secretase mostly sticks to each major pathway , producing Aβ40 or Aβ42 after initiating cleavage of APP at either the L49 or T48 ε sites , respectively .","Additionally , given that substrate movement in the form of helical unwinding is thought to be a required step in the poorly defined intramembrane protease cleavage mechanism ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) , the three S’ pockets could potentially provide a means for γ-secretase to stabilize its helical substrate in a more cleavable conformation , thereby lowering the activation energy required for catalysis .","To test this hypothesis , we took advantage of the fact that the putative S2’ pocket of γ-secretase is apparently small and has a reduced ability to accommodate a bulky amino acid such as phenylalanine .","We predicted that we should be able to selectively decrease cleavage of either the Aβ49 → 40 pathway or the Aβ48 → 42 pathway simply by placing an aromatic amino acid at the P2’ position of APP at the initial ε cut site .","In other words , an aromatic amino acid placed at V50 should reduce ε cleavage after T48 , thereby lowering the amount of Aβ42 produced , thus decreasing the Aβ42/40 ratio .","Conversely , an aromatic amino acid at M51 should reduce ε cleavage after L49 , lowering the amount of Aβ40 produced and therefore increase the Aβ42/40 ratio ( Figure 2A ) . 10 . 7554/eLife . 17578 . 004Figure 2 . Selective blocking of the Aβ40 or Aβ42 pathways with aromatic amino acids placed at the P2’ position of ε cleavage .","( A ) Schematic diagram of the TMD of APP with pathway blocking aromatic amino acid mutations at the P2’ position for the T48 or L49 ε cleavage .","( B ) In vitro Aβ42/40 ratios with Phe or Trp mutations at V50 or M51 .","Aβ measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .","Mean ± SD , n = 3 , t-test **<0 . 01 , ****<0 . 0001 .","( C ) MALDI/TOF MS of the AICD fragments generated from in vitro cleavage of C100: WT ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6907 . 4; AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7021 . 3 ) .","V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6949 . 8; AICD 48–99 , expected mass: 7167 . 9 , observed mass: 7163 . 5 ) .","M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7030 . 1; AICD 48–99 , expected mass: 7135 . 8 , observed mass: 7131 . 8 ) .","( D ) Aβ42/40 ratios measured from the media of HEK cells transfected with V50 mutants .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .","( E ) Aβ42/40 ratios measured from the media of HEK cells transfected with M51 mutants .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 .","( F ) Aβ40 and Aβ42 levels for aromatic substitutions at V50 and M51 normalized to WT .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 , t-test *<0 . 05 , ***<0 . 001 , ****<0 . 0001 .","( G ) Total secreted Aβ levels ( see Materials and methods ) from the aromatic mutations at V50 and M51 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 004 In an in vitro assay using purified γ-secretase to cleave recombinant C100-FLAG APP-based substrate , V50F and V50W both decreased the Aβ42/40 ratio , while the same substitutions at M51 increased the Aβ42/40 ratio as predicted ( Figure 2B ) .","MALDI/TOF mass spectrometry ( MS ) analysis of the corresponding AICD fragment revealed the complete elimination of AICD 49–99 for V50F and of AICD 50–99 for M51F ( Figure 2C ) .","Interestingly , in addition to the expected AICD fragments , both V50F and M51F are also cleaved to a minor extent after I47 , producing AICD 48–99 .","The reason for this is unknown , although γ-secretase may be compensating for reduced cleavage through one of the two major pathways .","Previous MS studies have demonstrated that the majority of Aβ47 is eventually processed to Aβ40 , through an Aβ43 intermediate ( Matsumura et al . , 2014 ) .","We obtained similar results measuring secreted Aβ after transiently transfecting HEK cells with full-length APP containing mutations at V50 or M51 .","Here , all mutations tested at V50 caused a reduction in the Aβ42/40 ratio , although none as robustly as the bulky aromatic amino acids Phe , Tyr and Trp ( Figure 2D ) .","And while mutations of smaller amino acids at M51 caused modest reductions in the Aβ42/40 ratio , aromatic substitutions here all caused substantial increases in the Aβ42/40 ratio ( Figure 2E ) .","As predicted by our model , there was a significant reduction in Aβ42 production for the V50 aromatic substitutions , with little change in the Aβ40 levels ( Figure 2F ) .","There was a similar predicted reduction in Aβ40 for the M51 aromatic mutants; however , we also see an increase in Aβ42 levels here ( Figure 2F ) , likely because the normally less used Aβ48 → 42 pathway is compensating for the reduced flux through the preferred Aβ49 → 40 pathway , as these mutants all produced roughly equivalent amounts of total Aβ ( Figure 2G ) .","Because each tripeptide cleavage event requires the reading of three amino acids of substrate at a time ( as dictated by the three S’ pockets ) , we reasoned we should be able to predictably shift the Aβ42/40 ratio by placing a Phe in the P2’ position at each tripeptide cleavage event along the two major pathways ( Figure 3A ) .","As expected , Phe substitutions at V44 and I47 decreased the Aβ42/40 ratio , while Phe mutations at I45 and T48 increased the Aβ42/40 ratio ( Figure 3B and C ) .","The predicted Aβ42/40 shifts were nearly identical whether Aβ levels were measured from an in vitro assay ( Figure 3B ) or from a cell-based assay ( Figure 3C ) .","These results are of particular note , as I45F is a known FAD mutation ( Guerreiro et al . , 2010 ) , likely indicating that the mechanism of this mutation is the Phe positioned in the P2’ position at the Aβ43 cut site , blocking cleavage of APP through the less pathogenic Aβ49 → 40 pathway and favoring the pathogenic Aβ48 → 42 pathway .","The Phe at I45 also lies in the S3’ position for Aβ42 cleavage , likely making the precursor to Aβ42 production a better substrate for γ-secretase through a favorable P3’-S3’ interaction .","Mutating A42 to Phe completely blocked the formation of Aβ40 in agreement with our model ( Figure 3D ) , while still allowing for the production of other Aβ species ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 17578 . 005Figure 3 . Phenylalanine mutations at P2’ positions predictively shift the Aβ42/40 ratio .","( A ) Schematic diagram of Phe mutations at P2’ positions at cut sites within the TMD of APP and the expected Aβ42/40 changes compared to WT .","( B ) In vitro Aβ42/40 ratios from γ-secretase cleavage of recombinant C100-FLAG substrates .","Aβ measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .","Mean ± SD , n = 3 , t-test *<0 . 05 , **<0 . 01 .","( C ) Aβ42/40 ratios from Aβ secreted from HEK cells .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .","( D ) Aβ40 levels measured from the conditioned media of HEK cells transfected with WT or A42F APP .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 .","( E ) Western blot analysis of the AICD fragments generated from V44F , I45F , I47F and T48F in vitro .","Total AICD was measured with anti-FLAG M2 antibody ( green ) .","Aβ49 → Aβ40 pathway preference was measured with an antibody specifically recognizing the N-terminus of AICD 50–99 fragment ( red ) .","( F ) MALDI/TOF MS confirmation of the AICD fragments measured in ( E ) : V44F ( AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7020 . 8 ) , V45F ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6906 . 2; AICD 49–99 , expected mass: 7018 . 8 , observed mass 7020 . 1 ) , I47F ( AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7019 . 5 ) , T48F ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6907 . 4; AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7019 . 5 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 00510 . 7554/eLife . 17578 . 006Figure 3—figure supplement 1 . Total Aβ from the A42F mutant .","( A ) Total secreted Aβ was measured by ELISA from transiently transfected HEK cells with full length WT and A42F APP .","Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 006 Although we expected the Phe substitutions at V44 , I45 , I47 and T48 to be acting independently of the pathway chosen at initial ε cleavage , an alternative explanation for the above results is that these mutations instead influence ε cleavage in favor of the final Aβ products measured .","To investigate this possibility , we utilized an antibody that specifically recognizes the free N-terminus of AICD 50–99 ( Chávez-Gutiérrez et al . , 2012 ) , and therefore the initiation of the Aβ49 → 40 pathway .","Surprisingly , all four of these mutants actually caused a shift in ε cleavage toward the initiation of the opposite pathway .","Although V44F and I47F cause reductions in the Aβ42/40 ratio , they shifted initial ε cleavage away from the Aβ49 → 40 pathway—nearly completely eliminating AICD 50–99 .","And while I45F and T48F increased the Aβ42/40 ratio , they shifted ε cleavage more in favor of AICD 50–99 compared to WT ( Figure 3E ) .","These AICD species were confirmed by mass spectrometry ( Figure 3F ) .","To explore further the apparent ability of tripeptide cleavage preference to dissociate the normal connection between initial ε cleavage and final γ cleavages , we made double Phe mutants in which the ε cut site is controlled with a Phe at either V50 or M51 , while placing a conflicting Phe at V44 , I45 , I47 or T48 in the initial pathway .","Measuring Aβ secreted from transfected HEK cells , we clearly saw the double mutants behave almost identically to the single point mutants N-terminal to the ε cut site ( Figure 4A and B ) .","MS of the AICD fragments revealed the expected and complete blocking of AICD 49–99 or AICD 50–99 for the V50F and M51F containing double mutants , respectively ( Figure 4C ) .","Together these data suggest final γ cleavages can be completely uncoupled from initial ε cleavages . 10 . 7554/eLife . 17578 . 007Figure 4 . Phenylalanine mutations in the P2’ position of the last read tripeptide segment dictates final pathway preference .","( A ) Aβ42/40 ratios from HEK cells of V44F-M51F and I47F-M51F double mutants behave like single Phe mutants V44F and I47F , respectively .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 .","( B ) Aβ42/40 ratios from HEK cells of I45F-V50F and T48F-V50F double mutants behave like single Phe mutants I45F and T48F , respectively .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test *<0 . 05 , **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .","( C ) MALDI/TOF MS conformation of the elimination of AICD 49–99 and AICD 50–99 for the V50F and M51F containing double Phe mutants , respectively .","V44F-M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7030 . 7; AICD 47–99 expected mass: 7249 . 0 , observed mass: 7253 . 7 ) , I45F-V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6950 . 1; AICD 48–99 , expected mass: 7167 . 9 , observed mass: 7164 . 6 ) , I47F-M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7032 . 2; AICD 47–99 , expected mass: 7283 . 0 , observed mass: 7280 . 1 ) , T48F-V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6949 . 4; AICD 48–99 , expected mass: 7214 . 0 , observed mass: 7209 . 4 ) .","( D ) Aβ42/40 ratios from HEK cells transfected with double Phe mutations in tandem .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 007 Single Phe mutations at V46 and L49 both caused modest increases in the Aβ42/40 ratio .","We predicted that since these mutations do not occupy the S2’ pocket for either major pathway , V46F and L49F when paired with a neighboring Phe mutation should behave like its neighboring Phe mutant alone .","Indeed , L49F-V50F had a reduced Aβ42/40 ratio compared to WT like V50F alone , while T48F-L49F had an elevated Aβ42/40 ratio comparable to T48F ( Figure 4D ) .","Likewise , V46F-I47F displayed a reduced Aβ42/40 ratio similar to I47F alone , while I45F-V46F had a drastically increased Aβ42/40 ratio like I45F alone ( Figure 4D ) .","To this point , aromatic amino acids placed in the P2’ position at each tripeptide cleavage site within the TMD of APP caused a predictable outcome without exception .","We therefore reasoned that placing two phenylalanines in tandem , such that there is a Phe in the P2’ position at both major ε cut sites , should reduce overall cleavage .","As predicted , a double mutant of V50F-M51F caused a sharp reduction in total AICD formation compared to WT and the V50F and M51F single point mutations alone ( Figure 5A ) .","However , AICD formation from V50F-M51F cleavage was not completely abolished; rather , its rate of production was markedly reduced ( Figure 5B ) .","This suggests γ-secretase has a means by which to overcome two aromatic amino acids in a row , which would seemingly conflict with its cleavage preferences . 10 . 7554/eLife . 17578 . 008Figure 5 . Phenylalanine blocking mutations at both ε cleavage sites reduces APP cleavage but not binding to γ-secretase .","( A ) Western blot of γ-secretase cleavage of WT , V50F , M51F and V50F-M51F C100-FLAG .","Duplicates from each substrate represent separate independent data points .","* denotes a degradation product which co-purified with the substrate .","( B ) Cleavage of WT and V50F-M51F C100-FLAG over time .","( C ) Co-immunoprecipitation of Myc-tagged WT or V50F-M51F C100 substrate .","Duplicates are from separate pull-down experiments .","* antibody light chain .","( D ) Competitive cleavage of WT C100-FLAG by WT C100-Myc or V50F-M51F C100-Myc .","( E ) Aβ42/40 ratio of the V50F-M51F double mutant .","Mean ± SD , n = 3 , t-test , ****<0 . 0001 .","( F ) MALDI/TOF MS of the AICD fragment from the V50F-M51F mutant: ( AICD 51–99 , expected mass: 6822 . 5 , observed mass: 6817 . 1; * unknown peak , observed mass: 7030 . 4; AICD 48–99 , expected mass: 7183 . 9 , observed mass: 7179 . 2; AICD 47–99 , expected mass: 7297 . 1 , observed mass: 7292 . 5 , AICD 46–99 , expected mass: 7396 . 2 , observed mass: 7396 . 1 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 008 The observation that both tripeptide cleavage products of APP and transition-state analog inhibitors are noncompetitive inhibitors of γ-secretase suggests that the subsites on γ-secretase for initial substrate binding and subsequent catalysis are spatially separate and distinct , meaning substrate movement is likely required after initial substrate binding but prior to catalysis .","This would be in agreement with previous reports suggesting there may be an exosite on γ-secretase to which substrate initially binds prior to translocating to the active site for cleavage ( Kornilova et al . , 2003 , 2005 ) .","It would also agree with studies of other intramembrane cleaving proteases , which have proposed substrate movement in the form of helical unwinding is a prerequisite for catalysis ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) .","Given that the S’ pockets within the active site are likely the second binding site for substrate , we reasoned that although the V50F-M51F mutant cannot be hydrolyzed as efficiently as WT , it should still be effectively bound by γ-secretase in an initial docking site .","Co-IP of WT and V50F-M51F C100 in complex with γ-secretase reveals equal amounts of substrate bound , suggesting both substrates have a similar binding affinity for the enzyme ( Figure 5C ) .","Furthermore , myc-tagged V50F-M51F was just as effective as myc-tagged WT C100 at competing for γ-secretase cleavage of FLAG-tagged WT C100 ( Figure 5D ) , again indicating V50F-M51F and WT substrate initially interact with γ-secretase in a similar manner .","Together this suggests that the two Phe mutations in V50F-M51F do not affect initial recognition or binding of the C100 substrate , but rather may reduce the stabilization of a cleavable intermediate enzyme-substrate complex by clashing with the S’ pockets in the presenilin active site .","We noticed that in addition to being processed more slowly , V50F-M51F also produced a sharply lower Aβ42/40 ratio compared to WT ( Figure 5E ) .","To determine why this was occurring , we performed MS on the AICD fragment .","Surprisingly , we found that ε cleavage was initiated almost exclusively after I47 ( the very next available cleavage site ) , generating a 48–99 AICD fragment ( Figure 5F ) , indicating γ-secretase is capable of skipping the two phenylalanines to initiate cleavage , albeit at a slower rate .","Aβ47 is primarily processed to Aβ40 ( Aβ47 → Aβ43 → Aβ40 ) ( Matsumura et al . , 2014 ) , accounting for the decreased Aβ42/40 ratio .","Although γ-secretase usually cleaves APP in increments of three amino acids to produce predominantly Aβ40 and Aβ42 in the mechanism outlined in this study , a seemingly unusual cleavage deviates from the tripeptide preference to produce Aβ38 in appreciable quantities ( Okochi et al . , 2013; Takami et al . , 2009 ) .","To determine if production of Aβ38 occurs through the same three S’ pockets used by γ-secretase to achieve tripeptide cleavage , we transiently transfected HEK cells with mutant APP containing a single Phe point mutant at V39 , V40 , I41 or A42 .","Measuring secreted Aβ38 revealed that V40F almost completely eliminated Aβ38 production ( Figure 6A ) , demonstrating occupancy of the three S’ pockets is required for Aβ38 formation .","V39F and I41F both sharply increased Aβ38 , a result we interpret as the phenylalanine mutations making the precursor to Aβ38 a better substrate for γ-secretase , as the S1’ and S3’ pockets are large and prefer phenylalanine over smaller amino acids ( Esler et al . , 2004 ) .","A42F produces about as much Aβ38 as WT , indicative of the lack of a contributory S4’ pocket . 10 . 7554/eLife . 17578 . 009Figure 6 . γ-Secretase preferentially cleaves APP near the helix-destabilizing Gly-Gly motif .","( A ) Aβ38 levels from HEK cells transiently transfected with V39F , V40F , I41F or A42F APP .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 .","( B ) Aβ42/40 ratios from V44F-I47F-V50F and I45F-T48F-M51F triple mutants from transiently transfected HEK cells .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test , ****<0 . 0001 .","( C ) Aβ38 , Aβ40 and Aβ42 levels from HEK cells transfected with V44F-I47F-V50F and I45F-T48F-M51F triple mutants , the V44F-I45F double mutant and the hexa-mutant V44F-I45F-I47F-T48F-V50F-M51F .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 .","( D ) Schematic diagram of sequential Phe mutants in the TMD of APP .","( E ) Aβ38 + 40 + 42 secreted from HEK cells transiently transfected with the mutants from ( D ) .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 .","( F ) Aβ42/40 ratios from ( E ) .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test *<0 . 05 , ****<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 00910 . 7554/eLife . 17578 . 010Figure 6—figure supplement 1 . HEK cell expression and Aβ production from consecutive phenylalanine APP mutants .","( A ) Aβ38 , Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines ( see Figure 6D ) .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 .","( B ) Expression levels of consecutive phenylalanine APP mutants from Figure 6D . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 010 During the course of this study , we found that three phenylalanines sequentially mutated in the P2’ positions of each major pathway ( V44F-I47F-V50F and I45F-T48F-M51F ) caused a very strong reduction or elevation in the Aβ42/40 ratio in the predicted direction ( Figure 6B ) .","As expected , the shifts were caused by the near complete elimination of Aβ42 for the V44F-I47F-V50F mutant and of Aβ40 for the I45F-T48F-M51F mutant ( Figure 6C ) .","Interestingly , in each case , Aβ38 levels were produced in amounts comparable to or greater than WT .","This demonstrates that Aβ38 is capable of being produced from both the Aβ49 → 40 and Aβ48 → 42 pathways , in perfect agreement with recent MS studies that identified the precursors to Aβ38 as being either Aβ43 or Aβ42 ( Matsumura et al . , 2014; Olsson et al . , 2014 ) .","Surprisingly , when we blocked the production of both Aβ43 and Aβ42 at the same time with a V44F-I45F double mutant we not only did not prevent the production of Aβ38 , but rather Aβ38 levels were drastically increased ( Figure 6C ) .","Even after blocking the first six major cleavage sites ( V44F-I45F-I47F-T48F-V50F-M51F ) of APP , we still observe elevated Aβ38 compared to WT , demonstrating γ-secretase is fully capable of traversing multiple phenylalanines within APP to find the especially labile amide bond between G38 and V39 .","It is likely that the helix-destabilizing Gly-Gly motif at G37 and G38 is the reason for this observation , making the G38-V39 bond particularly accessible for cleavage .","Next , we attempted to determine how many phenylalanines in a row γ-secretase was capable of skipping by taking advantage of the fact that the GG motif apparently allows for γ-secretase to deviate from normal sequential tripeptide cleavage .","Astonishingly , even after increasing the number of phenylalanines in a row to eight , γ-secretase was still able to produce Aβ above mock transfected levels ( Figure 6D , E ) .","The V44F⇒M51F mutant produced mostly Aβ38 ( Figure 6—figure supplement 1 ) , in what may be a single endoproteolytic cleavage event , although we have not been able to obtain enough AICD for MS confirmation .","Predictably , the Aβ42/40 ratios for these mutants follow a pattern expected if γ-secretase cleaves at the next available site after each additional Phe ( Figure 6F ) .","Using our newfound knowledge of the basic cleavage mechanism of γ-secretase , and our ability to precisely control it with phenylalanine mutations , we next decided to investigate the mechanism of FAD mutations within the TMD of APP , which all increase the Aβ42/40 ratio to different degrees .","There are more than a dozen missense FAD mutations targeting this region of APP ( Figure 1A ) , with the majority being located N-terminal and downstream of ε cleavage .","To date , the currently accepted explanations for how these mutations increase the Aβ42/40 ratio are:","1 ) affecting the positioning/helical stability of the γ-secretase bound APP TMD such that initial ε cleavage is shifted toward T48 , thus favoring the production of Aβ42 ( Chávez-Gutiérrez et al . , 2012; Chen et al . , 2014; Dimitrov et al . , 2013 ) ; or","2 ) influencing the reaction kinetics of γ-secretase’s processing of APP , leading to incomplete carboxy-trimming and therefore increased Aβ42 over the more processed Aβ38 ( Chávez-Gutiérrez et al . , 2012 ) ( Figure 7A ) .","As demonstrated above with the I45F FAD mutant , we now show that a third possible mechanism exists , in that sequence-specific cleavage preferences can uncouple initial ε cleavage and final γ cleavages of APP ( Figure 7A ) . 10 . 7554/eLife . 17578 . 011Figure 7 . The tripeptide cleavage mechanism of γ-secretase and the effect of APP transmembrane domain FAD mutations .","( A ) The three mechanisms by which FAD mutations within the TMD of APP increase the Aβ42/40 ratio .","1 ) Mutations shift initial ε cleavage towards the Aβ42 pathway .","2 ) Mutations reduce cleavage of the third cleavage event , producing more Aβ42 over the more processed Ab38 .","3 ) Cleavage specific preferences cause switching from the Aβ40 to the Ab42 pathway , as exemplified by the I45F FAD mutant .","( B ) The Aβ42/40 ratio of each FAD mutation with and without an additional V50F mutation to control the ε cleavage site .","The majority of mutations are rescued by the V50F substitution suggesting that these FAD mutations increase the Aβ42/40 ratio by influencing ε cleavage and/or affecting carboxy-trimming .","I45F , I45T and T48P retain significantly elevated ratios , indicating these mutants dissociate initial ε and final γ cleavages .","Aβ levels measured by 4G8 ELISA .","Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 . ( C ) The tripeptide cleavage mechanism of γ-secretase .","After initial substrate binding , we speculate that the helical TMD of substrate unwinds into the active site of presenilin ( PSEN ) where it is stabilized by the three S’ pockets in the catalytic pocket prior to cleavage .","Successive carboxy tripeptide trimming occurs until the eventual release of Aβ peptide .","( D ) γ-secretase cleavage of the transmembrane domain of Notch from Okochi et al , 2002 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01110 . 7554/eLife . 17578 . 012Figure 7—figure supplement 1 . APP FAD mutant panel measured by 6E10 ELISA .","( A ) The samples from Figure 7B were measured by ELISA with the 6E10 detection antibody , showing similar results to those of the 4G8 ELISA .","Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01210 . 7554/eLife . 17578 . 013Figure 7—figure supplement 2 . AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody .","( A ) Western blot of the AICD 50–99 fragment for I45F , I45V and I45T FAD mutations .","( B ) Quantification of western blot bands from ( A ) .","Mean ± SD , n = 3 , t-test **<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01310 . 7554/eLife . 17578 . 014Figure 7—figure supplement 3 . Secreted Aβ levels from V40F , A42F and V44F .","( A ) Aβ38 , Aβ40 and Aβ42 levels measured from V40F , A42F and V44F transfected HEK cells respectively , as well as mock transfected and WT .","Aβ levels measured by 6E10 ELISA .","Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 014 To determine the prevalence of the two previously proposed mechanisms and to possibly identify additional pathway uncoupling mutants , we made each FAD mutant within the APP TMD alone and as a double mutant with V50F .","The V50F mutation would be predicted to block ε cleavage after T48 independent of the FAD mutant’s affect on subsequent cleavage events .","We would therefore predict that if an FAD mutant causes an increase in the Aβ42/40 ratio by influencing initial ε cleavage or by affecting subsequent carboxy-trimming along the Aβ48 → 42 pathway , then that same FAD mutant when paired with V50F should produce a reduced Aβ42/40 ratio compared to WT , similar to V50F alone .","Conversely , if an FAD mutant affects subsequent cleavage events independent of ε cleavage , causing uncoupling of initial ε cleavage and final γ cleavages , then the FAD-V50F double mutant should retain an elevated Aβ42/40 ratio , similar to the FAD mutant alone .","Screening nearly all the FAD mutants within the TMD of APP by this method , we found that the majority were either completely or nearly completely rescued when paired with V50F ( Figure 7B , Figure 7—figure supplement 1 ) , resulting in 42/40 ratios significantly less than WT .","This suggests the predominant mechanism of elevating the Aβ42/40 ratio by FAD mutants within the TMD of APP is by shifting the preference of initial ε cleavage from the Aβ40 to the Aβ42 pathway , and/or by influencing carboxy-trimming .","This may have been predicted , given that I45F is the only aromatic amino acid mutation to fall in the S2’ pocket of one of the major cleavage pathways .","The only other FAD mutation containing an aromatic amino acid , V46F , falls within the S1’ and S3’ pockets for the Aβ42 and Aβ40 pathways , respectively , therefore never clashing with the S2’ pocket and not influencing the Aβ42/40 ratio as a major pathway blocker .","However , we found that in addition to I45F , there are two other mutants , I45T and T48P , which appear to dissociate the normal connection between initial pathway preference and final cleavage products .","V50F partially rescues the Aβ42/40 ratio when paired with I45T , but remains significantly elevated compared to WT , indicating the I45T mutant both influences initial ε cleavage and uncouples ε from γ cleavages .","The change in ε cleavage preference of I45T was verified using the AICD 50–99 specific antibody ( Figure 7—figure supplement 2 ) , showing a small reduction in AICD 50–99 .","Interestingly , the I45T mutant reduces the amount of AICD 50–99 comparable to I45V , even though these two mutants display very different Aβ42/40 ratios .","This again suggests I45T dissociates cleavage downstream of ε to achieve such a high Aβ42/40 ratio .","Exactly how I45T does this is currently unknown and requires further investigation .","The T48P-V50F double mutant behaves identically to T48P alone .","Given that proline is a helix-breaking amino acid , the T48P mutant may not undergo normal ε cleavage after P48 or L49 .","Determining how T48P increases the 42/40 ratio and overcomes the ε controlling V50F mutant will require further investigation ."],["In this study , we identify that three S’ amino acid binding pockets guide the productive positioning of substrate into the γ-secretase active site , providing the mechanism behind the enzyme’s preferred tripeptide cleavage of APP and pathogenic Aβ production .","Based on the data reported herein ( discussed further below ) and numerous other studies of γ-secretase ( Das et al . , 2003; Kornilova et al . , 2003 , 2005 ) and other intramembrane proteases ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) , we speculate that after initial binding to γ-secretase , substrate must undergo a translocation and/or conformational change in order to bind the three S’ pockets within the active site and subsequently be cleaved by the enzyme ( Figure 7C ) .","After initial endoproteolysis , the three S’ pockets guide further carboxy-trimming of the retained Aβ species until it is short enough to dissociate from the complex , producing predominantly Aβ38 , Aβ40 and Aβ42 .","More than a decade ago , SAR studies of γ-secretase-targeting transition-state analogs putatively assigned three S’ pockets to the active site of the enzyme ( Esler et al . , 2004 ) .","Transition-state analogs containing only two amino acids for S’ pocket binding inhibited γ-secretase less effectively , indicating that occupancy of the S3’ pocket is required for a strong interaction .","This likely provides the reason why γ-secretase only cleaves APP in segments of three or more amino acids , but never two .","The preference for cleaving three amino acids originates from the lack of a contributory fourth S’ pocket .","A fourth amino acid ( no matter the size ) added to a transition state inhibitor neither increased nor decreased the inhibitors potency ( Esler et al . , 2004 ) , suggesting a fourth S’ pocket doesn’t contribute to γ-secretase-inhibitor or -substrate interactions .","To support our proposed model , we exploited the fact that the second of the three S’ pockets is apparently too small to readily accommodate an aromatic amino acid .","We generated dozens of substrates containing aromatic amino acid substitutions in the P2’ positions at each cleavage site along the Aβ40 or Aβ42 pathways , selectively blocking each individual cleavage event .","Without exception we were able to predict the shift in the Aβ42/40 ratio .","This was accomplished both in vitro with purified γ-secretase and recombinant C100 substrate as well as in a cell-based assay , transiently transfecting mutant full-length APP in HEK cells and measuring secreted Aβ .","The same predicted Aβ42/40 ratio changes were observed in vitro and in the cell-based assay whether we measured Aβ ( 6E10 detection antibody ) or Aβ plus p3 products ( 4G8 detection antibody ) by ELISA .","Together this demonstrates we are probing the fundamental mechanism by which γ-secretase cleaves APP , irrespective of mutant effects on cellular localization , γ-secretase’s interaction with activity-modulating proteins/lipids within the cell or any artifacts that may arise from more artificial in vitro assays .","Although we are unable to measure every cleavage product from the ~70 mutant forms of APP we generated , we do note that every time a Phe was placed in the P2’ position of a cleavage product that we could readily and directly measure , there were almost negligible amounts of that product formed .","For example , in the cell-based assay , V40F , A42F and V44F generated levels of Aβ38 , Aβ40 and Aβ42 , respectively , that were actually less than mock transfected levels ( Figure 7—figure supplement 3 ) .","These Aβ levels are orders of magnitude less than that from WT APP transfected cells , although we cannot say whether these low levels of Aβ species were produced from endogenous HEK cell APP or from our transfected mutants .","Similarly , we were unable to detect by MS any AICD products containing a Phe in the P2’ position .","Together , these data suggest that aromatic amino acids may be completely excluded from the S2’ pocket and further demonstrates that substrate occupancy of the three S’ pockets is an absolute requirement for catalysis .","It is likely that phenyalanine substitutions at various positions along the transmembrane domain of APP influence the general structure and/or helical stability of the substrate .","This could affect the manner in which these substrates interact with γ-secretase .","Given that we are able to accurately predict the Aβ42/40 ratio without exception for the dozens of mutants used in this study , we expect that cleavage preferences dictated by the presence of aromatic amino acids in the P2’ position are overriding any affect these mutations have on substrate helical structure/stability and any altered manner in which these mutant substrates initially interact with the enzyme .","This is directly supported in Figure 3 where V44F , I45F , I47F and T48F all shift initial ε cleavage in favor of the opposite pathway relative to the final cleavage products measured and originally predicted by our model .","We present several lines of evidence suggesting that substrate movement and/or a substrate conformational change after initial enzyme binding is an important step in γ-secretase’s catalytic mechanism .","We identify that like transition-state analogs , tripeptide cleavage products are noncompetitive inhibitors of γ-secretase , albeit very weak inhibitors .","By definition this means the binding sites on γ-secretase for initial substrate binding and subsequent catalysis are spatially separate , requiring substrate movement after initial binding to be an integral part of γ-secretase’s catalytic mechanism .","For rhomboid , a recent study demonstrates that product-mimicking peptide aldehydes are non-competitive inhibitors of this serine intramembrane protease ( Cho et al . , 2016 ) , exactly like tripeptide products and transition-state analog inhibitors are for γ-secretase , suggesting a common two-step mechanism between these two intramembrane proteases .","Additionally , the double Phe mutant V50F-M51F , which is predicted by our model to sterically clash with the three S’ pockets in the active site , is still efficiently bound to γ-secretase even though it is processed less efficiently than WT .","Given that this mutant effectively competes for γ-secretase processing of other substrates , it must bind to the initial docking site on the enzyme for substrate .","This suggests that binding to the S’ pockets is the final step in substrate recognition and positioning within the enzyme prior to catalysis .","Furthermore , we are unable to prevent the formation of Aβ38 by specifically blocking the production of the known Aβ38 precursors Aβ42 and Aβ43; instead , paradoxically the V44F-I45F double mutant increases Aβ38 production .","It is likely that the helix-destabilizing Gly-Gly motif at G37 and G38 is the reason for this observation .","Local helical unwinding around this position probably makes the amide bond between G38 and V39 particularly accessible for cleavage , and this may be the reason for γ-secretase’s normal deviation from the preferred tripeptide cleavage for Aβ38 production .","However , further investigation , including quantification of the intramembrane peptide products by mass spectrometry , will be required to prove this .","In the absence of a γ-secretase—substrate co-complex structure , it will not be possible to definitively prove the existence of a partially unwound substrate intermediate .","Conceivably , it should be possible to capture such an intermediate with a transition-state analog covalently linked to the C-terminus of an APP- or notch-based helical substrate .","Given that tripeptide cleavage is dictated by the three S’ pockets in the active site of presenilin , we expect that other γ-secretase substrates will be similarly cleaved preferentially in increments of three amino acids , while skipping aromatic amino acids that fall in the S2’ pocket along the way , and with cleavage occurring preferentially in helix-destabilized regions .","This is important to note given that many of γ-secretase’s substrates naturally contain aromatic amino acids .","The TMD of notch , for example , naturally contains three phenylalanines .","We know from a previous MS study that these phenylalanines are skipped by γ-secretase in a pattern consistent with our model ( Figure 7D ) ( Okochi et al . , 2002 ) .","In a recent study , we have demonstrated that substrates with large ectodomains have a reduced binding affinity for γ-secretase due to steric clashing with the nicastrin component of the γ-secretase complex ( Bolduc et al . , 2016 ) .","Based on this , and results from our current study , we might expect that γ-secretase can chose its substrates through a complex interplay between ectodomain length , helical TMD stability and the sequence of amino acids ( specifically aromatic amino acids ) within substrate TMD .","There may even exist non-substrates containing short ectodomains but having stable helices that are further protected from cleavage by sequential stretches of aromatic amino acids .","Whether such non-substrates exist , and the relationship between ectodomain size , helical stability and amino acid sequence will require further investigation .","We show that the majority of FAD mutants within the TMD of APP primarily increase the Aβ42/40 ratio by changing ε cleavage , favoring the Aβ48 → 42 pathway .","This is mostly in agreement with previous studies ( Chávez-Gutiérrez et al . , 2012; Chen et al . , 2014; Dimitrov et al . , 2013; Quintero-Monzon et al . , 2011 ) .","However , we also identify a new mechanism by which certain FAD mutations can increase the production of pathogenic Aβ species .","Here , final γ cleavages are uncoupled from initial pathway preference determined by ε cleavage .","In the case of the I45F FAD mutation , the bulky Phe sterically clashes with the S2’ pocket of γ-secretase at the Aβ43 cleavage site , blocking its cleavage and the subsequent production of Aβ40 .","It is likely that the positioning of the Phe in the S3’ pocket of the Aβ42 cleavage also enhances γ-secretase proteolysis at this position through a favorable S3’-P3’ interaction .","These two interactions likely combine to account for the fact that no other mutation within the TMD of APP produces as much Aβ42 as I45F .","This is the most severe APP FAD mutation , with an onset of clinical AD at 31 years of age ( Guerreiro et al . , 2010 ) .","There are at least two additional FAD mutations , I45T and T48P , that appear to dissociate the ε and γ cleavages .","How these mutations accomplish this is currently unknown .","Our data also help explain previous observations in the literature .","Prior to the identification of presenilin as being the protease responsible for γ-secretase activity ( Wolfe et al . , 1999 ) , Lichtenthaler et al . performed a phenylalanine scanning study of the TMD of APP .","In their study , they observed the exact same Aβ42/40 shifts identified here for several of the same mutants ( Lichtenthaler et al . , 1999 ) .","Later , Sato et al . found that γ-secretase was unable to cleave through stretches of 3–5 consecutive tryptophans inserted into the TMD of APP ( Sato et al . , 2005 ) .","These observations are now explained by the determination that the small S2’ pocket of γ-secretase cannot accommodate aromatic amino acids .","With the identification of the key role the three S’ pockets play in γ-secretase’s cleavage mechanism , several important new questions are raised .","The exact locations of the three S’ pockets in presenilin are currently unknown .","Additionally , the identity of the initial substrate-binding site on presenilin is unknown .","The resolution of enzyme—substrate and/or -inhibitor co-complexes by cryo-EM will be informative in this regard .","All enzymatic assays in this study utilized purified γ-secretase complex containing presenilin-1 .","It will be interesting to see if presenilin-2 has similar substrate cleavage preferences .","A major unsolved question pertains to the mechanism by which substrate TMD is repositioned within γ-secretase after each cleavage event in order to be close enough to the active site for the next round of catalysis to occur .","Is this a ratcheting or sliding motion ?","Is this an active process or based on Brownian motion ?","At present , we do not know how γ-secretase is capable of skipping stretches of several phenylalanines in a row .","Determining how γ-secretase accomplishes this may help elucidate how substrate moves during normal sequential cleavage .","Now that we have identified several key aspects of γ-secretase’s substrate recognition and cleavage mechanisms , as well as provide valuable new tools for future structural and biochemical studies , we should be able design experiments to elucidate some of γ-secretase’s remaining unanswered functional questions .","The answers from which should have further broad implications for our understanding of Alzheimer’s disease and the development of safe and effective therapeutics targeting γ-secretase function ."],["The following antibodies were used: α-Myc ( 9E10 Santa Cruz #sc-40 ) , α-Flag ( M2 Sigma #F3165 ) , α-Nct ( Cell Signaling 3632S ) , α-GAPDH ( Cell Signaling Technology , ab125247 ) , α-APP ( C7 ) , α-AICD 50–99 ( Rb ) was a kind gift from Philip Szekeres at Eli Lilly , α-Ms 800nm ( Licor Bio 926–32212 ) and α-Rb 680nm ( Licor Bio 926–68021 ) .","Tripeptides were synthesized by Anaspec corp .","Total brain lipid extract was from Avanti Polar Lipids ( #131101 ) .","The following Aβ ELISA kits were used: 4G8 ( Meso Scale Diagnostics , K15199E ) or 6E10 ( Meso Scale Diagnostics , K15200E ) for cell-based assays; Aβ40 ( #KHB3482 ) and Aβ42 ( #KHB2442 ) ELISA kits from Invitrogen for in vitro assays .","All mutant forms of C100 FLAG or full-length APP were generated by site directed mutagenesis of either C100-FLAG in pET22b or full-length WT APP in the pCMV695 plasmid .","Adherent HEK cells were cultured in complete growth media: Dulbecco’s Modified Eagle’s Medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) , 2 mM L-glutamine , 10 Units/mL penicillin , and 10 µg/mL streptomycin .","For transfection , adherent HEK cells were seeded in six-well dishes at a density of 5x105 cells per well .","Transfection was carried out with Lipofectamine 3000 reagent in serum-free conditions with Opti-MEM I . Cells were incubated for 24 hr , at which time conditioned media was harvested for ELISA and cells were harvested for western blot .","Suspension HEK cells were cultured in 100 mL of unsupplemented Freestyle 293 media ( Life Technologies , 12338-018 ) with shaking at 125 rpm , and passaged at a density of 2 × 106 cells/mL .","For transfection , suspension HEK cultures were grown to a density of 2 × 106 cells/mL .","Media was replaced with fresh Freestyle 293 media .","5 mL Freestyle 293 , 150 µg of γ-secretase vector containing presenilin 1 ( provided by Yigong Shi ) , and 450 µg of 25-kDa linear polyethylenimines ( PEI ) was mixed and incubated for 30 min at room temperature .","The DNA/PEI solution was then added to the HEK culture and cells were grown for ~60 hrs prior to harvesting .","γ-secretase was purified as previously described ( Fraering et al . , 2004; Osenkowski et al . , 2009 ) C100-FLAG substrates were expressed in BL21 E . coli for 3 hrs at 37°C after induction with 1 mM IPTG .","Cells were then pelleted and lysed by French press in 50 mM HEPES pH 7 . 0 , 1% Triton X-100 detergent .","FLAG-tagged substrates were then isolated by immunoprecipitation for 3 hrs at 4°C with anti-FLAG M2 beads from Sigma .","Substrates were then eluted from the beads with 100 mM glycine pH 2 . 5 , 0 . 25% NP40 prior to being neutralized with tris buffer and stored at -80°C .","Purified γ-secretase was incorporated into vesicles by first dissolving total brain lipid extract ( 1 . 25 mM final ) in 50 mM HEPES pH 7 . 0 , 150 mM NaCl , 0 . 25% CHAPSO .","γ-Secretase ( 5–30 nM final concentration ) was then added to the solution and detergent removed by mixing SM-2 biobeads ( 62 mg/mL ) ( Bio-Rad ) with the lipid/detergent/enzyme solution for two hrs at 4°C .","Biobeads were removed from the newly formed proteoliposomes and reactions were initiated with the addition of purified recombinant substrate C100-FLAG substrate .","Reactions were quenched with SDS loading dye for western blot or centrifuged for ELISA or mass spectrometry on Aβ or AICD fragments , respectively .","For inhibition studies , tripeptide fragments or inhibitors were dissolved in DMSO prior to being diluted into the reaction buffer .","The concentration of C100-FLAG used in all in vitro assays was 500 nM unless otherwise stated .","Conditioned media from HEK cells transfected with WT or mutant APP was assayed for Aβ by 4G8 or 6E10 Aβ ELISA kits from Meso Scale Diagnostics .","Aβ levels were measured by both 6E10 and 4G8 ELISA for each data point , yielding nearly identical results .","In vitro assay Aβ was measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .","All ELISAs were performed according to the manufacture’s protocols .","Purified γ-secretase ( 5 nM final concentration ) was preincubated in assay buffer ( 50 mM HEPES pH 7 . 0 , 150 mM NaCl , 0 . 25% CHAPSO , 0 . 1% DOPC and 0 . 025% DOPE , 2% BSA ) in the presence of 2 μM III-31C for 1 hr at room temperature .","Purified WT or mutant C100-Myc ( 20 nM final concentration ) was then incubated with γ-secretase for 1 hr prior to pull down with anti-HA magnetic affinity beads for 4 hr with mixing at room temperature .","The immunoprecipitated complex was then washed three times and eluted with SDS loading buffer prior to western blot with an anti-Myc antibody .","Tripeptide IC50 inhibition was fit to:vivo=1/ ( 1+[ I ]IC50 ) where vi is the initial velocity in the presence of inhibitor at concentration [I] and vo is the initial velocity in the absence of inhibitor .","Tripeptide inhibition was globally fit to the following noncompetitive equation:v= Vmax[S][ S ] ( 1+[ I ]Kii ) +Km ( 1+[ I ]Ki ) where , v is the initial rate , Ki is dissociation constant for inhibitor binding to free enzyme , Kii is the dissociation constant for inhibitor binding to the enzyme-substrate complex .","Inhibitor cross-competition was globally fit to:1vij=1/v0 ( 1+[ I ]Ki+[ J ]Kj+[ I ][ J ]αKiKj ) where , vij is the initial rate in the presence of inhibitors , v0 is the initial rate in the absence of inhibitor , Ki and Kj are the dissociation constants for inhibitors I and J , respectively .","α = ∞ for inhibitors which bind in a mutually exclusive fashion , while α = 1 for inhibitors which have distinct binding sites .","Conditioned media was collected from transiently-transfected adherent HEK cells .","Each well of an uncoated 96-well multi-array plate ( Meso Scale Discovery , #L15XA-3 ) was coated with 30 µL of a PBS solution containing 3 µg/mL of 266 capture antibody ( Elan ) , and incubated at room temperature overnight .","A detection antibody solution was prepared with 3D6B detection antibody ( Elan ) , 100 ng/mL Streptavidin Sulfo-TAG ( Meso Scale Discovery , #R32AD-5 ) , and 1% MSD Blocker A ( #R93BA-4 ) in wash buffer ( #R61TX-1 ) .","Following overnight incubation , 25 µL/well of sample , followed by 25 µL/well of detection antibody solution were incubated for 2 hr at room temperature with shaking at >300 rpm , washing wells with wash buffer between incubations .","Plate was read and analyzed according to manufacturer protocol .","Following an in vitro proteoliposome activity assay , AICD-FLAG products were isolated by immunoprecipitiation with anti-FLAG M2 magnetic beads from Sigma .","Completed reactions were incubated with 50 µL of M2 beads in 10 mM MES pH 6 . 5 , 10 mM NaCl , 0 . 05% DDM detergent in 500 µL volumes overnight at 4°C .","AICD was then eluted from the beads with acetonitrile:water ( 1:1 ) with 0 . 1% trifluoroacetic acid .","MALDI/TOF mass spectrometry was performed with sinapinic acid matrix on a calibrated ultraflextreme MALDI/TOF/TOF from Bruker in linear mode ."]],"string":"[\n [\n \"Alzheimer’s disease ( AD ) is the most common form of dementia and currently the sixth leading cause of death in the United States , with no disease-modifying therapeutics available .\",\n \"A central and pathological hallmark of AD is the deposition of amyloid-beta ( Aβ ) plaques in the brain ( Hardy and Selkoe , 2002 ) .\",\n \"These plaques are comprised of aggregates of Aβ peptides , which are formed by the sequential cleavage of the membrane embedded amyloid precursor protein ( APP ) by two proteases—β-secretase first removes the ectodomain of APP , then γ-secretase cleaves the remaining C-terminal fragment within its transmembrane domain ( TMD ) to liberate Aβ from cellular membranes .\",\n \"Via its proteolytic component presenilin ( Li et al . , 2000; Wolfe et al . , 1999 ) , γ-secretase processes the TMD of APP into Aβ peptides of differing lengths , mostly producing a more benign Aβ species 40 amino acids in length , termed Aβ40 , as well as lesser amounts of a longer , more aggregation prone and pathogenic species , Aβ42 .\",\n \"The total amount of Aβ42 , as well as the ratio of Aβ42/40 , are thought to be key mediators of Aβ pathogenesis , a hypothesis strongly supported by the fact that nearly all the 200-plus autosomal-dominant familial Alzheimer’s disease ( FAD ) mutations in presenilin-1 , -2 and APP increase the Aβ42/40 ratio ( see www . alzforum . org/mutations ) .\",\n \"Due to the strong link between γ-secretase catalyzed Aβ formation and AD pathogenesis , the development of γ-secretase targeting therapeutics has been of high interest over the past two decades ( Golde et al . , 2013; De Strooper , 2014 ) .\",\n \"Both γ-secretase inhibitors ( GSIs ) and a γ-secretase modulator ( GSM ) , the latter working by an unknown mechanism to influence γ-secretase to produce shorter , presumably less pathogenic Aβ species , have failed in recent clinical trials .\",\n \"The failure of GSIs is due at least in part to toxicities from cleavage inhibition of other γ-secretase substrates such as Notch ( Doody et al . , 2013; Golde et al . , 2013 ) .\",\n \"Little is known about how γ-secretase recognizes the transmembrane domain of substrates , given that no consensus amino acid cleavage motif has been identified for the more than one hundred γ-secretase substrates discovered to date ( Haapasalo and Kovacs , 2011 ) .\",\n \"Unfortunately , the further development of safe and effective γ-secretase targeting therapeutics has been held back by a fundamental lack of understanding of how γ-secretase recognizes and cleaves the TMDs of its many substrates , especially APP .\",\n \"Elucidation of this basic mechanism should at the very least add to our understanding of how Aβ is produced and may also aid in the development of safe and effective disease-modifying therapeutics .\",\n \"Mass spectrometry studies have identified a complex mixture of products generated from γ-secretase’s cleavage of the transmembrane domain of APP ( Matsumura et al . , 2014 ) .\",\n \"Looking at the formation of these products over time , it is apparent that the TMD of APP is mostly processed via two major pathways .\",\n \"γ-secretase predominantly initiates endoproteolysis at a so-called epsilon ( ε ) cleavage site—after Leu49 or Thr48 , generating Aβ49 or Aβ48 and two different APP intracellular domains ( AICD ) , AICD 50–99 or AICD 49–99 , respectively ( Kakuda et al . , 2006; Sato et al . , 2003 ) .\",\n \"Aβ49 and Aβ48 are then sequentially cleaved in increments of three amino acids to produce mostly Aβ40 and Aβ42 , respectively ( Takami et al . , 2009 ) .\",\n \"The two major pathways are therefore Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 ( Figure 1A ) ( Fernandez et al . , 2014; Takami et al . , 2009 ) .\",\n \"There are , however , other Aβ species generated by γ-secretase through usually minor and sometimes overlapping , alternative pathways ( Matsumura et al . , 2014; Olsson et al . , 2014 ) .\",\n \"Importantly a shorter peptide , Aβ38 , can be formed from both major pathways , originating from Aβ42 or Aβ43 ( Okochi et al . , 2013 ) .\",\n \"Additionally , a third , sparingly used site of ε cleavage can lead to the production of Aβ47 , which rather than being processed to Aβ44 is instead mostly cleaved to Aβ43 , subsequently generating Aβ40 ( Aβ47 → Aβ43 → Aβ40 ) ( Matsumura et al . , 2014 ) . 10 . 7554/eLife . 17578 . 003Figure 1 . Tripeptide fragments of APP inhibit γ-secretase .\",\n \"( A ) Schematic diagram of the major sequential cleavage pathways of the transmembrane domain of APP ( Aβ49 → Aβ46 → Aβ43 → Aβ40 in red and Aβ48 → Aβ45 → Aβ42 in blue ) .\",\n \"Mutations causing Familial Alzheimer’s disease are below the APP TMD in blue .\",\n \"( B ) IC50 curves from the inhibition of γ-secretase activity by APP product tripeptide fragments .\",\n \"Mean ± SD , n = 2 .\",\n \"( C ) Noncompetitive inhibition of γ-secretase with VIV tripeptide , R2 = 0 . 98 .\",\n \"( D ) Yonetani-Theorell plot for the mutually exclusive binding of VIV and the noncompetitive transition-state analog inhibitor III-31-C , R2 = 0 . 98 .\",\n \"( E ) Cartoon representation of the three S’ pockets of presenilin ( PSEN ) along with three P’ amino acids of substrate and the transition-state analog L685 , 458 .\",\n \"( F ) IC50 curves from the inhibition of γ-secretase activity with FAF and AFA synthetic tripeptides .\",\n \"Mean ± SD , n = 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 003 Normally , γ-secretase uses the Aβ49 → Aβ40 and the Aβ48 → Aβ42 pathways to produce mostly Aβ40 and Aβ42 via a stepwise , tripeptide cleavage process .\",\n \"The mechanism that dictates this preferred tripeptide cleavage ( and thus the driving force behind γ-secretase catalysis and Aβ formation ) is completely unknown .\",\n \"In this study , we report that γ-secretase tripeptide cleavage is driven by three S’ pockets within the active site of the enzyme .\",\n \"We identify specific substrate cleavage preferences dictated by the three S’ pockets and exploit these preferences to determine the predominant mechanism of each FAD mutation within the transmembrane domain of APP , including a novel mechanism in which final cleavage products are uncoupled from initial ε pathway preference .\"\n ],\n [\n \"When studying enzyme catalysis much focus is appropriately placed on determining how an enzyme interacts with its substrate .\",\n \"However , oftentimes the manner in which an enzyme interacts with product ( in the form of product inhibition ) can be equally informative with regard to its catalytic mechanism .\",\n \"To this end , we asked whether the naturally produced tripeptide fragments of APP are inhibitors of γ-secretase .\",\n \"We found that all five tripeptides produced from the TMD of APP are indeed capable of inhibiting γ-secretase activity , albeit rather weakly with IC50 values ranging from ~150 μM to several mM ( Figure 1B ) .\",\n \"Although these binding affinities are too low for the tripeptides to be involved in any form of biologically relevant feedback inhibition , we imagined the manner in which they inhibit γ-secretase could be instructive in elucidating the basic cleavage mechanism of the protease .\",\n \"We characterized the mode of inhibition of the most potent of the tripeptides , VIV , finding that these data fit well to a noncompetitive inhibition model , with a global R2 of 0 . 98 ( Figure 1C ) .\",\n \"Given that the tripeptide segments of the TMD of APP must occupy the active site of γ-secretase during catalysis , we hypothesized that VIV may compete for the same binding site on the enzyme as transition-state analogs .\",\n \"An inhibitor cross-competition analysis reveals this is likely true , with a series of parallel lines resulting from a Yonetani-Theorell plot demonstrating mutually exclusive binding of VIV and the transition-state analog III-31-C ( Figure 1D ) .\",\n \"We noticed that nearly all of the γ-secretase targeting transition-state analog inhibitors developed to date ( e . g . L685 , 458 , III-31-C ) contain essentially a tripeptide fragment C-terminal to the transition-state-mimicking hydroxyl isostere ( Figure 1E ) .\",\n \"Structure-activity relationship ( SAR ) studies have demonstrated that transition-state analog inhibitors containing only two amino acids here are relatively weak inhibitors compared to those comprised of three amino acids , while adding a fourth amino acid does not achieve additional potency ( Esler et al . , 2004 ) .\",\n \"This suggests that there are three , and only three , putative S’ pockets in the presenilin active site that contribute to inhibitor binding .\",\n \"Of note , the SAR studies also suggest that while the putative S1’ and S3’ pockets are large and can accommodate amino acids of varying size , the S2’ pocket is small and inhibitors with an aromatic amino acid ( phenylalanine ) at this position have decreased potency by two orders of magnitude compared to those with less bulky aliphatic amino acids ( Esler et al . , 2004 ) .\",\n \"We imagined that the tripeptide APP TMD products may be binding these three putative S’ pockets to achieve inhibition .\",\n \"In agreement with this hypothesis , a synthetic tripeptide of the predicted optimal binding sequence ( FAF , to fit in the large-small-large S1’-S2’-S3’ pockets ) was a more potent inhibitor of γ-secretase activity than any of the naturally occurring APP-derived tripeptides , while a peptide predicted to clash with this binding site ( AFA ) is a very weak inhibitor , even at mM concentrations ( Figure 1F ) .\",\n \"Based on these results , we reasoned that the three putative S’ pockets within the γ-secretase active site are likely occupied by substrate prior to hydrolysis of the scissile bond as a means for γ-secretase to stabilize a transition-state-like scission complex with substrate .\",\n \"This would provide a simple mechanism for the preferred cleavage of APP in three amino acid increments , as well as provide an explanation for why γ-secretase mostly sticks to each major pathway , producing Aβ40 or Aβ42 after initiating cleavage of APP at either the L49 or T48 ε sites , respectively .\",\n \"Additionally , given that substrate movement in the form of helical unwinding is thought to be a required step in the poorly defined intramembrane protease cleavage mechanism ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) , the three S’ pockets could potentially provide a means for γ-secretase to stabilize its helical substrate in a more cleavable conformation , thereby lowering the activation energy required for catalysis .\",\n \"To test this hypothesis , we took advantage of the fact that the putative S2’ pocket of γ-secretase is apparently small and has a reduced ability to accommodate a bulky amino acid such as phenylalanine .\",\n \"We predicted that we should be able to selectively decrease cleavage of either the Aβ49 → 40 pathway or the Aβ48 → 42 pathway simply by placing an aromatic amino acid at the P2’ position of APP at the initial ε cut site .\",\n \"In other words , an aromatic amino acid placed at V50 should reduce ε cleavage after T48 , thereby lowering the amount of Aβ42 produced , thus decreasing the Aβ42/40 ratio .\",\n \"Conversely , an aromatic amino acid at M51 should reduce ε cleavage after L49 , lowering the amount of Aβ40 produced and therefore increase the Aβ42/40 ratio ( Figure 2A ) . 10 . 7554/eLife . 17578 . 004Figure 2 . Selective blocking of the Aβ40 or Aβ42 pathways with aromatic amino acids placed at the P2’ position of ε cleavage .\",\n \"( A ) Schematic diagram of the TMD of APP with pathway blocking aromatic amino acid mutations at the P2’ position for the T48 or L49 ε cleavage .\",\n \"( B ) In vitro Aβ42/40 ratios with Phe or Trp mutations at V50 or M51 .\",\n \"Aβ measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 , ****<0 . 0001 .\",\n \"( C ) MALDI/TOF MS of the AICD fragments generated from in vitro cleavage of C100: WT ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6907 . 4; AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7021 . 3 ) .\",\n \"V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6949 . 8; AICD 48–99 , expected mass: 7167 . 9 , observed mass: 7163 . 5 ) .\",\n \"M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7030 . 1; AICD 48–99 , expected mass: 7135 . 8 , observed mass: 7131 . 8 ) .\",\n \"( D ) Aβ42/40 ratios measured from the media of HEK cells transfected with V50 mutants .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .\",\n \"( E ) Aβ42/40 ratios measured from the media of HEK cells transfected with M51 mutants .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 .\",\n \"( F ) Aβ40 and Aβ42 levels for aromatic substitutions at V50 and M51 normalized to WT .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 , t-test *<0 . 05 , ***<0 . 001 , ****<0 . 0001 .\",\n \"( G ) Total secreted Aβ levels ( see Materials and methods ) from the aromatic mutations at V50 and M51 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 004 In an in vitro assay using purified γ-secretase to cleave recombinant C100-FLAG APP-based substrate , V50F and V50W both decreased the Aβ42/40 ratio , while the same substitutions at M51 increased the Aβ42/40 ratio as predicted ( Figure 2B ) .\",\n \"MALDI/TOF mass spectrometry ( MS ) analysis of the corresponding AICD fragment revealed the complete elimination of AICD 49–99 for V50F and of AICD 50–99 for M51F ( Figure 2C ) .\",\n \"Interestingly , in addition to the expected AICD fragments , both V50F and M51F are also cleaved to a minor extent after I47 , producing AICD 48–99 .\",\n \"The reason for this is unknown , although γ-secretase may be compensating for reduced cleavage through one of the two major pathways .\",\n \"Previous MS studies have demonstrated that the majority of Aβ47 is eventually processed to Aβ40 , through an Aβ43 intermediate ( Matsumura et al . , 2014 ) .\",\n \"We obtained similar results measuring secreted Aβ after transiently transfecting HEK cells with full-length APP containing mutations at V50 or M51 .\",\n \"Here , all mutations tested at V50 caused a reduction in the Aβ42/40 ratio , although none as robustly as the bulky aromatic amino acids Phe , Tyr and Trp ( Figure 2D ) .\",\n \"And while mutations of smaller amino acids at M51 caused modest reductions in the Aβ42/40 ratio , aromatic substitutions here all caused substantial increases in the Aβ42/40 ratio ( Figure 2E ) .\",\n \"As predicted by our model , there was a significant reduction in Aβ42 production for the V50 aromatic substitutions , with little change in the Aβ40 levels ( Figure 2F ) .\",\n \"There was a similar predicted reduction in Aβ40 for the M51 aromatic mutants; however , we also see an increase in Aβ42 levels here ( Figure 2F ) , likely because the normally less used Aβ48 → 42 pathway is compensating for the reduced flux through the preferred Aβ49 → 40 pathway , as these mutants all produced roughly equivalent amounts of total Aβ ( Figure 2G ) .\",\n \"Because each tripeptide cleavage event requires the reading of three amino acids of substrate at a time ( as dictated by the three S’ pockets ) , we reasoned we should be able to predictably shift the Aβ42/40 ratio by placing a Phe in the P2’ position at each tripeptide cleavage event along the two major pathways ( Figure 3A ) .\",\n \"As expected , Phe substitutions at V44 and I47 decreased the Aβ42/40 ratio , while Phe mutations at I45 and T48 increased the Aβ42/40 ratio ( Figure 3B and C ) .\",\n \"The predicted Aβ42/40 shifts were nearly identical whether Aβ levels were measured from an in vitro assay ( Figure 3B ) or from a cell-based assay ( Figure 3C ) .\",\n \"These results are of particular note , as I45F is a known FAD mutation ( Guerreiro et al . , 2010 ) , likely indicating that the mechanism of this mutation is the Phe positioned in the P2’ position at the Aβ43 cut site , blocking cleavage of APP through the less pathogenic Aβ49 → 40 pathway and favoring the pathogenic Aβ48 → 42 pathway .\",\n \"The Phe at I45 also lies in the S3’ position for Aβ42 cleavage , likely making the precursor to Aβ42 production a better substrate for γ-secretase through a favorable P3’-S3’ interaction .\",\n \"Mutating A42 to Phe completely blocked the formation of Aβ40 in agreement with our model ( Figure 3D ) , while still allowing for the production of other Aβ species ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 17578 . 005Figure 3 . Phenylalanine mutations at P2’ positions predictively shift the Aβ42/40 ratio .\",\n \"( A ) Schematic diagram of Phe mutations at P2’ positions at cut sites within the TMD of APP and the expected Aβ42/40 changes compared to WT .\",\n \"( B ) In vitro Aβ42/40 ratios from γ-secretase cleavage of recombinant C100-FLAG substrates .\",\n \"Aβ measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .\",\n \"Mean ± SD , n = 3 , t-test *<0 . 05 , **<0 . 01 .\",\n \"( C ) Aβ42/40 ratios from Aβ secreted from HEK cells .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .\",\n \"( D ) Aβ40 levels measured from the conditioned media of HEK cells transfected with WT or A42F APP .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 .\",\n \"( E ) Western blot analysis of the AICD fragments generated from V44F , I45F , I47F and T48F in vitro .\",\n \"Total AICD was measured with anti-FLAG M2 antibody ( green ) .\",\n \"Aβ49 → Aβ40 pathway preference was measured with an antibody specifically recognizing the N-terminus of AICD 50–99 fragment ( red ) .\",\n \"( F ) MALDI/TOF MS confirmation of the AICD fragments measured in ( E ) : V44F ( AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7020 . 8 ) , V45F ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6906 . 2; AICD 49–99 , expected mass: 7018 . 8 , observed mass 7020 . 1 ) , I47F ( AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7019 . 5 ) , T48F ( AICD 50–99 , expected mass: 6905 . 6 , observed mass: 6907 . 4; AICD 49–99 , expected mass: 7018 . 8 , observed mass: 7019 . 5 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 00510 . 7554/eLife . 17578 . 006Figure 3—figure supplement 1 . Total Aβ from the A42F mutant .\",\n \"( A ) Total secreted Aβ was measured by ELISA from transiently transfected HEK cells with full length WT and A42F APP .\",\n \"Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 006 Although we expected the Phe substitutions at V44 , I45 , I47 and T48 to be acting independently of the pathway chosen at initial ε cleavage , an alternative explanation for the above results is that these mutations instead influence ε cleavage in favor of the final Aβ products measured .\",\n \"To investigate this possibility , we utilized an antibody that specifically recognizes the free N-terminus of AICD 50–99 ( Chávez-Gutiérrez et al . , 2012 ) , and therefore the initiation of the Aβ49 → 40 pathway .\",\n \"Surprisingly , all four of these mutants actually caused a shift in ε cleavage toward the initiation of the opposite pathway .\",\n \"Although V44F and I47F cause reductions in the Aβ42/40 ratio , they shifted initial ε cleavage away from the Aβ49 → 40 pathway—nearly completely eliminating AICD 50–99 .\",\n \"And while I45F and T48F increased the Aβ42/40 ratio , they shifted ε cleavage more in favor of AICD 50–99 compared to WT ( Figure 3E ) .\",\n \"These AICD species were confirmed by mass spectrometry ( Figure 3F ) .\",\n \"To explore further the apparent ability of tripeptide cleavage preference to dissociate the normal connection between initial ε cleavage and final γ cleavages , we made double Phe mutants in which the ε cut site is controlled with a Phe at either V50 or M51 , while placing a conflicting Phe at V44 , I45 , I47 or T48 in the initial pathway .\",\n \"Measuring Aβ secreted from transfected HEK cells , we clearly saw the double mutants behave almost identically to the single point mutants N-terminal to the ε cut site ( Figure 4A and B ) .\",\n \"MS of the AICD fragments revealed the expected and complete blocking of AICD 49–99 or AICD 50–99 for the V50F and M51F containing double mutants , respectively ( Figure 4C ) .\",\n \"Together these data suggest final γ cleavages can be completely uncoupled from initial ε cleavages . 10 . 7554/eLife . 17578 . 007Figure 4 . Phenylalanine mutations in the P2’ position of the last read tripeptide segment dictates final pathway preference .\",\n \"( A ) Aβ42/40 ratios from HEK cells of V44F-M51F and I47F-M51F double mutants behave like single Phe mutants V44F and I47F , respectively .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 .\",\n \"( B ) Aβ42/40 ratios from HEK cells of I45F-V50F and T48F-V50F double mutants behave like single Phe mutants I45F and T48F , respectively .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test *<0 . 05 , **<0 . 01 , ***<0 . 001 , ****<0 . 0001 .\",\n \"( C ) MALDI/TOF MS conformation of the elimination of AICD 49–99 and AICD 50–99 for the V50F and M51F containing double Phe mutants , respectively .\",\n \"V44F-M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7030 . 7; AICD 47–99 expected mass: 7249 . 0 , observed mass: 7253 . 7 ) , I45F-V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6950 . 1; AICD 48–99 , expected mass: 7167 . 9 , observed mass: 7164 . 6 ) , I47F-M51F ( AICD 49–99 , expected mass: 7034 . 8 , observed mass: 7032 . 2; AICD 47–99 , expected mass: 7283 . 0 , observed mass: 7280 . 1 ) , T48F-V50F ( AICD 50–99 , expected mass: 6953 . 8 , observed mass: 6949 . 4; AICD 48–99 , expected mass: 7214 . 0 , observed mass: 7209 . 4 ) .\",\n \"( D ) Aβ42/40 ratios from HEK cells transfected with double Phe mutations in tandem .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 , ***<0 . 001 , ****<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 007 Single Phe mutations at V46 and L49 both caused modest increases in the Aβ42/40 ratio .\",\n \"We predicted that since these mutations do not occupy the S2’ pocket for either major pathway , V46F and L49F when paired with a neighboring Phe mutation should behave like its neighboring Phe mutant alone .\",\n \"Indeed , L49F-V50F had a reduced Aβ42/40 ratio compared to WT like V50F alone , while T48F-L49F had an elevated Aβ42/40 ratio comparable to T48F ( Figure 4D ) .\",\n \"Likewise , V46F-I47F displayed a reduced Aβ42/40 ratio similar to I47F alone , while I45F-V46F had a drastically increased Aβ42/40 ratio like I45F alone ( Figure 4D ) .\",\n \"To this point , aromatic amino acids placed in the P2’ position at each tripeptide cleavage site within the TMD of APP caused a predictable outcome without exception .\",\n \"We therefore reasoned that placing two phenylalanines in tandem , such that there is a Phe in the P2’ position at both major ε cut sites , should reduce overall cleavage .\",\n \"As predicted , a double mutant of V50F-M51F caused a sharp reduction in total AICD formation compared to WT and the V50F and M51F single point mutations alone ( Figure 5A ) .\",\n \"However , AICD formation from V50F-M51F cleavage was not completely abolished; rather , its rate of production was markedly reduced ( Figure 5B ) .\",\n \"This suggests γ-secretase has a means by which to overcome two aromatic amino acids in a row , which would seemingly conflict with its cleavage preferences . 10 . 7554/eLife . 17578 . 008Figure 5 . Phenylalanine blocking mutations at both ε cleavage sites reduces APP cleavage but not binding to γ-secretase .\",\n \"( A ) Western blot of γ-secretase cleavage of WT , V50F , M51F and V50F-M51F C100-FLAG .\",\n \"Duplicates from each substrate represent separate independent data points .\",\n \"* denotes a degradation product which co-purified with the substrate .\",\n \"( B ) Cleavage of WT and V50F-M51F C100-FLAG over time .\",\n \"( C ) Co-immunoprecipitation of Myc-tagged WT or V50F-M51F C100 substrate .\",\n \"Duplicates are from separate pull-down experiments .\",\n \"* antibody light chain .\",\n \"( D ) Competitive cleavage of WT C100-FLAG by WT C100-Myc or V50F-M51F C100-Myc .\",\n \"( E ) Aβ42/40 ratio of the V50F-M51F double mutant .\",\n \"Mean ± SD , n = 3 , t-test , ****<0 . 0001 .\",\n \"( F ) MALDI/TOF MS of the AICD fragment from the V50F-M51F mutant: ( AICD 51–99 , expected mass: 6822 . 5 , observed mass: 6817 . 1; * unknown peak , observed mass: 7030 . 4; AICD 48–99 , expected mass: 7183 . 9 , observed mass: 7179 . 2; AICD 47–99 , expected mass: 7297 . 1 , observed mass: 7292 . 5 , AICD 46–99 , expected mass: 7396 . 2 , observed mass: 7396 . 1 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 008 The observation that both tripeptide cleavage products of APP and transition-state analog inhibitors are noncompetitive inhibitors of γ-secretase suggests that the subsites on γ-secretase for initial substrate binding and subsequent catalysis are spatially separate and distinct , meaning substrate movement is likely required after initial substrate binding but prior to catalysis .\",\n \"This would be in agreement with previous reports suggesting there may be an exosite on γ-secretase to which substrate initially binds prior to translocating to the active site for cleavage ( Kornilova et al . , 2003 , 2005 ) .\",\n \"It would also agree with studies of other intramembrane cleaving proteases , which have proposed substrate movement in the form of helical unwinding is a prerequisite for catalysis ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) .\",\n \"Given that the S’ pockets within the active site are likely the second binding site for substrate , we reasoned that although the V50F-M51F mutant cannot be hydrolyzed as efficiently as WT , it should still be effectively bound by γ-secretase in an initial docking site .\",\n \"Co-IP of WT and V50F-M51F C100 in complex with γ-secretase reveals equal amounts of substrate bound , suggesting both substrates have a similar binding affinity for the enzyme ( Figure 5C ) .\",\n \"Furthermore , myc-tagged V50F-M51F was just as effective as myc-tagged WT C100 at competing for γ-secretase cleavage of FLAG-tagged WT C100 ( Figure 5D ) , again indicating V50F-M51F and WT substrate initially interact with γ-secretase in a similar manner .\",\n \"Together this suggests that the two Phe mutations in V50F-M51F do not affect initial recognition or binding of the C100 substrate , but rather may reduce the stabilization of a cleavable intermediate enzyme-substrate complex by clashing with the S’ pockets in the presenilin active site .\",\n \"We noticed that in addition to being processed more slowly , V50F-M51F also produced a sharply lower Aβ42/40 ratio compared to WT ( Figure 5E ) .\",\n \"To determine why this was occurring , we performed MS on the AICD fragment .\",\n \"Surprisingly , we found that ε cleavage was initiated almost exclusively after I47 ( the very next available cleavage site ) , generating a 48–99 AICD fragment ( Figure 5F ) , indicating γ-secretase is capable of skipping the two phenylalanines to initiate cleavage , albeit at a slower rate .\",\n \"Aβ47 is primarily processed to Aβ40 ( Aβ47 → Aβ43 → Aβ40 ) ( Matsumura et al . , 2014 ) , accounting for the decreased Aβ42/40 ratio .\",\n \"Although γ-secretase usually cleaves APP in increments of three amino acids to produce predominantly Aβ40 and Aβ42 in the mechanism outlined in this study , a seemingly unusual cleavage deviates from the tripeptide preference to produce Aβ38 in appreciable quantities ( Okochi et al . , 2013; Takami et al . , 2009 ) .\",\n \"To determine if production of Aβ38 occurs through the same three S’ pockets used by γ-secretase to achieve tripeptide cleavage , we transiently transfected HEK cells with mutant APP containing a single Phe point mutant at V39 , V40 , I41 or A42 .\",\n \"Measuring secreted Aβ38 revealed that V40F almost completely eliminated Aβ38 production ( Figure 6A ) , demonstrating occupancy of the three S’ pockets is required for Aβ38 formation .\",\n \"V39F and I41F both sharply increased Aβ38 , a result we interpret as the phenylalanine mutations making the precursor to Aβ38 a better substrate for γ-secretase , as the S1’ and S3’ pockets are large and prefer phenylalanine over smaller amino acids ( Esler et al . , 2004 ) .\",\n \"A42F produces about as much Aβ38 as WT , indicative of the lack of a contributory S4’ pocket . 10 . 7554/eLife . 17578 . 009Figure 6 . γ-Secretase preferentially cleaves APP near the helix-destabilizing Gly-Gly motif .\",\n \"( A ) Aβ38 levels from HEK cells transiently transfected with V39F , V40F , I41F or A42F APP .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 .\",\n \"( B ) Aβ42/40 ratios from V44F-I47F-V50F and I45F-T48F-M51F triple mutants from transiently transfected HEK cells .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test , ****<0 . 0001 .\",\n \"( C ) Aβ38 , Aβ40 and Aβ42 levels from HEK cells transfected with V44F-I47F-V50F and I45F-T48F-M51F triple mutants , the V44F-I45F double mutant and the hexa-mutant V44F-I45F-I47F-T48F-V50F-M51F .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 .\",\n \"( D ) Schematic diagram of sequential Phe mutants in the TMD of APP .\",\n \"( E ) Aβ38 + 40 + 42 secreted from HEK cells transiently transfected with the mutants from ( D ) .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 .\",\n \"( F ) Aβ42/40 ratios from ( E ) .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test *<0 . 05 , ****<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 00910 . 7554/eLife . 17578 . 010Figure 6—figure supplement 1 . HEK cell expression and Aβ production from consecutive phenylalanine APP mutants .\",\n \"( A ) Aβ38 , Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines ( see Figure 6D ) .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 .\",\n \"( B ) Expression levels of consecutive phenylalanine APP mutants from Figure 6D . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 010 During the course of this study , we found that three phenylalanines sequentially mutated in the P2’ positions of each major pathway ( V44F-I47F-V50F and I45F-T48F-M51F ) caused a very strong reduction or elevation in the Aβ42/40 ratio in the predicted direction ( Figure 6B ) .\",\n \"As expected , the shifts were caused by the near complete elimination of Aβ42 for the V44F-I47F-V50F mutant and of Aβ40 for the I45F-T48F-M51F mutant ( Figure 6C ) .\",\n \"Interestingly , in each case , Aβ38 levels were produced in amounts comparable to or greater than WT .\",\n \"This demonstrates that Aβ38 is capable of being produced from both the Aβ49 → 40 and Aβ48 → 42 pathways , in perfect agreement with recent MS studies that identified the precursors to Aβ38 as being either Aβ43 or Aβ42 ( Matsumura et al . , 2014; Olsson et al . , 2014 ) .\",\n \"Surprisingly , when we blocked the production of both Aβ43 and Aβ42 at the same time with a V44F-I45F double mutant we not only did not prevent the production of Aβ38 , but rather Aβ38 levels were drastically increased ( Figure 6C ) .\",\n \"Even after blocking the first six major cleavage sites ( V44F-I45F-I47F-T48F-V50F-M51F ) of APP , we still observe elevated Aβ38 compared to WT , demonstrating γ-secretase is fully capable of traversing multiple phenylalanines within APP to find the especially labile amide bond between G38 and V39 .\",\n \"It is likely that the helix-destabilizing Gly-Gly motif at G37 and G38 is the reason for this observation , making the G38-V39 bond particularly accessible for cleavage .\",\n \"Next , we attempted to determine how many phenylalanines in a row γ-secretase was capable of skipping by taking advantage of the fact that the GG motif apparently allows for γ-secretase to deviate from normal sequential tripeptide cleavage .\",\n \"Astonishingly , even after increasing the number of phenylalanines in a row to eight , γ-secretase was still able to produce Aβ above mock transfected levels ( Figure 6D , E ) .\",\n \"The V44F⇒M51F mutant produced mostly Aβ38 ( Figure 6—figure supplement 1 ) , in what may be a single endoproteolytic cleavage event , although we have not been able to obtain enough AICD for MS confirmation .\",\n \"Predictably , the Aβ42/40 ratios for these mutants follow a pattern expected if γ-secretase cleaves at the next available site after each additional Phe ( Figure 6F ) .\",\n \"Using our newfound knowledge of the basic cleavage mechanism of γ-secretase , and our ability to precisely control it with phenylalanine mutations , we next decided to investigate the mechanism of FAD mutations within the TMD of APP , which all increase the Aβ42/40 ratio to different degrees .\",\n \"There are more than a dozen missense FAD mutations targeting this region of APP ( Figure 1A ) , with the majority being located N-terminal and downstream of ε cleavage .\",\n \"To date , the currently accepted explanations for how these mutations increase the Aβ42/40 ratio are:\",\n \"1 ) affecting the positioning/helical stability of the γ-secretase bound APP TMD such that initial ε cleavage is shifted toward T48 , thus favoring the production of Aβ42 ( Chávez-Gutiérrez et al . , 2012; Chen et al . , 2014; Dimitrov et al . , 2013 ) ; or\",\n \"2 ) influencing the reaction kinetics of γ-secretase’s processing of APP , leading to incomplete carboxy-trimming and therefore increased Aβ42 over the more processed Aβ38 ( Chávez-Gutiérrez et al . , 2012 ) ( Figure 7A ) .\",\n \"As demonstrated above with the I45F FAD mutant , we now show that a third possible mechanism exists , in that sequence-specific cleavage preferences can uncouple initial ε cleavage and final γ cleavages of APP ( Figure 7A ) . 10 . 7554/eLife . 17578 . 011Figure 7 . The tripeptide cleavage mechanism of γ-secretase and the effect of APP transmembrane domain FAD mutations .\",\n \"( A ) The three mechanisms by which FAD mutations within the TMD of APP increase the Aβ42/40 ratio .\",\n \"1 ) Mutations shift initial ε cleavage towards the Aβ42 pathway .\",\n \"2 ) Mutations reduce cleavage of the third cleavage event , producing more Aβ42 over the more processed Ab38 .\",\n \"3 ) Cleavage specific preferences cause switching from the Aβ40 to the Ab42 pathway , as exemplified by the I45F FAD mutant .\",\n \"( B ) The Aβ42/40 ratio of each FAD mutation with and without an additional V50F mutation to control the ε cleavage site .\",\n \"The majority of mutations are rescued by the V50F substitution suggesting that these FAD mutations increase the Aβ42/40 ratio by influencing ε cleavage and/or affecting carboxy-trimming .\",\n \"I45F , I45T and T48P retain significantly elevated ratios , indicating these mutants dissociate initial ε and final γ cleavages .\",\n \"Aβ levels measured by 4G8 ELISA .\",\n \"Mean ± SD , n = 3 , t-test ***<0 . 001 , ****<0 . 0001 . ( C ) The tripeptide cleavage mechanism of γ-secretase .\",\n \"After initial substrate binding , we speculate that the helical TMD of substrate unwinds into the active site of presenilin ( PSEN ) where it is stabilized by the three S’ pockets in the catalytic pocket prior to cleavage .\",\n \"Successive carboxy tripeptide trimming occurs until the eventual release of Aβ peptide .\",\n \"( D ) γ-secretase cleavage of the transmembrane domain of Notch from Okochi et al , 2002 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01110 . 7554/eLife . 17578 . 012Figure 7—figure supplement 1 . APP FAD mutant panel measured by 6E10 ELISA .\",\n \"( A ) The samples from Figure 7B were measured by ELISA with the 6E10 detection antibody , showing similar results to those of the 4G8 ELISA .\",\n \"Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01210 . 7554/eLife . 17578 . 013Figure 7—figure supplement 2 . AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody .\",\n \"( A ) Western blot of the AICD 50–99 fragment for I45F , I45V and I45T FAD mutations .\",\n \"( B ) Quantification of western blot bands from ( A ) .\",\n \"Mean ± SD , n = 3 , t-test **<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 01310 . 7554/eLife . 17578 . 014Figure 7—figure supplement 3 . Secreted Aβ levels from V40F , A42F and V44F .\",\n \"( A ) Aβ38 , Aβ40 and Aβ42 levels measured from V40F , A42F and V44F transfected HEK cells respectively , as well as mock transfected and WT .\",\n \"Aβ levels measured by 6E10 ELISA .\",\n \"Mean ± SD , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 17578 . 014 To determine the prevalence of the two previously proposed mechanisms and to possibly identify additional pathway uncoupling mutants , we made each FAD mutant within the APP TMD alone and as a double mutant with V50F .\",\n \"The V50F mutation would be predicted to block ε cleavage after T48 independent of the FAD mutant’s affect on subsequent cleavage events .\",\n \"We would therefore predict that if an FAD mutant causes an increase in the Aβ42/40 ratio by influencing initial ε cleavage or by affecting subsequent carboxy-trimming along the Aβ48 → 42 pathway , then that same FAD mutant when paired with V50F should produce a reduced Aβ42/40 ratio compared to WT , similar to V50F alone .\",\n \"Conversely , if an FAD mutant affects subsequent cleavage events independent of ε cleavage , causing uncoupling of initial ε cleavage and final γ cleavages , then the FAD-V50F double mutant should retain an elevated Aβ42/40 ratio , similar to the FAD mutant alone .\",\n \"Screening nearly all the FAD mutants within the TMD of APP by this method , we found that the majority were either completely or nearly completely rescued when paired with V50F ( Figure 7B , Figure 7—figure supplement 1 ) , resulting in 42/40 ratios significantly less than WT .\",\n \"This suggests the predominant mechanism of elevating the Aβ42/40 ratio by FAD mutants within the TMD of APP is by shifting the preference of initial ε cleavage from the Aβ40 to the Aβ42 pathway , and/or by influencing carboxy-trimming .\",\n \"This may have been predicted , given that I45F is the only aromatic amino acid mutation to fall in the S2’ pocket of one of the major cleavage pathways .\",\n \"The only other FAD mutation containing an aromatic amino acid , V46F , falls within the S1’ and S3’ pockets for the Aβ42 and Aβ40 pathways , respectively , therefore never clashing with the S2’ pocket and not influencing the Aβ42/40 ratio as a major pathway blocker .\",\n \"However , we found that in addition to I45F , there are two other mutants , I45T and T48P , which appear to dissociate the normal connection between initial pathway preference and final cleavage products .\",\n \"V50F partially rescues the Aβ42/40 ratio when paired with I45T , but remains significantly elevated compared to WT , indicating the I45T mutant both influences initial ε cleavage and uncouples ε from γ cleavages .\",\n \"The change in ε cleavage preference of I45T was verified using the AICD 50–99 specific antibody ( Figure 7—figure supplement 2 ) , showing a small reduction in AICD 50–99 .\",\n \"Interestingly , the I45T mutant reduces the amount of AICD 50–99 comparable to I45V , even though these two mutants display very different Aβ42/40 ratios .\",\n \"This again suggests I45T dissociates cleavage downstream of ε to achieve such a high Aβ42/40 ratio .\",\n \"Exactly how I45T does this is currently unknown and requires further investigation .\",\n \"The T48P-V50F double mutant behaves identically to T48P alone .\",\n \"Given that proline is a helix-breaking amino acid , the T48P mutant may not undergo normal ε cleavage after P48 or L49 .\",\n \"Determining how T48P increases the 42/40 ratio and overcomes the ε controlling V50F mutant will require further investigation .\"\n ],\n [\n \"In this study , we identify that three S’ amino acid binding pockets guide the productive positioning of substrate into the γ-secretase active site , providing the mechanism behind the enzyme’s preferred tripeptide cleavage of APP and pathogenic Aβ production .\",\n \"Based on the data reported herein ( discussed further below ) and numerous other studies of γ-secretase ( Das et al . , 2003; Kornilova et al . , 2003 , 2005 ) and other intramembrane proteases ( Akiyama et al . , 2015; Dickey et al . , 2013; Fluhrer et al . , 2012; Moin and Urban , 2012; Urban and Freeman , 2003; Ye et al . , 2000 ) , we speculate that after initial binding to γ-secretase , substrate must undergo a translocation and/or conformational change in order to bind the three S’ pockets within the active site and subsequently be cleaved by the enzyme ( Figure 7C ) .\",\n \"After initial endoproteolysis , the three S’ pockets guide further carboxy-trimming of the retained Aβ species until it is short enough to dissociate from the complex , producing predominantly Aβ38 , Aβ40 and Aβ42 .\",\n \"More than a decade ago , SAR studies of γ-secretase-targeting transition-state analogs putatively assigned three S’ pockets to the active site of the enzyme ( Esler et al . , 2004 ) .\",\n \"Transition-state analogs containing only two amino acids for S’ pocket binding inhibited γ-secretase less effectively , indicating that occupancy of the S3’ pocket is required for a strong interaction .\",\n \"This likely provides the reason why γ-secretase only cleaves APP in segments of three or more amino acids , but never two .\",\n \"The preference for cleaving three amino acids originates from the lack of a contributory fourth S’ pocket .\",\n \"A fourth amino acid ( no matter the size ) added to a transition state inhibitor neither increased nor decreased the inhibitors potency ( Esler et al . , 2004 ) , suggesting a fourth S’ pocket doesn’t contribute to γ-secretase-inhibitor or -substrate interactions .\",\n \"To support our proposed model , we exploited the fact that the second of the three S’ pockets is apparently too small to readily accommodate an aromatic amino acid .\",\n \"We generated dozens of substrates containing aromatic amino acid substitutions in the P2’ positions at each cleavage site along the Aβ40 or Aβ42 pathways , selectively blocking each individual cleavage event .\",\n \"Without exception we were able to predict the shift in the Aβ42/40 ratio .\",\n \"This was accomplished both in vitro with purified γ-secretase and recombinant C100 substrate as well as in a cell-based assay , transiently transfecting mutant full-length APP in HEK cells and measuring secreted Aβ .\",\n \"The same predicted Aβ42/40 ratio changes were observed in vitro and in the cell-based assay whether we measured Aβ ( 6E10 detection antibody ) or Aβ plus p3 products ( 4G8 detection antibody ) by ELISA .\",\n \"Together this demonstrates we are probing the fundamental mechanism by which γ-secretase cleaves APP , irrespective of mutant effects on cellular localization , γ-secretase’s interaction with activity-modulating proteins/lipids within the cell or any artifacts that may arise from more artificial in vitro assays .\",\n \"Although we are unable to measure every cleavage product from the ~70 mutant forms of APP we generated , we do note that every time a Phe was placed in the P2’ position of a cleavage product that we could readily and directly measure , there were almost negligible amounts of that product formed .\",\n \"For example , in the cell-based assay , V40F , A42F and V44F generated levels of Aβ38 , Aβ40 and Aβ42 , respectively , that were actually less than mock transfected levels ( Figure 7—figure supplement 3 ) .\",\n \"These Aβ levels are orders of magnitude less than that from WT APP transfected cells , although we cannot say whether these low levels of Aβ species were produced from endogenous HEK cell APP or from our transfected mutants .\",\n \"Similarly , we were unable to detect by MS any AICD products containing a Phe in the P2’ position .\",\n \"Together , these data suggest that aromatic amino acids may be completely excluded from the S2’ pocket and further demonstrates that substrate occupancy of the three S’ pockets is an absolute requirement for catalysis .\",\n \"It is likely that phenyalanine substitutions at various positions along the transmembrane domain of APP influence the general structure and/or helical stability of the substrate .\",\n \"This could affect the manner in which these substrates interact with γ-secretase .\",\n \"Given that we are able to accurately predict the Aβ42/40 ratio without exception for the dozens of mutants used in this study , we expect that cleavage preferences dictated by the presence of aromatic amino acids in the P2’ position are overriding any affect these mutations have on substrate helical structure/stability and any altered manner in which these mutant substrates initially interact with the enzyme .\",\n \"This is directly supported in Figure 3 where V44F , I45F , I47F and T48F all shift initial ε cleavage in favor of the opposite pathway relative to the final cleavage products measured and originally predicted by our model .\",\n \"We present several lines of evidence suggesting that substrate movement and/or a substrate conformational change after initial enzyme binding is an important step in γ-secretase’s catalytic mechanism .\",\n \"We identify that like transition-state analogs , tripeptide cleavage products are noncompetitive inhibitors of γ-secretase , albeit very weak inhibitors .\",\n \"By definition this means the binding sites on γ-secretase for initial substrate binding and subsequent catalysis are spatially separate , requiring substrate movement after initial binding to be an integral part of γ-secretase’s catalytic mechanism .\",\n \"For rhomboid , a recent study demonstrates that product-mimicking peptide aldehydes are non-competitive inhibitors of this serine intramembrane protease ( Cho et al . , 2016 ) , exactly like tripeptide products and transition-state analog inhibitors are for γ-secretase , suggesting a common two-step mechanism between these two intramembrane proteases .\",\n \"Additionally , the double Phe mutant V50F-M51F , which is predicted by our model to sterically clash with the three S’ pockets in the active site , is still efficiently bound to γ-secretase even though it is processed less efficiently than WT .\",\n \"Given that this mutant effectively competes for γ-secretase processing of other substrates , it must bind to the initial docking site on the enzyme for substrate .\",\n \"This suggests that binding to the S’ pockets is the final step in substrate recognition and positioning within the enzyme prior to catalysis .\",\n \"Furthermore , we are unable to prevent the formation of Aβ38 by specifically blocking the production of the known Aβ38 precursors Aβ42 and Aβ43; instead , paradoxically the V44F-I45F double mutant increases Aβ38 production .\",\n \"It is likely that the helix-destabilizing Gly-Gly motif at G37 and G38 is the reason for this observation .\",\n \"Local helical unwinding around this position probably makes the amide bond between G38 and V39 particularly accessible for cleavage , and this may be the reason for γ-secretase’s normal deviation from the preferred tripeptide cleavage for Aβ38 production .\",\n \"However , further investigation , including quantification of the intramembrane peptide products by mass spectrometry , will be required to prove this .\",\n \"In the absence of a γ-secretase—substrate co-complex structure , it will not be possible to definitively prove the existence of a partially unwound substrate intermediate .\",\n \"Conceivably , it should be possible to capture such an intermediate with a transition-state analog covalently linked to the C-terminus of an APP- or notch-based helical substrate .\",\n \"Given that tripeptide cleavage is dictated by the three S’ pockets in the active site of presenilin , we expect that other γ-secretase substrates will be similarly cleaved preferentially in increments of three amino acids , while skipping aromatic amino acids that fall in the S2’ pocket along the way , and with cleavage occurring preferentially in helix-destabilized regions .\",\n \"This is important to note given that many of γ-secretase’s substrates naturally contain aromatic amino acids .\",\n \"The TMD of notch , for example , naturally contains three phenylalanines .\",\n \"We know from a previous MS study that these phenylalanines are skipped by γ-secretase in a pattern consistent with our model ( Figure 7D ) ( Okochi et al . , 2002 ) .\",\n \"In a recent study , we have demonstrated that substrates with large ectodomains have a reduced binding affinity for γ-secretase due to steric clashing with the nicastrin component of the γ-secretase complex ( Bolduc et al . , 2016 ) .\",\n \"Based on this , and results from our current study , we might expect that γ-secretase can chose its substrates through a complex interplay between ectodomain length , helical TMD stability and the sequence of amino acids ( specifically aromatic amino acids ) within substrate TMD .\",\n \"There may even exist non-substrates containing short ectodomains but having stable helices that are further protected from cleavage by sequential stretches of aromatic amino acids .\",\n \"Whether such non-substrates exist , and the relationship between ectodomain size , helical stability and amino acid sequence will require further investigation .\",\n \"We show that the majority of FAD mutants within the TMD of APP primarily increase the Aβ42/40 ratio by changing ε cleavage , favoring the Aβ48 → 42 pathway .\",\n \"This is mostly in agreement with previous studies ( Chávez-Gutiérrez et al . , 2012; Chen et al . , 2014; Dimitrov et al . , 2013; Quintero-Monzon et al . , 2011 ) .\",\n \"However , we also identify a new mechanism by which certain FAD mutations can increase the production of pathogenic Aβ species .\",\n \"Here , final γ cleavages are uncoupled from initial pathway preference determined by ε cleavage .\",\n \"In the case of the I45F FAD mutation , the bulky Phe sterically clashes with the S2’ pocket of γ-secretase at the Aβ43 cleavage site , blocking its cleavage and the subsequent production of Aβ40 .\",\n \"It is likely that the positioning of the Phe in the S3’ pocket of the Aβ42 cleavage also enhances γ-secretase proteolysis at this position through a favorable S3’-P3’ interaction .\",\n \"These two interactions likely combine to account for the fact that no other mutation within the TMD of APP produces as much Aβ42 as I45F .\",\n \"This is the most severe APP FAD mutation , with an onset of clinical AD at 31 years of age ( Guerreiro et al . , 2010 ) .\",\n \"There are at least two additional FAD mutations , I45T and T48P , that appear to dissociate the ε and γ cleavages .\",\n \"How these mutations accomplish this is currently unknown .\",\n \"Our data also help explain previous observations in the literature .\",\n \"Prior to the identification of presenilin as being the protease responsible for γ-secretase activity ( Wolfe et al . , 1999 ) , Lichtenthaler et al . performed a phenylalanine scanning study of the TMD of APP .\",\n \"In their study , they observed the exact same Aβ42/40 shifts identified here for several of the same mutants ( Lichtenthaler et al . , 1999 ) .\",\n \"Later , Sato et al . found that γ-secretase was unable to cleave through stretches of 3–5 consecutive tryptophans inserted into the TMD of APP ( Sato et al . , 2005 ) .\",\n \"These observations are now explained by the determination that the small S2’ pocket of γ-secretase cannot accommodate aromatic amino acids .\",\n \"With the identification of the key role the three S’ pockets play in γ-secretase’s cleavage mechanism , several important new questions are raised .\",\n \"The exact locations of the three S’ pockets in presenilin are currently unknown .\",\n \"Additionally , the identity of the initial substrate-binding site on presenilin is unknown .\",\n \"The resolution of enzyme—substrate and/or -inhibitor co-complexes by cryo-EM will be informative in this regard .\",\n \"All enzymatic assays in this study utilized purified γ-secretase complex containing presenilin-1 .\",\n \"It will be interesting to see if presenilin-2 has similar substrate cleavage preferences .\",\n \"A major unsolved question pertains to the mechanism by which substrate TMD is repositioned within γ-secretase after each cleavage event in order to be close enough to the active site for the next round of catalysis to occur .\",\n \"Is this a ratcheting or sliding motion ?\",\n \"Is this an active process or based on Brownian motion ?\",\n \"At present , we do not know how γ-secretase is capable of skipping stretches of several phenylalanines in a row .\",\n \"Determining how γ-secretase accomplishes this may help elucidate how substrate moves during normal sequential cleavage .\",\n \"Now that we have identified several key aspects of γ-secretase’s substrate recognition and cleavage mechanisms , as well as provide valuable new tools for future structural and biochemical studies , we should be able design experiments to elucidate some of γ-secretase’s remaining unanswered functional questions .\",\n \"The answers from which should have further broad implications for our understanding of Alzheimer’s disease and the development of safe and effective therapeutics targeting γ-secretase function .\"\n ],\n [\n \"The following antibodies were used: α-Myc ( 9E10 Santa Cruz #sc-40 ) , α-Flag ( M2 Sigma #F3165 ) , α-Nct ( Cell Signaling 3632S ) , α-GAPDH ( Cell Signaling Technology , ab125247 ) , α-APP ( C7 ) , α-AICD 50–99 ( Rb ) was a kind gift from Philip Szekeres at Eli Lilly , α-Ms 800nm ( Licor Bio 926–32212 ) and α-Rb 680nm ( Licor Bio 926–68021 ) .\",\n \"Tripeptides were synthesized by Anaspec corp .\",\n \"Total brain lipid extract was from Avanti Polar Lipids ( #131101 ) .\",\n \"The following Aβ ELISA kits were used: 4G8 ( Meso Scale Diagnostics , K15199E ) or 6E10 ( Meso Scale Diagnostics , K15200E ) for cell-based assays; Aβ40 ( #KHB3482 ) and Aβ42 ( #KHB2442 ) ELISA kits from Invitrogen for in vitro assays .\",\n \"All mutant forms of C100 FLAG or full-length APP were generated by site directed mutagenesis of either C100-FLAG in pET22b or full-length WT APP in the pCMV695 plasmid .\",\n \"Adherent HEK cells were cultured in complete growth media: Dulbecco’s Modified Eagle’s Medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) , 2 mM L-glutamine , 10 Units/mL penicillin , and 10 µg/mL streptomycin .\",\n \"For transfection , adherent HEK cells were seeded in six-well dishes at a density of 5x105 cells per well .\",\n \"Transfection was carried out with Lipofectamine 3000 reagent in serum-free conditions with Opti-MEM I . Cells were incubated for 24 hr , at which time conditioned media was harvested for ELISA and cells were harvested for western blot .\",\n \"Suspension HEK cells were cultured in 100 mL of unsupplemented Freestyle 293 media ( Life Technologies , 12338-018 ) with shaking at 125 rpm , and passaged at a density of 2 × 106 cells/mL .\",\n \"For transfection , suspension HEK cultures were grown to a density of 2 × 106 cells/mL .\",\n \"Media was replaced with fresh Freestyle 293 media .\",\n \"5 mL Freestyle 293 , 150 µg of γ-secretase vector containing presenilin 1 ( provided by Yigong Shi ) , and 450 µg of 25-kDa linear polyethylenimines ( PEI ) was mixed and incubated for 30 min at room temperature .\",\n \"The DNA/PEI solution was then added to the HEK culture and cells were grown for ~60 hrs prior to harvesting .\",\n \"γ-secretase was purified as previously described ( Fraering et al . , 2004; Osenkowski et al . , 2009 ) C100-FLAG substrates were expressed in BL21 E . coli for 3 hrs at 37°C after induction with 1 mM IPTG .\",\n \"Cells were then pelleted and lysed by French press in 50 mM HEPES pH 7 . 0 , 1% Triton X-100 detergent .\",\n \"FLAG-tagged substrates were then isolated by immunoprecipitation for 3 hrs at 4°C with anti-FLAG M2 beads from Sigma .\",\n \"Substrates were then eluted from the beads with 100 mM glycine pH 2 . 5 , 0 . 25% NP40 prior to being neutralized with tris buffer and stored at -80°C .\",\n \"Purified γ-secretase was incorporated into vesicles by first dissolving total brain lipid extract ( 1 . 25 mM final ) in 50 mM HEPES pH 7 . 0 , 150 mM NaCl , 0 . 25% CHAPSO .\",\n \"γ-Secretase ( 5–30 nM final concentration ) was then added to the solution and detergent removed by mixing SM-2 biobeads ( 62 mg/mL ) ( Bio-Rad ) with the lipid/detergent/enzyme solution for two hrs at 4°C .\",\n \"Biobeads were removed from the newly formed proteoliposomes and reactions were initiated with the addition of purified recombinant substrate C100-FLAG substrate .\",\n \"Reactions were quenched with SDS loading dye for western blot or centrifuged for ELISA or mass spectrometry on Aβ or AICD fragments , respectively .\",\n \"For inhibition studies , tripeptide fragments or inhibitors were dissolved in DMSO prior to being diluted into the reaction buffer .\",\n \"The concentration of C100-FLAG used in all in vitro assays was 500 nM unless otherwise stated .\",\n \"Conditioned media from HEK cells transfected with WT or mutant APP was assayed for Aβ by 4G8 or 6E10 Aβ ELISA kits from Meso Scale Diagnostics .\",\n \"Aβ levels were measured by both 6E10 and 4G8 ELISA for each data point , yielding nearly identical results .\",\n \"In vitro assay Aβ was measured using Aβ40 and Aβ42 ELISA kits from Invitrogen .\",\n \"All ELISAs were performed according to the manufacture’s protocols .\",\n \"Purified γ-secretase ( 5 nM final concentration ) was preincubated in assay buffer ( 50 mM HEPES pH 7 . 0 , 150 mM NaCl , 0 . 25% CHAPSO , 0 . 1% DOPC and 0 . 025% DOPE , 2% BSA ) in the presence of 2 μM III-31C for 1 hr at room temperature .\",\n \"Purified WT or mutant C100-Myc ( 20 nM final concentration ) was then incubated with γ-secretase for 1 hr prior to pull down with anti-HA magnetic affinity beads for 4 hr with mixing at room temperature .\",\n \"The immunoprecipitated complex was then washed three times and eluted with SDS loading buffer prior to western blot with an anti-Myc antibody .\",\n \"Tripeptide IC50 inhibition was fit to:vivo=1/ ( 1+[ I ]IC50 ) where vi is the initial velocity in the presence of inhibitor at concentration [I] and vo is the initial velocity in the absence of inhibitor .\",\n \"Tripeptide inhibition was globally fit to the following noncompetitive equation:v= Vmax[S][ S ] ( 1+[ I ]Kii ) +Km ( 1+[ I ]Ki ) where , v is the initial rate , Ki is dissociation constant for inhibitor binding to free enzyme , Kii is the dissociation constant for inhibitor binding to the enzyme-substrate complex .\",\n \"Inhibitor cross-competition was globally fit to:1vij=1/v0 ( 1+[ I ]Ki+[ J ]Kj+[ I ][ J ]αKiKj ) where , vij is the initial rate in the presence of inhibitors , v0 is the initial rate in the absence of inhibitor , Ki and Kj are the dissociation constants for inhibitors I and J , respectively .\",\n \"α = ∞ for inhibitors which bind in a mutually exclusive fashion , while α = 1 for inhibitors which have distinct binding sites .\",\n \"Conditioned media was collected from transiently-transfected adherent HEK cells .\",\n \"Each well of an uncoated 96-well multi-array plate ( Meso Scale Discovery , #L15XA-3 ) was coated with 30 µL of a PBS solution containing 3 µg/mL of 266 capture antibody ( Elan ) , and incubated at room temperature overnight .\",\n \"A detection antibody solution was prepared with 3D6B detection antibody ( Elan ) , 100 ng/mL Streptavidin Sulfo-TAG ( Meso Scale Discovery , #R32AD-5 ) , and 1% MSD Blocker A ( #R93BA-4 ) in wash buffer ( #R61TX-1 ) .\",\n \"Following overnight incubation , 25 µL/well of sample , followed by 25 µL/well of detection antibody solution were incubated for 2 hr at room temperature with shaking at >300 rpm , washing wells with wash buffer between incubations .\",\n \"Plate was read and analyzed according to manufacturer protocol .\",\n \"Following an in vitro proteoliposome activity assay , AICD-FLAG products were isolated by immunoprecipitiation with anti-FLAG M2 magnetic beads from Sigma .\",\n \"Completed reactions were incubated with 50 µL of M2 beads in 10 mM MES pH 6 . 5 , 10 mM NaCl , 0 . 05% DDM detergent in 500 µL volumes overnight at 4°C .\",\n \"AICD was then eluted from the beads with acetonitrile:water ( 1:1 ) with 0 . 1% trifluoroacetic acid .\",\n \"MALDI/TOF mass spectrometry was performed with sinapinic acid matrix on a calibrated ultraflextreme MALDI/TOF/TOF from Bruker in linear mode .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["γ-secretase is responsible for the proteolysis of amyloid precursor protein ( APP ) into short , aggregation-prone amyloid-beta ( Aβ ) peptides , which are centrally implicated in the pathogenesis of Alzheimer’s disease ( AD ) .","Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD , the precise mechanism by which γ-secretase produces Aβ has remained elusive .","Herein , we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme’s active site , providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments .","Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis , suggesting a conformational change in substrate may be required for cleavage .","We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer’s disease mutations within APP increase the production of pathogenic Aβ species ."],"string":"[\n \"γ-secretase is responsible for the proteolysis of amyloid precursor protein ( APP ) into short , aggregation-prone amyloid-beta ( Aβ ) peptides , which are centrally implicated in the pathogenesis of Alzheimer’s disease ( AD ) .\",\n \"Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD , the precise mechanism by which γ-secretase produces Aβ has remained elusive .\",\n \"Herein , we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme’s active site , providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments .\",\n \"Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis , suggesting a conformational change in substrate may be required for cleavage .\",\n \"We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer’s disease mutations within APP increase the production of pathogenic Aβ species .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Individuals with Alzheimer’s disease generally have deposits known as “amyloid plaques” in the brain .","These plaques are made up of a mixture of molecules called amyloid beta peptides that clump together and are thought to be a key cause of the disease .","The amyloid beta peptides vary in size; the larger peptides tend to be more prone to forming clumps than the smaller ones and are thus more toxic to the brain .","An enzyme called gamma-secretase makes amyloid beta peptides by cutting up a protein called APP .","Proteins are made of chains of building blocks called amino acids and studies using a technique called mass spectrometry show that gamma-secretase cuts APP in segments of three amino acids at a time .","The size of the amyloid beta peptides produced is determined by the positions in APP that gamma-secretase selects to cut .","Therefore , understanding how the enzyme works could provide new opportunities for developing drugs to treat Alzheimer’s disease .","Here , Bolduc et al . found that the human gamma-secretase enzyme has sites that amino acids in APP can bind to that help to guide the enzyme to cut APP by three amino acids at a time .","These binding sites control where the enzyme cuts APP and therefore determines which amyloid peptides are produced .","Previous studies have linked several naturally occurring mutations in the gene encoding APP to inherited forms of Alzheimer’s disease .","Bolduc et al . now reveal that several of these mutations affect the places that gamma-secretase cuts APP to produce amyloid peptides .","These findings may be helpful for developing drugs that could manipulate gamma-secretase to produce smaller , less harmful amyloid peptides .","Gamma-secretase can cut many other proteins , and so a future challenge will be to find out if the enzyme cuts these other proteins in the same way that it cuts APP ."],"string":"[\n \"Individuals with Alzheimer’s disease generally have deposits known as “amyloid plaques” in the brain .\",\n \"These plaques are made up of a mixture of molecules called amyloid beta peptides that clump together and are thought to be a key cause of the disease .\",\n \"The amyloid beta peptides vary in size; the larger peptides tend to be more prone to forming clumps than the smaller ones and are thus more toxic to the brain .\",\n \"An enzyme called gamma-secretase makes amyloid beta peptides by cutting up a protein called APP .\",\n \"Proteins are made of chains of building blocks called amino acids and studies using a technique called mass spectrometry show that gamma-secretase cuts APP in segments of three amino acids at a time .\",\n \"The size of the amyloid beta peptides produced is determined by the positions in APP that gamma-secretase selects to cut .\",\n \"Therefore , understanding how the enzyme works could provide new opportunities for developing drugs to treat Alzheimer’s disease .\",\n \"Here , Bolduc et al . found that the human gamma-secretase enzyme has sites that amino acids in APP can bind to that help to guide the enzyme to cut APP by three amino acids at a time .\",\n \"These binding sites control where the enzyme cuts APP and therefore determines which amyloid peptides are produced .\",\n \"Previous studies have linked several naturally occurring mutations in the gene encoding APP to inherited forms of Alzheimer’s disease .\",\n \"Bolduc et al . now reveal that several of these mutations affect the places that gamma-secretase cuts APP to produce amyloid peptides .\",\n \"These findings may be helpful for developing drugs that could manipulate gamma-secretase to produce smaller , less harmful amyloid peptides .\",\n \"Gamma-secretase can cut many other proteins , and so a future challenge will be to find out if the enzyme cuts these other proteins in the same way that it cuts APP .\"\n]","isConversational":false},"year":{"kind":"string","value":"2016"}}},{"rowIdx":286,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"RGS7/Gβ5/R7BP complex regulates synaptic plasticity and memory by modulating hippocampal GABABR-GIRK signaling"},"id":{"kind":"string","value":"elife-02053-v1"},"sections":{"kind":"list like","value":[["Signaling through G protein-coupled receptors for the inhibitory neurotransmitter GABA ( GABABR ) has been recognized to play key roles in mood , nociception , memory , reward , and movement ( Bowery , 2006; Padgett and Slesinger , 2010 ) .","In the hippocampus , activation of postsynaptic GABABR on pyramidal neurons produces slow inhibitory postsynaptic currents ( sIPSCs ) , which counteract the excitatory influence of ionotropic glutamate receptors to shape neuronal output ( Ulrich and Bettler , 2007; Luscher and Slesinger , 2010 ) .","As a result , GABABR signaling profoundly affects hippocampal synaptic plasticity and has marked effects on memory formation ( Davies et al . , 1991; Wagner and Alger , 1995; Schuler et al . , 2001 ) .","A large share of the postsynaptic inhibitory effect of GABABR stimulation in the hippocampus is mediated by activation G protein-gated inwardly-rectifying K+ ( GIRK/Kir3 ) channels , which inhibit neuronal excitability via hyperpolarizing K+ efflux ( Luscher and Slesinger , 2010 ) .","In the hippocampus , GIRK channels are predominantly formed by GIRK1 and GIRK2 subunits , which co-localize and may interact directly with GABABR protomers ( Koyrakh et al . , 2005; Fajardo-Serrano et al . , 2013 ) .","Activation of GABABR releases G protein βγ subunits , which bind to GIRK channels and increase channel gating ( Padgett and Slesinger , 2010 ) .","Blockade of GABABR or GIRK channels by either pharmacological manipulations or genetic knockout ablates the slow IPSC , and blunts a form of hippocampal synaptic plasticity known as depotentiation ( Luscher et al . , 1997; Chung et al . , 2009 ) .","Conversely , enhanced GABABR-GIRK signaling seen in a mouse model of Down syndrome disrupts both excitatory and inhibitory synaptic plasticity , and is linked to cognitive impairment ( Kleschevnikov et al . , 2004; Cramer et al . , 2010; Cooper et al . , 2012 ) .","GABABR-GIRK signaling is negatively modulated by the Regulators of G protein Signaling ( RGS ) proteins , which accelerate G protein inactivation ( Hollinger and Hepler , 2002; Padgett and Slesinger , 2010 ) .","Among more than 30 RGS genes found in mammalian genomes , the R7 family of RGS proteins ( R7 RGS ) stands out for its prominent roles in a range of fundamental neuronal processes , from vision to motor control to reward-related behavior ( Anderson et al . , 2009 ) .","The four members of this group ( RGS6 , RGS7 , RGS9 and RGS11 ) form heterotrimers with two subunits ( Gβ5 and R7BP ) , and these interactions regulate the localization and/or expression of the complexes ( Anderson et al . , 2009; Jayaraman et al . , 2009 ) .","Previous studies have shown that Gβ5 serves as a central scaffold that bridges the catalytic ( RGS ) and targeting ( R7BP ) subunits and ensures the stability of R7 RGS protein ( Cheever et al . , 2008; Sandiford et al . , 2010; Masuho et al . , 2011 ) .","Elimination of Gβ5 also resulted in dramatic slowing of GIRK channel deactivation kinetics , prolongation of synaptically-evoked slow IPSCs in hippocampal pyramidal neurons , and increased behavioral sensitivity to GABABR stimulation ( Xie et al . , 2010 ) .","However , the identity of the RGS isoform that modulates GABABR-GIRK signaling in hippocampus , as well as the relative impact of the R7BP subunit , are unknown .","Furthermore , the relevance of RGS-dependent modulation of GIRK-dependent signaling to hippocampal circuit function , plasticity , and behavior remain unclear .","In this study , we examined the importance of RGS and R7BP subunits to hippocampal physiology and hippocampal-dependent behavior .","We report that ablation of Rgs7 results in alterations of GABABR-GIRK signaling , disrupts synaptic plasticity in the hippocampus , and impairs contextual learning and memory .","Moreover , the function of the RGS7/Gβ5 complex is fine-tuned by R7BP , which sets the sensitivity range of GABABR-GIRK signaling ."],["We began by characterizing the expression of RGS complex subunits in the mouse hippocampus .","We detected robust expression of RGS6 , RGS7 , R7BP and Gβ5 by western blotting ( Figure 1A ) .","To understand the contribution of individual subunits to GABABR-GIRK signaling , we studied the effects of selective knockout of Rgs6 , Rgs7 , and R7bp in mice .","Elimination of RGS7 dramatically reduced levels of Gβ5 and R7BP in the hippocampus ( Figure 1A , B ) .","In contrast , elimination of RGS6 had no significant effect on the expression of R7BP or Gβ5 .","Similarly , loss of one RGS protein did not affect the expression of the other , or the GIRK channel subunit GIRK2 .","Given the interdependence of subunit expression in R7 RGS complexes ( Chen et al . , 2003; Anderson et al . , 2007; Grabowska et al . , 2008 ) , these results suggested that RGS7 was likely the dominant catalytic subunit in the hippocampus . 10 . 7554/eLife . 02053 . 003Figure 1 . RGS7 and R7BP modulate GABABR-GIRK signaling in cultured hippocampal neurons .","( A ) Western blot analysis of protein expression in hippocampi extracted from wild-type ( WT ) mice , or mice lacking RGS7 ( Rgs7−/− ) , RGS6 ( Rgs6−/− ) or R7BP ( R7bp−/− ) .","( B ) Quantification of Western blotting data , with protein levels arrayed as a function of genotype , ***p<0 . 01 , One-Way ANOVA n = 3 mice .","ND-undetectable .","( C ) Representative normalized traces of GIRK currents evoked by a saturating concentration of the GABABR agonist baclofen ( 100 µM ) .","( D–E )","Rgs6 , Rgs7 , and R7bp knockouts do not affect GIRK current amplitudes ( D ) , desensitization ( E ) and current onset kinetics ( F ) evoked by 100 μM Baclofen .","p>0 . 05 , One-Way ANOVA , n = 8–29 cells for each genotype .","( G ) Current deactivation rate following removal of baclofen was slower in neurons from Rgs7−/− and R7bp−/− mice as compared to WT controls , *p<0 . 05 and **p<0 . 01 vs WT , †††p<0 . 001 Rgs7−/− vs R7bp−/− .","One-Way ANOVA , Bonferroni’s post hoc test , n = 8–29 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 003 We next compared GABABR-GIRK responses in hippocampal pyramidal neurons from Rgs6 , Rgs7 , and R7bp knockout ( −/− ) and wild-type ( WT ) mice ( Figure 1C–F ) .","Application of a saturating concentration ( 100 μM ) of the GABABR agonist baclofen elicited currents with similar maximal amplitudes in neurons of all genotypes ( Figure 1D ) .","We also measured the activation and deactivation kinetics of the baclofen-induced currents .","At this saturating concentration of baclofen , we observed no significant differences between genotypes in the activation phase of the response ( Figure 1C , F ) .","Desensitization of current during the timeframe of agonist application was negligible and similar across genotypes , suggesting that the response at the steady-state is not compounded by the GIRK channel inactivation ( Figure 1C , E ) .","While no change in the current deactivation kinetics was observed in Rgs6−/− neurons , elimination of RGS7 markedly slowed response deactivation ( Figure 1C , G ) .","The rate of GIRK current deactivation in R7bp–/– neurons was also slower than in wild-type neurons , but the effect was substantially smaller than seen in neurons from Rgs7−/− mice ( Figure 1C , G ) .","Importantly , there were no detectable changes in any of the measured response parameters in Rgs6−/− neurons as compared to wild-type , arguing that GABABR-GIRK signaling in hippocampal neurons is modulated by RGS7 and R7BP , but not RGS6 .","Given the observed changes in the kinetics of GIRK channel modulation by GABABR , we next sought to determine the impact of ablating Rgs7 and R7bp on the sensitivity of the GIRK response to GABABR activation .","Increasing baclofen concentrations caused a progressive enhancement in GIRK-mediated currents in all genotypes ( Figure 2A ) .","However , pronounced differences in the concentration-response relationship were evident ( Figure 2B ) .","First , in both Rgs7−/− and R7bp−/− neurons , curves were shifted to the left relative to wild-type neurons , indicating that ablation of RGS7 or R7BP increased GABABR-GIRK coupling sensitivity .","Second , while RGS7 ablation resulted in a largely parallel leftward shift of the curve , its shape in R7bp−/− neurons was markedly steeper .","Indeed , response amplitudes resembled those seen in wild-type neurons at lower agonist concentrations , but at higher concentrations , the responses more resembled those seen in Rgs7−/− neurons . 10 . 7554/eLife . 02053 . 004Figure 2 . Timing and sensitivity of GABABR-GIRK signaling is differentially controlled by RGS7 and R7BP .","( A ) Representative traces of GIRK currents evoked by increasing concentrations of baclofen .","( B ) Dose-response curves fit by Hill equation .","EC50 values are 1 . 48–2 . 14 μM for WT , 0 . 35–0 . 58 μM for Rgs7−/− , and 0 . 65–0 . 81 μM for R7bp−/− ( 95% CI ) .","Hill coefficients were 1 . 48 ± 0 . 17 in WT , 1 . 23 ± 0 . 21 in Rgs7−/− , and 2 . 04 ± 0 . 28 in R7bp−/− .","p<0 . 0001 for difference in EC50 or each curve; p=0 . 04 for difference in Hill coefficients , Extra sum-of-squares F test , n = 10–22 cells .","( C ) Rgs7−/− cultured hippocampal neurons show slower current activation at lower concentrations of baclofen .","***p<0 . 001 vs WT , †††p<0 . 001 for Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells .","( D ) Dependence of GIRK current deactivation kinetics on agonist concentration .","*p<0 . 05 and ***p<0 . 001 vs WT; ††p<0 . 01 , †††p<0 . 001 for Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells .","( E–G )","Differences in the lag times before the onset of response deactivation ( Lag Deactivation ) .","( E ) Representative traces of currents evoked by 100 µM baclofen show Lag time measurement as time between the onset of agonist removal and the point at which 10% of the current deactivated: 1560 , 4900 and 3000 ms for WT , Rgs7−/− , R7bp−/− , respectively .","( F ) Semi-log plot of lag time dependence on concentration .","The data were fitted with a linear regression , R2 = 0 . 66 , 0 . 67 , and 0 . 84; and slopes 619 ± 69 , 1440 ± 151 , and 952 ± 84 for WT , Rgs7−/− , R7bp−/− , correspondingly .","p<0 . 0001 for differences in slopes , two way ANOVA , n = 10–22 cells .","( G ) Comparison of lag times at baclofen concentrations that generated equivalent responses .","Lag times were significantly different in genotypes at saturating ( EC90 ) but not submaximal ( EC60 ) concentrations , *p<0 . 05 and ***p<0 . 001 vs WT , ††p<0 . 01 Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 004 We next tested whether GABABR-GIRK current kinetics exhibited a similar dependence on agonist concentration .","Comparing activation rates across different concentrations of baclofen revealed that onset kinetics were affected by RGS7 ablation only at low baclofen concentrations , whereas no effect of R7BP elimination was seen at any level of GABABR stimulation ( Figure 2C ) .","Since GIRK activation kinetics could be influenced by the G protein deactivation cycle ( Doupnik et al . , 1997; Lambert et al . , 2010 ) , we did not put significant emphasis on the analysis of the differences in the raising phases of the response .","The impact of RGS7 ablation on deactivation rates was consistent at different agonist concentrations ( Figure 2D ) .","In contrast , R7BP ablation significantly affected deactivation kinetics only at higher agonist concentrations , consistent with the effects on response sensitivity ( Figure 2D ) .","Analysis of the responses also revealed that elimination of RGS7 and R7BP resulted in a significant delay between agonist removal and the beginning of current deactivation ( Figure 2E ) .","This lag time showed an exponential dependence on agonist concentration that continued to develop past the saturation point for the maximal GIRK response ( ∼10 μM baclofen ) in all genotypes ( Figure 2F ) .","While there was no difference in lag time between genotypes at non-saturating baclofen concentrations ( ∼EC60 ) , it became pronounced as the current response reached saturation ( ∼EC90 ) ( Figure 2G ) .","Combined with the observations that GABABR continues to increase the amount of activated G proteins past the saturation point of the GIRK channel response ( Hensler et al . , 2012 ) , these data suggest that the lag time reflects the clearance of free βγ subunit produced above the stoichiometric level relative to the GIRK channel .","To provide an independent evidence that increase in the lag time reflects changes in stoichiometry of Gβγ subunits relative to their effector molecule , we utilized a bioluminescence resonance energy transfer ( BRET ) -based approach that monitors interactions of Gβγ with a reporter derived from an effector ( GRK3 ) upon reconstitution in transfected cells ( Figure 3A ) .","In this assay , we induced production of free Gβγ subunits via GABABR activation and then measured delay time between antagonizing GABABR and the onset of signal decay , while changing the Gβγ to GRK3 effector ratio ( Figure 3B ) .","In agreement with the electrophysiological recordings of GIRK channel activity , BRET experiments showed that the increase in Gβγ stoichiometry over an effector results in prolongation of the response deactivation lag ( Figure 3C , D ) . 10 . 7554/eLife . 02053 . 005Figure 3 . Changing the ratio of Gβγ to an effector affects response deactivation lag in a reconstituted system .","( A ) Schematic representation of the assay principle .","The assay measures interaction of YFP-tagged Gβγ with its effector reporter GRK , tagged with N-luc , producing BRET signal .","Gβγ subunits are released upon GABABR stimulation with GABA increasing BRET signal .","Inactivation of GABABR with an antagonist CGP 54626 results in dissociation of Gβγ from GRK and re-association with Gαo to form inactive heterotrimer .","( B ) Time course of changes in BRET signal upon stimulation of cells with GABA and subsequent deactivation by CGP 54626 .","Cells were transfected with varying amounts of constructs encoding Gβγ and GRK reporter ( from black to green ) .","( C ) Deactivation phase of the response showing kinetics of signal decay .","The lag deactivation time ( dotted line for the trace in black ) is defined as the time that it takes to quench the BRET signal by 10% from its steady state value in the presence of an agonist .","( D ) Quantification of a lag deactivation time as a function of Gβγ/GRK ratio .","Error bars are SEM values , n = 4 per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 005 In transfected cells , R7BP is essential for the membrane localization of RGS7 ( Drenan et al . , 2005; Narayanan et al . , 2007 ) .","Furthermore , we previously reported that knockout of R7bp resulted in a reduction of the total membrane-bound RGS7 protein in hippocampal tissue ( Panicker et al . , 2010 ) .","To analyze the effect of R7BP on RGS7 localization in hippocampal pyramidal neurons , we performed high-resolution immunoelectron microscopy .","Consistent with the earlier findings , immunoparticles for RGS7 were abundant on the extrasynaptic plasma membrane of dendritic spines and dendritic shafts of pyramidal cells , as well as at intracellular sites ( Fajardo-Serrano et al . , 2013 ) .","In the hippocampus of R7bp−/− animals , immunoparticles for RGS7 were distributed similarly to the wild-type , but they were more frequently observed just beneath the plasma membrane and more broadly distributed in somata ( Figure 4 ) .","Indeed , quantitative analysis indicated that in R7bp−/− mice , RGS7 was less frequently detected in the plasma membrane and tended to accumulate within 100 nm of the plasma membrane .","We also detected a significant increase in RGS7 labelling in the rough endoplasmic reticulum ( rER ) in the soma of R7bp−/− pyramidal neurons ( 2489 vs 3371 immunoparticles in wild-type and R7bp−/− neurons , respectively ) .","These findings indicate that knockout of R7BP caused a modest but significant redistribution of RGS7 away from the plasma membrane . 10 . 7554/eLife . 02053 . 006Figure 4 . Change in subcellular localization of RGS7 in the hippocampus of the R7bp−/− mice . Electron micrographs of the stratum radiatum of the hippocampal CA1 region showing immunoparticles for RGS7 , as detected using a pre-embedding immunogold method .","Dendritic spines ( s ) and axon terminals ( at ) are marked .","Arrows indicate locations of immunoparticles at the plasma membrane , while arrowheads identify RGS7 immunoparticles found just below the membrane .","Quantitative analysis showed that RGS7 is less frequently detected in the plasma membrane , and accumulates within the first 100 nm from the plasma membrane , samples from R7bp−/− mice , *p<0 . 05 , One-way ANOVA followed by the Bonferroni’s post hoc test , n = 3 mice .","Scale bar: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 006 The slower kinetics and increased sensitivity of GIRK channels to GABABR activation suggested that there was a net up-regulation of GIRK-dependent inhibition in hippocampal neurons lacking RGS7 .","Since GIRK channels significantly contribute to setting neuronal excitability ( Chen and Johnston , 2005 ) , we next determined how deletion of RGS7 influences the excitability of CA1 pyramidal neurons .","To characterize intrinsic electrophysiological properties of different genotypes , the responses of CA1 neurons to somatic current injections ranging from −150 pA to +300 pA were measured ( Figure 5A ) .","While membrane resistance was similar in both genotypes ( Rin = 120 ± 10 MΩ vs 105 ± 11 MΩ , p=0 . 31 , in wild-type and Rgs7−/− cells , respectively ) , resting membrane potential ( RMP ) was significantly hyperpolarized in Rgs7−/− neurons ( −64 . 7 ± 0 . 5 mV vs −68 . 5 ± 0 . 7 mV in wild-type and Rgs7−/− , respectively; Figure 5B ) .","In addition , the current required to elicit action potentials ( APs ) was significantly higher in RGS7−/− neurons ( I = 104 . 3 ± 8 . 8 pA vs 146 . 4 ± 17 . 0 , in WT and Rgs7−/− cells , respectively; Figure 5C ) .","Finally , Rgs7−/− neurons fired significantly fewer action potentials in response to depolarizing current as compared to wild-type controls ( Figure 5D ) .","Collectively , these observations argued that hippocampal pyramidal neurons from Rgs7−/− mice were less excitable than wild-type counterparts . 10 . 7554/eLife . 02053 . 007Figure 5 . Altered intrinsic excitability and excitatory transmission in CA1 hippocampal neurons from Rgs7−/− mice .","( A ) Representative traces of responses elicited by current injections of −150 , −50 , 0 , +50 , and +300 pA in WT and Rgs7−/− CA1 neurons .","( B ) Hyperpolarized resting membrane potential ( RMP ) in Rgs7−/− neurons , ***p=0 . 0001 , t test , n = 14–23 cells .","( C ) Current required to evoke an action potential ( firing threshold ) is higher in Rgs7−/− neurons , *p=0 . 02 , t test , n = 14–23 cells .","( D ) Lower intrinsic excitability in Rgs7−/− neurons .","**p<0 . 01 and *p<0 . 05 , two-way ANOVA with Bonferroni’s posttest , n = 14–23 cells .","( E ) Representative traces of slow excitatory synaptic currents ( sEPSCs ) in WT and Rgs7−/− cells .","( F ) Cumulative distribution and mean values for sEPSCs amplitudes ( 14 . 9 ± 1 . 9 vs 12 . 8 ± 0 . 9 pA in WT and Rgs7−/− correspondingly , p=0 . 3 , unpaired t test; n = 11 cells and 1100 events for each genotype ) .","( G ) Cumulative distribution and mean values for sEPSCs and frequencies ( 3 . 4 ± 0 . 6 vs 3 . 1 ± 0 . 6 Hz in WT and Rgs7−/− correspondingly , p=0 . 7 , unpaired t test , n = 11 cells and 1100 events for each genotype ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 007 We further examined whether decreased intrinsic excitability of Rgs7−/− neurons is caused by dysregulated net excitatory transmission .","For this purpose , we measured spontaneous EPSCs ( sEPSC ) in CA1 pyramidal neurons ( Figure 5E–G ) .","There were no significant differences in either amplitudes or frequencies of sEPSC events between Rgs7−/− and wild-type neurons ( Figure 5F–G ) .","These observations indicate that excitatory input into CA1 pyramidal neurons is unchanged by elimination of RGS7 and suggest that the observed decrease in neuronal excitability is caused by changes in the intrinsic membrane properties of CA1 neurons , likely stemming from enhanced activity of the postsynaptic GIRK channel .","Previous studies have implicated GIRK channels in several forms of hippocampal synaptic plasticity , including long-term potentiation ( LTP ) ( Cramer et al . , 2010 ) , long-term depression ( LTD ) ( Cooper et al . , 2012 ) and depotentiation ( DP ) ( Chung et al . , 2009; Cooper et al . , 2012 ) .","Because GABAB receptors play key roles in these processes and since GABAB-GIRK signaling is severely dysregulated in Rgs7−/− neurons , we next examined the impact of RGS7 ablation on hippocampal synaptic plasticity .","A high-frequency stimulation protocol of 2 tetanized stimuli ( TS , 100 Hz for 1s each ) produced robust LTP in both genotypes ( Figure 6A , D ) .","The extent of the potentiation in Rgs7−/− slices was not significantly different from that measured in wild-type slices , arguing that elimination of RGS7 has no effect on LTP .","A low-frequency stimulation ( LFS , 2 Hz for 10 min , 1200 pulses ) elicited LTD in both wild-type and Rgs7−/− hippocampal slices ( Figure 6B , D ) .","However , the extent of inhibition was significantly smaller in Rgs7−/− slices .","Interestingly , the magnitude of the first fEPSP after the train of LFS stimulation was significantly reduced in knockout mice ( 51 ± 6% and only 72 ± 6% in wild-type and Rgs7−/− slices correspondingly , *p=0 . 03 , t test , n = 6 ) , suggesting that the LTD impairment in Rgs7−/− is due to a deficit in an induction mechanism ( s ) .","The depotentiation produced by consecutive application of TS and LFS was also significantly impaired in Rgs7−/− mice ( Figure 6C , D ) . 10 . 7554/eLife . 02053 . 008Figure 6 . RGS7 ablation disrupts hippocampal synaptic plasticity . Field EPSP ( fEPSP ) slope change following induction of: ( A ) LTP in Rgs7−/− , 166 ± 9% vs wild-type , 155 ± 6% slices; p=0 . 33 , t test , n = 6–10; ( B ) LTD in Rgs7−/− , 90 ± 2% vs wild-type , 81 ± 2%; *p=0 . 01 , t test , n = 6–7; ( C ) depotentiation ( DP ) in Rgs7−/− , 111 ± 3% vs wild-type , 128 ± 6%; *p=0 . 013 , t test , n = 6–11 .","Insets show representative fEPSP traces at baseline and 1 hr following induction protocol in WT ( black and grey ) and Rgs7−/− ( red and pink ) slices .","( D ) Quantification of the EPSP slope change 55–60 min following induction of each form of plasticity after normalization to pre-induction baseline .","( E ) Paired Pulse Ratio ( PPR ) dependence on the inter stimulus interval for wild-type ( WT ) and Rgs7−/− .","Significant inter-stimulus interval but not genotype effect was observed , p<0 . 0001 and p=0 . 5 , correspondingly .","Two-way ANOVA , n = 4 slices per genotype .","( F–H )","Basal synaptic transmission properties in Rgs7−/− slices .","Dependence of fEPSP slope ( F ) and FV amplitude ( G ) on stimulus intensity .","( H ) Linear regression plot of fEPSP slope dependence on FV amplitude .","The data were fitted with a linear regression , R2 = 0 . 67 and 0 . 53; and slopes 1 . 9 ± 0 . 1 and 1 . 5 ± 0 . 2 for WT and Rgs7−/−correspondingly .","p=0 . 1 for differences in slopes , two way ANOVA , n = 12–13 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 008 We further investigated the impact of RGS7 ablation on paired-pulse facilitation ( PPF ) , a form of short-term plasticity that results from the enhancement of presynaptic vesicle release in response to two closely-spaced stimuli ( Zucker , 1989; Dobrunz and Stevens , 1997 ) .","We found that fEPSP facilitation was similar between genotypes across the examined range of 10–1000 ms interpulse intervals ( Figure 6E ) , indicating that presynaptic mechanisms are not likely to be the cause of the observed alterations in synaptic plasticity .","We next compared the dependence of fEPSP slopes ( Figure 6F ) and presynaptic fibre volley ( FV ) amplitudes ( Figure 6G ) on stimulus intensity .","Analysis of the relationship between FV amplitudes and fEPSP slopes by linear fitting reveals their nearly perfect correspondence between wild-type and Rgs7−/− slices , indicating preservation of basal synaptic transmission properties ( Figure 6H ) .","Together , these results suggest that RGS7 elimination selectively impaired LTD and DP forms of synaptic plasticity , likely by a post-synaptic mechanism .","Changes in the intrinsic excitability of CA1 pyramidal neurons , together with deficits in hippocampal synaptic plasticity , suggested that RGS7 complexes play a role in spatial learning and memory .","To test this possibility , we first studied the impact of RGS or R7BP ablation in contextual fear conditioning , a test that requires hippocampal processing for memory formation ( Maren , 2001 ) .","We found significant deficits in context recognition 24-hr after training in Rgs7−/− mice , but not in Rgs6−/− or R7bp−/− mice ( Figure 7A–C ) .","Importantly , we observed no difference in baseline freezing behavior before associative training between the genotypes .","Furthermore , there were no significant differences between wild-type mice and mutant mice in amygdala-dependent cue recognition . 10 . 7554/eLife . 02053 . 009Figure 7 . RGS ablation affects hippocampal-dependent learning and memory .","( A–C )","Evaluation of mouse behavior in fear conditioning paradigm .","( A ) Rgs7−/− mice show deficits in hippocampal-dependent contextual , but not cue , memory .","*p<0 . 05 , t test; n = 12 per genotype .","( B ) Normal contextual and cue memory of Rgs6−/− mice in the fear conditioning test as compared to wild-type ( WT ) littermates .","n = 8 per genotype .","( C ) R7bp−/− mice showed the same contextual and cue memory in fear conditioning test as compared to WT littermates , n = 8 per genotype .","( D–F )","Evaluation of mouse behavior in Morris water maze .","Rgs7−/− mice ( n = 13 ) and their WT littermates ( n = 12 ) were trained for 6 d with 4 trials/d with an inter-trial interval of approximately 15 min .","Performance during the acquisition phase was monitored and plotted as average time ( D ) or success rate ( E ) to reach the hidden platform .","Mice showed improvement with training .","There was a significant effect of a genotype in both escape latencies ( *p<0 . 05 ) and success rates ( *p<0 . 05 ) using two-way ANOVA analysis .","Post hoc comparison revealed a significant impairment of Rgs7−/− mice during the last two acquisition days ( *p<0 . 05 , Tukey’s test ) .","( F ) Results of a probe trial given 24 hr after 6 d of training .","The latency to the first crossing of the former location of the platform and the total number of crossing are shown , *p<0 . 05 , t test .","( G ) Evaluation of mouse behavior in novel object recognition paradigm .","Rgs7−/− mice showed significant impairment during the test trial with a novel object in comparison with wild-type littermates .","*p<0 . 05 , t test; n = 12–13 . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 009 To clarify the impairment of hippocampus dependent learning and memory in Rgs7−/− mice , we conducted other tests that rely on hippocampal function .","In the Morris water maze test , Rgs7−/− mice showed delayed escape latencies and reduced success rates during acquisition trials ( Figure 7D , E ) .","In the probe trial , when the platform was removed 24 hr after training , it took Rgs7−/− mice significantly longer to reach the target area where the platform was located during training as compared to their wild-type littermates ( Figure 7F ) .","Mice lacking RGS7 also made fewer crossings over the target area during the probe trial .","Importantly , despite a pronounced spatial impairment , Rgs7−/− mice performed normally in the visible platform version of water maze .","In the novel object recognition test , wild-type littermates spent significantly more time exploring novel objects over familiar objects ( Figure 7G ) .","However , Rgs7−/− mice spent approximately equal time exploring familiar and novel objects .","Furthermore , there was a significant difference in time exploring novel objects between Rgs7−/− mice and their wild-type littermates .","No genotype differences in object exploratory behavior were observed during the training day , when the mice were first introduced to identical objects .","Taken together , these results indicate that elimination of RGS7 leads to a disruption of spatial learning and memory in mice ."],["The results of this study , together with prior investigations , establish the RGS7/Gβ5/R7BP complex as an essential regulator of GABABR-GIRK signaling in the hippocampus .","In hippocampal neurons , RGS7 is closely co-localized with both GABABR and GIRK2-containing channels ( Fajardo-Serrano et al . , 2013 ) .","Furthermore , in transfected cells , RGS7 can directly interact with GIRK channel subunits as demonstrated by both co-immunoprecipitation and bioluminescence energy transfer approaches ( Xie et al . , 2010; Zhou et al . , 2012 ) .","The complex may further involve GABABR as well as G protein subunits ( Kovoor and Lester , 2002; Fajardo-Serrano et al . , 2013 ) , supporting the contention that components of the entire pathway are scaffolded into a larger macromolecular assembly .","The interaction between RGS7 with GIRK channels is mediated by the Gβ5 subunit , which mimics signal-transducing Gβγ subunits in binding to the channel ( Xie et al . , 2010; Zhou et al . , 2012 ) .","Knockout of Gβ5 greatly slows GABABR-GIRK response deactivation rates and dramatically increases agonist sensitivity of the signaling pathway ( Xie et al . , 2010 ) .","In addition to mediating interactions with the channel , Gβ5 is essential for the expression of all four R7 RGS proteins ( Chen et al . , 2003 ) .","With several R7 RGS proteins expressed in hippocampus , the identity of the exact isoform that participates in GIRK channel regulation in the region , through complex formation with Gβ5 , remained unknown .","The elimination of RGS7 , reported in this study , largely phenocopies the loss of Gβ5 , both in terms of kinetics and agonist sensitivity .","This argues that in hippocampal pyramidal neurons , GIRK signaling is regulated by RGS7 and not by other R7 RGS proteins .","Indeed , while RGS6 regulates GIRK signaling in cerebellar neurons ( Maity et al . , 2012 ) and sinoatrial pacemaking cells ( Posokhova et al . , 2010; Yang et al . , 2010 ) , RGS6 ablation had no effect on any GIRK response parameters in hippocampal pyramidal neurons .","The function of R7 RGS proteins is regulated by the membrane anchor R7BP , which augments the ability of R7 RGS proteins to deactivate G protein signaling in reconstituted systems ( Drenan et al . , 2006; Masuho et al . , 2013 ) .","Consistent with these observations , we show that knockout of R7BP reduces plasma membrane localization of RGS7 and slows GABABR-GIRK current deactivation in hippocampal pyramidal neurons .","Our results also agree well with a recent study reporting similar deceleration of GIRK deactivation kinetics in R7bp−/− hippocampal neurons ( Zhou et al . , 2012 ) .","However , the consequences of RGS7 and R7BP elimination are different in two important ways .","First , relative to the loss of RGS7 or Gβ5 , elimination of R7BP had a very modest effect on GABABR-GIRK deactivation kinetics , and was seen only at high GABABR agonist concentrations .","This suggests that the RGS7/Gβ5 complex can regulate GABABR-GIRK signaling without R7BP .","Secondly , loss of R7BP and RGS7 has different effects on GABABR-GIRK coupling sensitivity .","While elimination of RGS7 ( or Gβ5 ) results in a parallel leftward shift in the concentration-response relationship , the concentration-response measured in R7bp−/− neurons shows markedly greater cooperativity .","Thus , at low levels of GABABR stimulation , R7BP is dispensable and its elimination fails to affect response amplitude .","As the response reaches saturation , R7BP becomes indispensable and its elimination has the same effect on response amplitude as loss of RGS7 .","These observations are inconsistent with the proposed role for R7BP as a critical factor in the assembly of RGS7-GIRK complexes ( Zhou et al . , 2012 ) , which postulates the equivalence of R7BP effects across agonist concentrations and a similar impact of R7BP and RGS7 elimination on GIRK channel kinetics , and certainly not an increase in cooperativity of GIRK channel activation .","Based on our findings , we propose an alternative model , where R7BP sets GABABR-GIRK coupling efficiency .","In this model , at low levels of GIRK activation , when released Gβγ subunits do not saturate the GIRK channel , deactivation kinetics are primarily mediated by RGS7/Gβ5 directly associated with the GIRK subunits .","Under these conditions , there are virtually no genotype-dependent differences in the lag time of GIRK deactivation upon agonist removal .","With higher agonist concentrations , as GABABR produces more Gβγ subunits , the amount of available free Gβγ exceeds that of activatable GIRK channels , evidenced by a delay that precedes GIRK current deactivation upon agonist removal .","Under these conditions , R7BP elimination affects response sensitivity as much as elimination of RGS7 .","While response deactivation kinetics reflect Gβγ inactivation after it had a chance to interact with the channel , response sensitivity likely relates to the efficiency of the Gβγ reaching the GIRK channel .","Considering that R7BP affects both of these parameters only when free Gβγ is produced in excess , we think that the RGS7/Gβ5 complex exists in two states: ( 1 ) anchored to the GIRK complex where it affects deactivation kinetics , and ( 2 ) anchored via R7BP to the plasma membrane outside of GIRK complex where it primarily determines response sensitivity .","In agreement with this model , we observe an increase in cooperativity of GIRK channel activation upon R7BP loss that suggests that RGS7/Gβ5 alone , but not in complex with R7BP , promotes more productive association of Gβγ with the channel .","On an intuitive level , this may reflect larger proportion of Gβγ subunits that reach GIRK while being deactivated in its vicinity by RGS7/Gβ5 .","In contrast , complexes of RGS7/Gβ5 with R7BP may predominantly act elsewhere on the plasma membrane , thereby decreasing the number of Gβγ subunits capable of reaching the channel .","By physically binding to the GIRK channel , RGS7/Gβ5 may further act to promote productive interactions of Gβγ ( and/or Gα ) with the channel , thus increasing cooperativity of its activation .","This model is also consistent with the observed decrease in RGS7 on the plasma membrane but largely preserved localization at specific postsynaptic sites where it might be anchored via complex formation with GIRK channels .","Thus , the main role of R7BP appears to be in tuning the sensitivity of the response by endowing RGS7/Gβ5 complexes with an ability to deactivate G proteins before they reach the GIRK channel .","RGS proteins are potent negative regulators of both the extent and duration of neurotransmitter signaling via G protein-coupled receptors ( Sjogren , 2011; Xie and Martemyanov , 2011 ) .","As a result , knockout of individual RGS genes in mice is frequently associated with augmented GPCR signaling .","This makes the analysis of changes in sensitivity of behavioral or cellular reactions to neurotransmitter actions associated with elimination of individual RGS proteins a powerful strategy that allows for establishing physiological receptor-RGS pairings .","Using this strategy , two RGS proteins have been previously implicated in controlling GABABR signaling in vivo: RGS6 in cerebellar neurons ( Maity et al . , 2012 ) and RGS2 in the ventral tegmental area ( Labouebe et al . , 2007 ) .","Now , our findings establish that in hippocampal neurons , GABABR signaling is negatively regulated by RGS7 .","These observations complement earlier findings with RGS14 , the only other RGS protein implicated in hippocampal synaptic plasticity and spatial learning ( Lee et al . , 2010 ) .","However , in contrast to RGS7 that acts in the CA1 region , RGS14 functions in CA2 synapses and its ablation augments LTP and improves spatial learning in mice .","The major forms of synaptic plasticity ( LTP , LTD and depotentiation ) in the hippocampus have been implicated in learning and memory ( Martin et al . , 2000 ) .","Recent models propose that all three forms cooperate to affect distinct aspects of spatial information storage ( Kemp and Manahan-Vaughan , 2007 ) .","In relevance to this thinking , we found a selective disruption of LTD and depotentiation in RGS7−/− mice , but normal LTP .","There is a mounting evidence suggesting an active and selective role of LTD in object recognition and creation of spatial representation of memory ( Kemp and Manahan-Vaughan , 2007 ) .","For example , LTD but not LTP was found to be important for spatial memory consolidation and memory enhancement for novelty acquisition ( Ge et al . , 2010; Dong et al . , 2012 ) .","Consistent with this , electrophysiological recordings in behaving animals show facilitation of LTD during novelty exploration ( Xu et al . , 1997 , 1998; Manahan-Vaughan and Braunewell , 1999; Dong et al . , 2012 ) .","Disruption of LTD via genetic mutations often results in memory deficits , particularly affecting behavioral flexibility ( Nicholls et al . , 2008 ) , while LTD enhancement can lead to improvement in spatial reversal learning ( Duffy et al . , 2008 ) .","In particular , our findings resemble the phenotype of SRF−/− mice , which exhibited a selective disruption of LTD in hippocampal CA1 neurons paralleled by an inability to learn hippocampus-dependent tasks ( Etkin et al . , 2006 ) .","While specific knowledge on how depotentiation contributes to memory is still lacking , it has been observed that exposure to novel environment and spatial exploration depotentiates previously-induced LTP ( Xu et al . , 1998 ) .","Together , our observations in mice lacking RGS7 reinforce the idea that normal inhibitory synaptic plasticity ( LTD and depotentiation ) is required for hippocampal-dependent learning and memory .","Although , it has never been systematically addressed in a single study , GIRK channel elimination was reported to enhance LTP ( Cramer et al . , 2010 ) .","Given that the Girk2−/− neurons are relatively depolarized , the enhanced LTP likely results from a general increase in excitability .","Conversely , GIRK2 over-expression in transgenic models of Down syndrome leads to reduced LTP ( Kleschevnikov et al . , 2004; Siarey et al . , 2005 ) , but enhanced LTD ( Siarey et al . , 2005; Cooper et al . , 2012 ) .","Interestingly , GIRK2 over-expression and ablation both resulted in decreased depotentiation ( Chung et al . , 2009; Cooper et al . , 2012 ) .","Similar to Down syndrome mouse models , Rgs7−/− mice also exhibited augmented GABABR-GIRK signaling .","However , in the case of Rgs7−/− mice , this augmentation resulted in yet another distinct phenotype: selective deficits in LTD and depotentiation , with normal LTP .","Several lines of evidence argue that these changes in synaptic plasticity are driven by a postsynaptic mechanism whereby RGS7 controls inhibitory GIRK-dependent signaling in the CA1 pyramidal neurons .","First , CA1 pyramidal neurons lacking RGS7 are hyperpolarized and less excitable relative to wild-type neurons , a property that is significantly shaped by the GIRK channels .","Second , basal excitatory transmission is unaltered in Rgs7−/− slices .","Third , direct measurements of events that reflect excitatory presynaptic function ( e . g . , FV and PPR ) reveal no changes caused by the elimination of RGS7 .","Given these observations , we propose that GIRK signaling sets the general excitability of postsynaptic pyramidal neurons , which in turn determines a sliding scale window for the induction of different forms of synaptic plasticity .","By adjusting signaling strength in the hippocampal GABABR-GIRK pathway , the RGS7/Gβ5/R7BP complex influences the range of neuronal responses necessary for memory formation .","While dysregulation of GABABR-GIRK signaling in hippocampus may be sufficient for explaining the effects of RGS7 on learning an memory , at this point we cannot rule out a contribution of other brain regions and/or signaling pathways in the process .","In any event , we believe that the results of this study establish RGS7 complex as an important molecule for understanding and/or correcting the pathology of neuropsychiatric disorders associated with disruptions in synaptic plasticity and imbalances in inhibitory signaling ."],["All studies were carried out in accordance with the National Institute of Health guidelines and were granted formal approval by the Institutional Animal Care and Use Committee of the Scripps Research Institute .","The generation of Rgs6−/− ( Posokhova et al . , 2010 ) , Rgs7−/− ( Cao et al . , 2012 ) , R7bp−/− ( Anderson et al . , 2007 ) mice were described earlier .","All animals used for comparing genotypes were littermates derived from heterozygous breeding pairs .","Mice were housed in groups on a 12 hr light–dark cycle with food and water available ad libitum .","Males and females ( 2–5 months ) were used for all experiments .","Lysates were prepared by homogenizing hippocampal tissue from age-matched littermates by sonication in the lysis buffer ( 1 × PBS , 150 mm NaCl , 1% Triton X-100 , protease inhibitors ) followed centrifugation at 14 , 000×g for 10 min .","The resulting extract was used for protein concentration determination by the BCA protein assay ( Pierce , Rockford , IL ) .","The lysates were adjusted to equalize total protein content by adding lysis buffer and 2x SDS sample buffer .","Samples were boiled for 5 min , resolved on SDS-PAGE gels , transferred onto PVDF membrane and subjected to western blot analysis using HRP conjugated secondary antibodies and ECL West Pico ( Pierce ) detection system .","Signals were captured on film and scanned by densitometer , and band intensities were determined using NIH ImageJ software .","Rabbit anti-R7BP ( TRS ) and rabbit Gβ5 ( ADTG ) were generous gifts from Dr William Simonds ( NIDDK/NIH ) .","Anti-RGS6 was generated and used previously ( Posokhova et al . , 2010 ) .","Chicken IgY anti-RGS7 antibody was from Thermo Scientific ( Waltham , MA ) , anti-GIRK2 was purchased from Alomone labs ( Jerusalem , Israel ) and anti-β-actin was from Sigma ( St . Lois , MO ) .","Primary cultures of hippocampal neurons were prepared using a modified version of a published protocol ( Xie et al . , 2010 ) .","Briefly , hippocampi were extracted from neonatal ( P1-3 ) pups and placed into an ice-cold HBSS/FBS solution: Hank’s Balanced Salt Solution ( Sigma; St . Louis , MO ) , 4 . 2 mM NaHCO3 , 1 mM HEPES , and 20% FBS .","The tissue was washed twice with HBSS/FBS , and then three times with HBSS alone .","Hippocampi were digested at room temperature for 5 min with 10 mg/ml Trypsin Type XI ( Sigma; St . Louis , MO ) in a solution that contained ( in mM ) : 137 NaCl , 5 KCl , 7 Na2HPO4 , and 25 HEPES ( pH 7 . 2 ) .","The tissue was washed twice with HBSS/FBS and three times with HBSS alone , and then hippocampi were mechanically-dissociated in HBSS ( supplemented with 12 mM MgSO4 ) using Pasteur pipettes of decreasing diameter .","The neurons were pelleted by centrifugation ( 600×g for 10 min at 4°C ) and plated onto 8-mm glass coverslips pre-treated with Matrigel ( BD Biosciences; San Jose , CA ) in 48-well plate .","Neurons were allowed to adhere for 30 min prior to adding 0 . 3 ml of pre-warmed culture medium consisting of Neurobasal A ( Life Technologies; Carlsbad , CA ) , 2 mM GlutaMAX-I ( Life Technologies , Carlsbad , CA ) , 2% B-27 supplement , and 5% FBS .","After 4–12 hr , culture media was completely replaced with the same media without FBS .","Neurons were incubated at 37°C/5% CO2 , and half of the medium was replaced with fresh medium on each of the first 3 days of culture .","Neurons were kept in culture for 10–14 days prior to experiments .","Coverslips containing neurons were transferred to a chamber containing a low-K+ bath solution ( in mM ) : 145 NaCl , 4 KCl , 1 . 8 CaCl2 , 1 MgCl2 , 5 . 5 D-glucose , 5 HEPES/NaOH ( pH 7 . 4 ) .","Borosilicate patch pipettes ( 3–5 MΩ ) were filled with ( in mM ) : 130 KCl , 10 NaCl , 1 EGTA/KOH ( pH 7 . 2 ) , 0 . 5 MgCl2 , 10 HEPES/KOH ( pH 7 . 2 ) , 2 Na2ATP , 5 phosphocreatine , 0 . 3 GTP .","Baclofen ( R- ( + ) -b- ( aminomethyl ) -4-chlorobenzenepropanoic acid hydrochloride ) was purchased from Sigma ( St . Louis , MO ) .","Baclofen-induced currents were measured at room temperature using a high-K+ bath solution ( in mM ) : 120 NaCl , 25 KCl , 1 . 8 CaCl2 , 1 MgCl2 , 5 . 5 D-glucose , 5 HEPES/NaOH ( pH 7 . 4 ) .","The high-K+ bath solution ( +/− baclofen ) was applied directly to the soma and proximal dendrites with an SF-77B rapid perfusion system ( Warner Instruments , Inc . ; Hamden , CT ) .","Current responses to the application of the high-K+ solution ( +/− baclofen ) were measured at a holding potential of −80 mV .","Membrane potentials and whole-cell currents were measured in large neurons ( >75 pF ) with hardware ( Axopatch-700B amplifier , Digidata 1440A ) and software ( pCLAMP v . 10 . 3 ) from Molecular Devices ( Sunnyvale , CA ) .","All currents were low-pass filtered at 2 kHz , sampled at 5 kHz , and stored on computer hard disk for subsequent analysis .","Peak and steady-state current amplitudes were measured for each experiment .","Current activation rates were extracted from a standard exponential fit of the current trace corresponding to the onset of drug effect and the peak evoked current , while deactivation rates were extracted from an exponential fit of the trace corresponding to the return of current to baseline following removal of drug ( Clampfit v . 10 . 3 software ) .","Current desensitization was defined as % change in steady state current from the maximal baclofen-evoked response amplitude during 10 s of continuous drug application .","Only experiments where access resistances were stable and low ( <20 MΩ ) were included in the analysis .","Immunohistochemical reactions were carried out using the pre-embedding immunogold method as described earlier ( Lujan et al . , 1996 ) .","Briefly , after blocking with 10% serum for 1 hr at room temperature free-floating sections were incubated for 48 hr with anti-RGS7 antibodies ( 1–2 mg/ml ) .","Sections were washed and incubated for 3 hr with goat anti-rabbit IgG coupled to 1 . 4 nm gold ( Nanoprobes Inc ) at 1:100 dilution .","Sections were washed , postfixed in 1% glutaraldehyde and processed for silver enhancement of the gold particles with an HQ Silver kit ( Nanoprobes Inc . ) .","The reacted sections were treated with osmium tetraoxide ( 1% in 0 . 1 M PB ) , block-stained with uranyl acetate , dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan ( Fluka ) resin .","Regions of interest were cut at 70–90 nm on an ultramicrotome ( Reichert Ultracut E; Leica ) .","Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds’s lead citrate .","Ultrastructural analyses were performed in a Jeol-1010 electron microscope .","To establish the relative the abundance of RGS7 immunoreactivity along the plasma membrane of pyramidal cells , we used 60-µm coronal slices processed for pre-embedding immunogold immunohistochemistry .","The procedure was similar to that used previously ( Lujan et al . , 1996 ) .","Briefly , for each of three animals from different postnatal ages and adult , three samples of tissue were obtained for preparation of embedding blocks ( totalling nine blocks for each age ) .","To minimize false negatives , electron microscopic serial ultrathin sections were cut close to the surface of each block , as immunoreactivity decreased with depth .","We estimated the quality of immunolabelling by always selecting areas with optimal gold labelling at approximately the same distance from the cutting surface .","Randomly selected areas were then photographed from the selected ultrathin sections and printed with a final magnification of 45 000X .","Quantification of immunogold labelling was carried out in reference areas totalling approx .","1 , 800 µm2 for each age .","Immunoparticles identified in each reference area and present in different subcellular compartments ( dendritic spines , dendritic shafts and somata ) were counted .","We measured the radial distance of each immunoparticle to the plasma membrane , being 0 for those just located in the plasma membrane .","The data was expressed as percentage of immunoparticles along the radial distance from the plasma membrane expressed in nanometers .","Mice were sacrificed under isoflurane anesthesia , and brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid ( aCSF ) without CaCl2 , composed of ( mM ) : 124 NaCl , 3 KCl , 24 NaHCO3 , 1 . 25 NaH2PO4 , 1 MgSO4 , and 10 D-Glucose , equilibrated with 95% O2 and 5% CO2 .","The tissue was cut in 350–400 µm thick sections with a Vibrating microtome ( Leica VT1200S , Germany ) .","The slices were warmed to 35°C for 25–45 min in aCSF supplemented with 2 mM CaCl2 , and equilibrated with 95% O2 and 5% CO2 .","Then slices were maintained in gassed aCSF at room temperature until being transferred to submerged-type recording chambers of volume ∼1 . 5 ml .","Here , the slices were constantly superfused ( 1–2 ml/min ) with warmed ( 30–31°C ) , gassed aCSF .","All measurements were performed by an experimenter blind to genotype .","CA1 neurons were visually identified in the hippocampal transverse slices of 350 µM thickness using Scientifica SliceScope system .","Glass microelectrodes with an open-tip resistance of 3 . 5–6 . 5 MΩ were used .","The following internal solution was used ( mM ) : 130 K-Gluconate , 20 KCl , 10 K-HEPES , 0 . 2 EGTA , 0 . 3 Na-GTP and 4 Mg-ATP ( pH 7 . 3 ) .","To determine intrinsic cellular properties such as resting membrane potential , input resistance and spike numbers , 500 ms , 50 pA , 10-step hyperpolarizing and depolarizing current injections were delivered every 10 s .","Cells with series resistance >20 MΩ or resting membrane potentials > −55 mV were excluded from analysis .","Liquid junction potential was −14 mV .","Spontaneous EPSCs ( sEPSCs ) were measured by holding the cells at −70 mV in normal aCSF .","At least 100 events , which are above 4 pA , were obtained during sEPSC measurements in each cell .","Field excitatory postsynaptic potentials ( fEPSPs ) were elicited by a concentric bipolar stimulating electrode ( inner diameter [ID]: 25 µm; outer diameter [OD]: 125 µm , FHC Inc . , Bowdoin , ME ) connected to a constant current isolated stimulator unit ( A-M Systems; Carlsborg , WA ) and recorded with low resistance ( 3–5 MΩ ) glass pipettes ( ID: 1 . 16 mm , OD: 1 . 5 mm , Harvard Apparatus , Holliston , MA ) filled with aCSF .","The electrodes were placed in the stratum radiatum of the CA1 area of the dorsal hippocampus slices .","Stimulation frequency was set to 0 . 05 Hz .","Input-output curves were generated by adjusting the stimulus intensity in increments of 10 µA , from 0 to 100 µA .","Paired pulse ratio ( PPR ) was assessed using a succession of paired pulses separated by time intervals ranging from 25 to 1000 ms , delivered every 20 s .","The degree of facilitation was determined by taking the ratio of the initial slope of the second fEPSP to the initial slope of the first fEPSP .","A PPR >1 was considered to reflect facilitation .","For synaptic plasticity experiments , a stable baseline for at least 30 min was achieved prior to induction .","LTP and LTD were recorded for 1 hr after HFS ( 2 tetanized stimuli ( TS ) of 100 Hz for 1s each ) or LFS ( 2 Hz for 10 min , 1200 pulses ) was applied .","Depotentiation was achieved by applying HFS followed by LFS after 1-2 min interval .","Spatial learning and memory were evaluated in the Morris water maze using a video tracking systems ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .","Mice first received 4 cued trials ( visible but variable platform location ) on the first day to determine if non-associative impairment in other behavioral responses affecting performance in this task , such as exploratory activity , motor coordination , vision and motivation .","After completing the cued trials , spatial learning acquisition was evaluated during the place condition ( hidden platform , constant location; 4 trials/day , 6 consecutive days ) .","Escape latency and success rates in finding hidden platform were calculated for all place trials .","Retention performance was evaluated during probe trials ( platform removed ) , which were conducted 24 hr after the last cued trial on the sixth day .","Latency to the first target platform area crossing and the number of crossings in the probe trial ( 1 min ) served as the dependent variables .","For fear conditioning experiments , mice were habituated in individual conditioning chambers to obtain freezing baseline on the first day .","On the second day , mice were trained in individual conditioning chambers ( Med Associates , St . Albans , VT ) .","Video images were recorded via video tracking systems ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .","In context A , visible light was turned on , a stainless steel grid floor inserted , and the chambers were cleaned with 70% ethanol prior to conditioning .","In context B , white plastic inserts were placed inside the chamber to change the shape , size and texture of the wall and the floor .","A small weight boat with orange extract is placed behind the wall inserts to provide a novel smell for the chambers .","The chambers were cleaned with 70% isopropanol .","During the training sessions , mice were placed in context A and allowed to explore for 3 min prior to the delivery shocks ( 0 . 75 mA , 1 s ) .","We used auditory tone ( 85 dB , 30 s duration ) as CS and an electric foot shock ( delivered through the grid floor , 0 . 75 mA AC current , 1 s duration ) as US .","Mice received 3 tone-shock stimuli ( with 1–2 min interval ) during the training day .","In every session , tone always co-terminated with electric shock .","Mice were removed from the conditioning chamber 30 s after the last shock .","Contextual test ( 5 min ) was done in context A 24 hr after training .","Cue test was performed 1 hr after contextual test in context B . The animals were brought into the test room in a different covered mover with different bedding inside to avoid contextual reminders .","The animal was immediately placed in the contextual B chamber and allowed to explore for 3 min prior to the delivery the auditory tone ( 85 dB ) for another 3 min .","The freezing response was measured with the automated tracking and analyzed offline .","For the novel object recognition test , mice were habituated to the arena for two consecutive days , where they were placed in a white plastic open-box of dimensions 43 . 2 cm2 by 30 . 5 cm height for 10 min each .","The acquisition phase of the object recognition assay involved placing each individual mouse in the test arena for 8 min in the presence of two identical objects ( either silver or black objects in different shape , size and texture , the order of which alternated between mice ) .","The video was recorded via a camera .","The time spent investigating both identical objects was recorded by automatic tracking system ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .","24 hr after the acquisition phase , each mouse was reintroduced to the test arena for 8 min .","During this phase ( retention testing phase ) the mouse was presented with one copy of the object that it was exposed to during the acquisition phase ( familiar object ) and one novel object ( either silver or black objects , depending on which was presented during the acquisition phase ) .","The time spent investigating each object was recorded .","All objects and the testing box were thoroughly cleaned with 70% methanol between mice to remove any odor cues .","Agonist-dependent cellular measurements of bioluminescence resonance energy transfer ( BRET ) between Venus-Gβ1γ2 and its effector fragment masGRK3ct-Nluc were performed upon reconstitution in living cells as previously described with slight modification ( Hollins et al . , 2009; Masuho et al . , 2013 ) .","The masGRK3ct-Nluc construct contained amino acids G495-L688 of bovine GRK3 ( NP_776925 ) , preceded by a myristic acid attachment peptide ( mas; MGSSKSKTSNS ) .","The stop codon of GRK3 was replaced with a GGGS linker , which was followed by the NanoLuc ( Nluc ) ( Hall et al . , 2012 ) .","Briefly , GABAB1R , GABAB2R , GαoA , Venus156-239-Gβ1 , and Venus1-155-Gγ2 constructs were transfected into HEK293T/17 cells at a 1:1:2:1:1 ratio with increasing masGRK3ct-Nluc from 0 . 125 to 6 in ratio .","5 µg total DNA was delivered per 4 × 106 cells in a 6-cm-dish .","16–24 hr post transfection cells were stimulated with 100 µM GABA followed by treatment with 100 µM CGP 54626 .","The BRET signal is determined by calculating the ration of the light emitted by the Venus-Gβ1γ2 ( 535 nm ) over the light emitted by the masGRKct-Rluc8 ( 475 nm ) .","The average baseline value recorded prior to agonist stimulation was subtracted from BRET signal values , and the resulting difference ( ΔBRET ) was obtained .","Statistical analyses were performed using Prism ( GraphPad Software , Inc . ; La Jolla , CA ) .","Data are presented throughout as the mean ± SEM .","Student t test , one-way or two-way ANOVA , followed by Bonferroni’s post hoc test were used as appropriate .","The minimal level of significance was set at p<0 . 05 ."]],"string":"[\n [\n \"Signaling through G protein-coupled receptors for the inhibitory neurotransmitter GABA ( GABABR ) has been recognized to play key roles in mood , nociception , memory , reward , and movement ( Bowery , 2006; Padgett and Slesinger , 2010 ) .\",\n \"In the hippocampus , activation of postsynaptic GABABR on pyramidal neurons produces slow inhibitory postsynaptic currents ( sIPSCs ) , which counteract the excitatory influence of ionotropic glutamate receptors to shape neuronal output ( Ulrich and Bettler , 2007; Luscher and Slesinger , 2010 ) .\",\n \"As a result , GABABR signaling profoundly affects hippocampal synaptic plasticity and has marked effects on memory formation ( Davies et al . , 1991; Wagner and Alger , 1995; Schuler et al . , 2001 ) .\",\n \"A large share of the postsynaptic inhibitory effect of GABABR stimulation in the hippocampus is mediated by activation G protein-gated inwardly-rectifying K+ ( GIRK/Kir3 ) channels , which inhibit neuronal excitability via hyperpolarizing K+ efflux ( Luscher and Slesinger , 2010 ) .\",\n \"In the hippocampus , GIRK channels are predominantly formed by GIRK1 and GIRK2 subunits , which co-localize and may interact directly with GABABR protomers ( Koyrakh et al . , 2005; Fajardo-Serrano et al . , 2013 ) .\",\n \"Activation of GABABR releases G protein βγ subunits , which bind to GIRK channels and increase channel gating ( Padgett and Slesinger , 2010 ) .\",\n \"Blockade of GABABR or GIRK channels by either pharmacological manipulations or genetic knockout ablates the slow IPSC , and blunts a form of hippocampal synaptic plasticity known as depotentiation ( Luscher et al . , 1997; Chung et al . , 2009 ) .\",\n \"Conversely , enhanced GABABR-GIRK signaling seen in a mouse model of Down syndrome disrupts both excitatory and inhibitory synaptic plasticity , and is linked to cognitive impairment ( Kleschevnikov et al . , 2004; Cramer et al . , 2010; Cooper et al . , 2012 ) .\",\n \"GABABR-GIRK signaling is negatively modulated by the Regulators of G protein Signaling ( RGS ) proteins , which accelerate G protein inactivation ( Hollinger and Hepler , 2002; Padgett and Slesinger , 2010 ) .\",\n \"Among more than 30 RGS genes found in mammalian genomes , the R7 family of RGS proteins ( R7 RGS ) stands out for its prominent roles in a range of fundamental neuronal processes , from vision to motor control to reward-related behavior ( Anderson et al . , 2009 ) .\",\n \"The four members of this group ( RGS6 , RGS7 , RGS9 and RGS11 ) form heterotrimers with two subunits ( Gβ5 and R7BP ) , and these interactions regulate the localization and/or expression of the complexes ( Anderson et al . , 2009; Jayaraman et al . , 2009 ) .\",\n \"Previous studies have shown that Gβ5 serves as a central scaffold that bridges the catalytic ( RGS ) and targeting ( R7BP ) subunits and ensures the stability of R7 RGS protein ( Cheever et al . , 2008; Sandiford et al . , 2010; Masuho et al . , 2011 ) .\",\n \"Elimination of Gβ5 also resulted in dramatic slowing of GIRK channel deactivation kinetics , prolongation of synaptically-evoked slow IPSCs in hippocampal pyramidal neurons , and increased behavioral sensitivity to GABABR stimulation ( Xie et al . , 2010 ) .\",\n \"However , the identity of the RGS isoform that modulates GABABR-GIRK signaling in hippocampus , as well as the relative impact of the R7BP subunit , are unknown .\",\n \"Furthermore , the relevance of RGS-dependent modulation of GIRK-dependent signaling to hippocampal circuit function , plasticity , and behavior remain unclear .\",\n \"In this study , we examined the importance of RGS and R7BP subunits to hippocampal physiology and hippocampal-dependent behavior .\",\n \"We report that ablation of Rgs7 results in alterations of GABABR-GIRK signaling , disrupts synaptic plasticity in the hippocampus , and impairs contextual learning and memory .\",\n \"Moreover , the function of the RGS7/Gβ5 complex is fine-tuned by R7BP , which sets the sensitivity range of GABABR-GIRK signaling .\"\n ],\n [\n \"We began by characterizing the expression of RGS complex subunits in the mouse hippocampus .\",\n \"We detected robust expression of RGS6 , RGS7 , R7BP and Gβ5 by western blotting ( Figure 1A ) .\",\n \"To understand the contribution of individual subunits to GABABR-GIRK signaling , we studied the effects of selective knockout of Rgs6 , Rgs7 , and R7bp in mice .\",\n \"Elimination of RGS7 dramatically reduced levels of Gβ5 and R7BP in the hippocampus ( Figure 1A , B ) .\",\n \"In contrast , elimination of RGS6 had no significant effect on the expression of R7BP or Gβ5 .\",\n \"Similarly , loss of one RGS protein did not affect the expression of the other , or the GIRK channel subunit GIRK2 .\",\n \"Given the interdependence of subunit expression in R7 RGS complexes ( Chen et al . , 2003; Anderson et al . , 2007; Grabowska et al . , 2008 ) , these results suggested that RGS7 was likely the dominant catalytic subunit in the hippocampus . 10 . 7554/eLife . 02053 . 003Figure 1 . RGS7 and R7BP modulate GABABR-GIRK signaling in cultured hippocampal neurons .\",\n \"( A ) Western blot analysis of protein expression in hippocampi extracted from wild-type ( WT ) mice , or mice lacking RGS7 ( Rgs7−/− ) , RGS6 ( Rgs6−/− ) or R7BP ( R7bp−/− ) .\",\n \"( B ) Quantification of Western blotting data , with protein levels arrayed as a function of genotype , ***p<0 . 01 , One-Way ANOVA n = 3 mice .\",\n \"ND-undetectable .\",\n \"( C ) Representative normalized traces of GIRK currents evoked by a saturating concentration of the GABABR agonist baclofen ( 100 µM ) .\",\n \"( D–E )\",\n \"Rgs6 , Rgs7 , and R7bp knockouts do not affect GIRK current amplitudes ( D ) , desensitization ( E ) and current onset kinetics ( F ) evoked by 100 μM Baclofen .\",\n \"p>0 . 05 , One-Way ANOVA , n = 8–29 cells for each genotype .\",\n \"( G ) Current deactivation rate following removal of baclofen was slower in neurons from Rgs7−/− and R7bp−/− mice as compared to WT controls , *p<0 . 05 and **p<0 . 01 vs WT , †††p<0 . 001 Rgs7−/− vs R7bp−/− .\",\n \"One-Way ANOVA , Bonferroni’s post hoc test , n = 8–29 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 003 We next compared GABABR-GIRK responses in hippocampal pyramidal neurons from Rgs6 , Rgs7 , and R7bp knockout ( −/− ) and wild-type ( WT ) mice ( Figure 1C–F ) .\",\n \"Application of a saturating concentration ( 100 μM ) of the GABABR agonist baclofen elicited currents with similar maximal amplitudes in neurons of all genotypes ( Figure 1D ) .\",\n \"We also measured the activation and deactivation kinetics of the baclofen-induced currents .\",\n \"At this saturating concentration of baclofen , we observed no significant differences between genotypes in the activation phase of the response ( Figure 1C , F ) .\",\n \"Desensitization of current during the timeframe of agonist application was negligible and similar across genotypes , suggesting that the response at the steady-state is not compounded by the GIRK channel inactivation ( Figure 1C , E ) .\",\n \"While no change in the current deactivation kinetics was observed in Rgs6−/− neurons , elimination of RGS7 markedly slowed response deactivation ( Figure 1C , G ) .\",\n \"The rate of GIRK current deactivation in R7bp–/– neurons was also slower than in wild-type neurons , but the effect was substantially smaller than seen in neurons from Rgs7−/− mice ( Figure 1C , G ) .\",\n \"Importantly , there were no detectable changes in any of the measured response parameters in Rgs6−/− neurons as compared to wild-type , arguing that GABABR-GIRK signaling in hippocampal neurons is modulated by RGS7 and R7BP , but not RGS6 .\",\n \"Given the observed changes in the kinetics of GIRK channel modulation by GABABR , we next sought to determine the impact of ablating Rgs7 and R7bp on the sensitivity of the GIRK response to GABABR activation .\",\n \"Increasing baclofen concentrations caused a progressive enhancement in GIRK-mediated currents in all genotypes ( Figure 2A ) .\",\n \"However , pronounced differences in the concentration-response relationship were evident ( Figure 2B ) .\",\n \"First , in both Rgs7−/− and R7bp−/− neurons , curves were shifted to the left relative to wild-type neurons , indicating that ablation of RGS7 or R7BP increased GABABR-GIRK coupling sensitivity .\",\n \"Second , while RGS7 ablation resulted in a largely parallel leftward shift of the curve , its shape in R7bp−/− neurons was markedly steeper .\",\n \"Indeed , response amplitudes resembled those seen in wild-type neurons at lower agonist concentrations , but at higher concentrations , the responses more resembled those seen in Rgs7−/− neurons . 10 . 7554/eLife . 02053 . 004Figure 2 . Timing and sensitivity of GABABR-GIRK signaling is differentially controlled by RGS7 and R7BP .\",\n \"( A ) Representative traces of GIRK currents evoked by increasing concentrations of baclofen .\",\n \"( B ) Dose-response curves fit by Hill equation .\",\n \"EC50 values are 1 . 48–2 . 14 μM for WT , 0 . 35–0 . 58 μM for Rgs7−/− , and 0 . 65–0 . 81 μM for R7bp−/− ( 95% CI ) .\",\n \"Hill coefficients were 1 . 48 ± 0 . 17 in WT , 1 . 23 ± 0 . 21 in Rgs7−/− , and 2 . 04 ± 0 . 28 in R7bp−/− .\",\n \"p<0 . 0001 for difference in EC50 or each curve; p=0 . 04 for difference in Hill coefficients , Extra sum-of-squares F test , n = 10–22 cells .\",\n \"( C ) Rgs7−/− cultured hippocampal neurons show slower current activation at lower concentrations of baclofen .\",\n \"***p<0 . 001 vs WT , †††p<0 . 001 for Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells .\",\n \"( D ) Dependence of GIRK current deactivation kinetics on agonist concentration .\",\n \"*p<0 . 05 and ***p<0 . 001 vs WT; ††p<0 . 01 , †††p<0 . 001 for Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells .\",\n \"( E–G )\",\n \"Differences in the lag times before the onset of response deactivation ( Lag Deactivation ) .\",\n \"( E ) Representative traces of currents evoked by 100 µM baclofen show Lag time measurement as time between the onset of agonist removal and the point at which 10% of the current deactivated: 1560 , 4900 and 3000 ms for WT , Rgs7−/− , R7bp−/− , respectively .\",\n \"( F ) Semi-log plot of lag time dependence on concentration .\",\n \"The data were fitted with a linear regression , R2 = 0 . 66 , 0 . 67 , and 0 . 84; and slopes 619 ± 69 , 1440 ± 151 , and 952 ± 84 for WT , Rgs7−/− , R7bp−/− , correspondingly .\",\n \"p<0 . 0001 for differences in slopes , two way ANOVA , n = 10–22 cells .\",\n \"( G ) Comparison of lag times at baclofen concentrations that generated equivalent responses .\",\n \"Lag times were significantly different in genotypes at saturating ( EC90 ) but not submaximal ( EC60 ) concentrations , *p<0 . 05 and ***p<0 . 001 vs WT , ††p<0 . 01 Rgs7−/− vs R7bp−/− , Two-Way ANOVA , Bonferroni’s post hoc test , n = 10–22 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 004 We next tested whether GABABR-GIRK current kinetics exhibited a similar dependence on agonist concentration .\",\n \"Comparing activation rates across different concentrations of baclofen revealed that onset kinetics were affected by RGS7 ablation only at low baclofen concentrations , whereas no effect of R7BP elimination was seen at any level of GABABR stimulation ( Figure 2C ) .\",\n \"Since GIRK activation kinetics could be influenced by the G protein deactivation cycle ( Doupnik et al . , 1997; Lambert et al . , 2010 ) , we did not put significant emphasis on the analysis of the differences in the raising phases of the response .\",\n \"The impact of RGS7 ablation on deactivation rates was consistent at different agonist concentrations ( Figure 2D ) .\",\n \"In contrast , R7BP ablation significantly affected deactivation kinetics only at higher agonist concentrations , consistent with the effects on response sensitivity ( Figure 2D ) .\",\n \"Analysis of the responses also revealed that elimination of RGS7 and R7BP resulted in a significant delay between agonist removal and the beginning of current deactivation ( Figure 2E ) .\",\n \"This lag time showed an exponential dependence on agonist concentration that continued to develop past the saturation point for the maximal GIRK response ( ∼10 μM baclofen ) in all genotypes ( Figure 2F ) .\",\n \"While there was no difference in lag time between genotypes at non-saturating baclofen concentrations ( ∼EC60 ) , it became pronounced as the current response reached saturation ( ∼EC90 ) ( Figure 2G ) .\",\n \"Combined with the observations that GABABR continues to increase the amount of activated G proteins past the saturation point of the GIRK channel response ( Hensler et al . , 2012 ) , these data suggest that the lag time reflects the clearance of free βγ subunit produced above the stoichiometric level relative to the GIRK channel .\",\n \"To provide an independent evidence that increase in the lag time reflects changes in stoichiometry of Gβγ subunits relative to their effector molecule , we utilized a bioluminescence resonance energy transfer ( BRET ) -based approach that monitors interactions of Gβγ with a reporter derived from an effector ( GRK3 ) upon reconstitution in transfected cells ( Figure 3A ) .\",\n \"In this assay , we induced production of free Gβγ subunits via GABABR activation and then measured delay time between antagonizing GABABR and the onset of signal decay , while changing the Gβγ to GRK3 effector ratio ( Figure 3B ) .\",\n \"In agreement with the electrophysiological recordings of GIRK channel activity , BRET experiments showed that the increase in Gβγ stoichiometry over an effector results in prolongation of the response deactivation lag ( Figure 3C , D ) . 10 . 7554/eLife . 02053 . 005Figure 3 . Changing the ratio of Gβγ to an effector affects response deactivation lag in a reconstituted system .\",\n \"( A ) Schematic representation of the assay principle .\",\n \"The assay measures interaction of YFP-tagged Gβγ with its effector reporter GRK , tagged with N-luc , producing BRET signal .\",\n \"Gβγ subunits are released upon GABABR stimulation with GABA increasing BRET signal .\",\n \"Inactivation of GABABR with an antagonist CGP 54626 results in dissociation of Gβγ from GRK and re-association with Gαo to form inactive heterotrimer .\",\n \"( B ) Time course of changes in BRET signal upon stimulation of cells with GABA and subsequent deactivation by CGP 54626 .\",\n \"Cells were transfected with varying amounts of constructs encoding Gβγ and GRK reporter ( from black to green ) .\",\n \"( C ) Deactivation phase of the response showing kinetics of signal decay .\",\n \"The lag deactivation time ( dotted line for the trace in black ) is defined as the time that it takes to quench the BRET signal by 10% from its steady state value in the presence of an agonist .\",\n \"( D ) Quantification of a lag deactivation time as a function of Gβγ/GRK ratio .\",\n \"Error bars are SEM values , n = 4 per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 005 In transfected cells , R7BP is essential for the membrane localization of RGS7 ( Drenan et al . , 2005; Narayanan et al . , 2007 ) .\",\n \"Furthermore , we previously reported that knockout of R7bp resulted in a reduction of the total membrane-bound RGS7 protein in hippocampal tissue ( Panicker et al . , 2010 ) .\",\n \"To analyze the effect of R7BP on RGS7 localization in hippocampal pyramidal neurons , we performed high-resolution immunoelectron microscopy .\",\n \"Consistent with the earlier findings , immunoparticles for RGS7 were abundant on the extrasynaptic plasma membrane of dendritic spines and dendritic shafts of pyramidal cells , as well as at intracellular sites ( Fajardo-Serrano et al . , 2013 ) .\",\n \"In the hippocampus of R7bp−/− animals , immunoparticles for RGS7 were distributed similarly to the wild-type , but they were more frequently observed just beneath the plasma membrane and more broadly distributed in somata ( Figure 4 ) .\",\n \"Indeed , quantitative analysis indicated that in R7bp−/− mice , RGS7 was less frequently detected in the plasma membrane and tended to accumulate within 100 nm of the plasma membrane .\",\n \"We also detected a significant increase in RGS7 labelling in the rough endoplasmic reticulum ( rER ) in the soma of R7bp−/− pyramidal neurons ( 2489 vs 3371 immunoparticles in wild-type and R7bp−/− neurons , respectively ) .\",\n \"These findings indicate that knockout of R7BP caused a modest but significant redistribution of RGS7 away from the plasma membrane . 10 . 7554/eLife . 02053 . 006Figure 4 . Change in subcellular localization of RGS7 in the hippocampus of the R7bp−/− mice . Electron micrographs of the stratum radiatum of the hippocampal CA1 region showing immunoparticles for RGS7 , as detected using a pre-embedding immunogold method .\",\n \"Dendritic spines ( s ) and axon terminals ( at ) are marked .\",\n \"Arrows indicate locations of immunoparticles at the plasma membrane , while arrowheads identify RGS7 immunoparticles found just below the membrane .\",\n \"Quantitative analysis showed that RGS7 is less frequently detected in the plasma membrane , and accumulates within the first 100 nm from the plasma membrane , samples from R7bp−/− mice , *p<0 . 05 , One-way ANOVA followed by the Bonferroni’s post hoc test , n = 3 mice .\",\n \"Scale bar: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 006 The slower kinetics and increased sensitivity of GIRK channels to GABABR activation suggested that there was a net up-regulation of GIRK-dependent inhibition in hippocampal neurons lacking RGS7 .\",\n \"Since GIRK channels significantly contribute to setting neuronal excitability ( Chen and Johnston , 2005 ) , we next determined how deletion of RGS7 influences the excitability of CA1 pyramidal neurons .\",\n \"To characterize intrinsic electrophysiological properties of different genotypes , the responses of CA1 neurons to somatic current injections ranging from −150 pA to +300 pA were measured ( Figure 5A ) .\",\n \"While membrane resistance was similar in both genotypes ( Rin = 120 ± 10 MΩ vs 105 ± 11 MΩ , p=0 . 31 , in wild-type and Rgs7−/− cells , respectively ) , resting membrane potential ( RMP ) was significantly hyperpolarized in Rgs7−/− neurons ( −64 . 7 ± 0 . 5 mV vs −68 . 5 ± 0 . 7 mV in wild-type and Rgs7−/− , respectively; Figure 5B ) .\",\n \"In addition , the current required to elicit action potentials ( APs ) was significantly higher in RGS7−/− neurons ( I = 104 . 3 ± 8 . 8 pA vs 146 . 4 ± 17 . 0 , in WT and Rgs7−/− cells , respectively; Figure 5C ) .\",\n \"Finally , Rgs7−/− neurons fired significantly fewer action potentials in response to depolarizing current as compared to wild-type controls ( Figure 5D ) .\",\n \"Collectively , these observations argued that hippocampal pyramidal neurons from Rgs7−/− mice were less excitable than wild-type counterparts . 10 . 7554/eLife . 02053 . 007Figure 5 . Altered intrinsic excitability and excitatory transmission in CA1 hippocampal neurons from Rgs7−/− mice .\",\n \"( A ) Representative traces of responses elicited by current injections of −150 , −50 , 0 , +50 , and +300 pA in WT and Rgs7−/− CA1 neurons .\",\n \"( B ) Hyperpolarized resting membrane potential ( RMP ) in Rgs7−/− neurons , ***p=0 . 0001 , t test , n = 14–23 cells .\",\n \"( C ) Current required to evoke an action potential ( firing threshold ) is higher in Rgs7−/− neurons , *p=0 . 02 , t test , n = 14–23 cells .\",\n \"( D ) Lower intrinsic excitability in Rgs7−/− neurons .\",\n \"**p<0 . 01 and *p<0 . 05 , two-way ANOVA with Bonferroni’s posttest , n = 14–23 cells .\",\n \"( E ) Representative traces of slow excitatory synaptic currents ( sEPSCs ) in WT and Rgs7−/− cells .\",\n \"( F ) Cumulative distribution and mean values for sEPSCs amplitudes ( 14 . 9 ± 1 . 9 vs 12 . 8 ± 0 . 9 pA in WT and Rgs7−/− correspondingly , p=0 . 3 , unpaired t test; n = 11 cells and 1100 events for each genotype ) .\",\n \"( G ) Cumulative distribution and mean values for sEPSCs and frequencies ( 3 . 4 ± 0 . 6 vs 3 . 1 ± 0 . 6 Hz in WT and Rgs7−/− correspondingly , p=0 . 7 , unpaired t test , n = 11 cells and 1100 events for each genotype ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 007 We further examined whether decreased intrinsic excitability of Rgs7−/− neurons is caused by dysregulated net excitatory transmission .\",\n \"For this purpose , we measured spontaneous EPSCs ( sEPSC ) in CA1 pyramidal neurons ( Figure 5E–G ) .\",\n \"There were no significant differences in either amplitudes or frequencies of sEPSC events between Rgs7−/− and wild-type neurons ( Figure 5F–G ) .\",\n \"These observations indicate that excitatory input into CA1 pyramidal neurons is unchanged by elimination of RGS7 and suggest that the observed decrease in neuronal excitability is caused by changes in the intrinsic membrane properties of CA1 neurons , likely stemming from enhanced activity of the postsynaptic GIRK channel .\",\n \"Previous studies have implicated GIRK channels in several forms of hippocampal synaptic plasticity , including long-term potentiation ( LTP ) ( Cramer et al . , 2010 ) , long-term depression ( LTD ) ( Cooper et al . , 2012 ) and depotentiation ( DP ) ( Chung et al . , 2009; Cooper et al . , 2012 ) .\",\n \"Because GABAB receptors play key roles in these processes and since GABAB-GIRK signaling is severely dysregulated in Rgs7−/− neurons , we next examined the impact of RGS7 ablation on hippocampal synaptic plasticity .\",\n \"A high-frequency stimulation protocol of 2 tetanized stimuli ( TS , 100 Hz for 1s each ) produced robust LTP in both genotypes ( Figure 6A , D ) .\",\n \"The extent of the potentiation in Rgs7−/− slices was not significantly different from that measured in wild-type slices , arguing that elimination of RGS7 has no effect on LTP .\",\n \"A low-frequency stimulation ( LFS , 2 Hz for 10 min , 1200 pulses ) elicited LTD in both wild-type and Rgs7−/− hippocampal slices ( Figure 6B , D ) .\",\n \"However , the extent of inhibition was significantly smaller in Rgs7−/− slices .\",\n \"Interestingly , the magnitude of the first fEPSP after the train of LFS stimulation was significantly reduced in knockout mice ( 51 ± 6% and only 72 ± 6% in wild-type and Rgs7−/− slices correspondingly , *p=0 . 03 , t test , n = 6 ) , suggesting that the LTD impairment in Rgs7−/− is due to a deficit in an induction mechanism ( s ) .\",\n \"The depotentiation produced by consecutive application of TS and LFS was also significantly impaired in Rgs7−/− mice ( Figure 6C , D ) . 10 . 7554/eLife . 02053 . 008Figure 6 . RGS7 ablation disrupts hippocampal synaptic plasticity . Field EPSP ( fEPSP ) slope change following induction of: ( A ) LTP in Rgs7−/− , 166 ± 9% vs wild-type , 155 ± 6% slices; p=0 . 33 , t test , n = 6–10; ( B ) LTD in Rgs7−/− , 90 ± 2% vs wild-type , 81 ± 2%; *p=0 . 01 , t test , n = 6–7; ( C ) depotentiation ( DP ) in Rgs7−/− , 111 ± 3% vs wild-type , 128 ± 6%; *p=0 . 013 , t test , n = 6–11 .\",\n \"Insets show representative fEPSP traces at baseline and 1 hr following induction protocol in WT ( black and grey ) and Rgs7−/− ( red and pink ) slices .\",\n \"( D ) Quantification of the EPSP slope change 55–60 min following induction of each form of plasticity after normalization to pre-induction baseline .\",\n \"( E ) Paired Pulse Ratio ( PPR ) dependence on the inter stimulus interval for wild-type ( WT ) and Rgs7−/− .\",\n \"Significant inter-stimulus interval but not genotype effect was observed , p<0 . 0001 and p=0 . 5 , correspondingly .\",\n \"Two-way ANOVA , n = 4 slices per genotype .\",\n \"( F–H )\",\n \"Basal synaptic transmission properties in Rgs7−/− slices .\",\n \"Dependence of fEPSP slope ( F ) and FV amplitude ( G ) on stimulus intensity .\",\n \"( H ) Linear regression plot of fEPSP slope dependence on FV amplitude .\",\n \"The data were fitted with a linear regression , R2 = 0 . 67 and 0 . 53; and slopes 1 . 9 ± 0 . 1 and 1 . 5 ± 0 . 2 for WT and Rgs7−/−correspondingly .\",\n \"p=0 . 1 for differences in slopes , two way ANOVA , n = 12–13 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 008 We further investigated the impact of RGS7 ablation on paired-pulse facilitation ( PPF ) , a form of short-term plasticity that results from the enhancement of presynaptic vesicle release in response to two closely-spaced stimuli ( Zucker , 1989; Dobrunz and Stevens , 1997 ) .\",\n \"We found that fEPSP facilitation was similar between genotypes across the examined range of 10–1000 ms interpulse intervals ( Figure 6E ) , indicating that presynaptic mechanisms are not likely to be the cause of the observed alterations in synaptic plasticity .\",\n \"We next compared the dependence of fEPSP slopes ( Figure 6F ) and presynaptic fibre volley ( FV ) amplitudes ( Figure 6G ) on stimulus intensity .\",\n \"Analysis of the relationship between FV amplitudes and fEPSP slopes by linear fitting reveals their nearly perfect correspondence between wild-type and Rgs7−/− slices , indicating preservation of basal synaptic transmission properties ( Figure 6H ) .\",\n \"Together , these results suggest that RGS7 elimination selectively impaired LTD and DP forms of synaptic plasticity , likely by a post-synaptic mechanism .\",\n \"Changes in the intrinsic excitability of CA1 pyramidal neurons , together with deficits in hippocampal synaptic plasticity , suggested that RGS7 complexes play a role in spatial learning and memory .\",\n \"To test this possibility , we first studied the impact of RGS or R7BP ablation in contextual fear conditioning , a test that requires hippocampal processing for memory formation ( Maren , 2001 ) .\",\n \"We found significant deficits in context recognition 24-hr after training in Rgs7−/− mice , but not in Rgs6−/− or R7bp−/− mice ( Figure 7A–C ) .\",\n \"Importantly , we observed no difference in baseline freezing behavior before associative training between the genotypes .\",\n \"Furthermore , there were no significant differences between wild-type mice and mutant mice in amygdala-dependent cue recognition . 10 . 7554/eLife . 02053 . 009Figure 7 . RGS ablation affects hippocampal-dependent learning and memory .\",\n \"( A–C )\",\n \"Evaluation of mouse behavior in fear conditioning paradigm .\",\n \"( A ) Rgs7−/− mice show deficits in hippocampal-dependent contextual , but not cue , memory .\",\n \"*p<0 . 05 , t test; n = 12 per genotype .\",\n \"( B ) Normal contextual and cue memory of Rgs6−/− mice in the fear conditioning test as compared to wild-type ( WT ) littermates .\",\n \"n = 8 per genotype .\",\n \"( C ) R7bp−/− mice showed the same contextual and cue memory in fear conditioning test as compared to WT littermates , n = 8 per genotype .\",\n \"( D–F )\",\n \"Evaluation of mouse behavior in Morris water maze .\",\n \"Rgs7−/− mice ( n = 13 ) and their WT littermates ( n = 12 ) were trained for 6 d with 4 trials/d with an inter-trial interval of approximately 15 min .\",\n \"Performance during the acquisition phase was monitored and plotted as average time ( D ) or success rate ( E ) to reach the hidden platform .\",\n \"Mice showed improvement with training .\",\n \"There was a significant effect of a genotype in both escape latencies ( *p<0 . 05 ) and success rates ( *p<0 . 05 ) using two-way ANOVA analysis .\",\n \"Post hoc comparison revealed a significant impairment of Rgs7−/− mice during the last two acquisition days ( *p<0 . 05 , Tukey’s test ) .\",\n \"( F ) Results of a probe trial given 24 hr after 6 d of training .\",\n \"The latency to the first crossing of the former location of the platform and the total number of crossing are shown , *p<0 . 05 , t test .\",\n \"( G ) Evaluation of mouse behavior in novel object recognition paradigm .\",\n \"Rgs7−/− mice showed significant impairment during the test trial with a novel object in comparison with wild-type littermates .\",\n \"*p<0 . 05 , t test; n = 12–13 . DOI: http://dx . doi . org/10 . 7554/eLife . 02053 . 009 To clarify the impairment of hippocampus dependent learning and memory in Rgs7−/− mice , we conducted other tests that rely on hippocampal function .\",\n \"In the Morris water maze test , Rgs7−/− mice showed delayed escape latencies and reduced success rates during acquisition trials ( Figure 7D , E ) .\",\n \"In the probe trial , when the platform was removed 24 hr after training , it took Rgs7−/− mice significantly longer to reach the target area where the platform was located during training as compared to their wild-type littermates ( Figure 7F ) .\",\n \"Mice lacking RGS7 also made fewer crossings over the target area during the probe trial .\",\n \"Importantly , despite a pronounced spatial impairment , Rgs7−/− mice performed normally in the visible platform version of water maze .\",\n \"In the novel object recognition test , wild-type littermates spent significantly more time exploring novel objects over familiar objects ( Figure 7G ) .\",\n \"However , Rgs7−/− mice spent approximately equal time exploring familiar and novel objects .\",\n \"Furthermore , there was a significant difference in time exploring novel objects between Rgs7−/− mice and their wild-type littermates .\",\n \"No genotype differences in object exploratory behavior were observed during the training day , when the mice were first introduced to identical objects .\",\n \"Taken together , these results indicate that elimination of RGS7 leads to a disruption of spatial learning and memory in mice .\"\n ],\n [\n \"The results of this study , together with prior investigations , establish the RGS7/Gβ5/R7BP complex as an essential regulator of GABABR-GIRK signaling in the hippocampus .\",\n \"In hippocampal neurons , RGS7 is closely co-localized with both GABABR and GIRK2-containing channels ( Fajardo-Serrano et al . , 2013 ) .\",\n \"Furthermore , in transfected cells , RGS7 can directly interact with GIRK channel subunits as demonstrated by both co-immunoprecipitation and bioluminescence energy transfer approaches ( Xie et al . , 2010; Zhou et al . , 2012 ) .\",\n \"The complex may further involve GABABR as well as G protein subunits ( Kovoor and Lester , 2002; Fajardo-Serrano et al . , 2013 ) , supporting the contention that components of the entire pathway are scaffolded into a larger macromolecular assembly .\",\n \"The interaction between RGS7 with GIRK channels is mediated by the Gβ5 subunit , which mimics signal-transducing Gβγ subunits in binding to the channel ( Xie et al . , 2010; Zhou et al . , 2012 ) .\",\n \"Knockout of Gβ5 greatly slows GABABR-GIRK response deactivation rates and dramatically increases agonist sensitivity of the signaling pathway ( Xie et al . , 2010 ) .\",\n \"In addition to mediating interactions with the channel , Gβ5 is essential for the expression of all four R7 RGS proteins ( Chen et al . , 2003 ) .\",\n \"With several R7 RGS proteins expressed in hippocampus , the identity of the exact isoform that participates in GIRK channel regulation in the region , through complex formation with Gβ5 , remained unknown .\",\n \"The elimination of RGS7 , reported in this study , largely phenocopies the loss of Gβ5 , both in terms of kinetics and agonist sensitivity .\",\n \"This argues that in hippocampal pyramidal neurons , GIRK signaling is regulated by RGS7 and not by other R7 RGS proteins .\",\n \"Indeed , while RGS6 regulates GIRK signaling in cerebellar neurons ( Maity et al . , 2012 ) and sinoatrial pacemaking cells ( Posokhova et al . , 2010; Yang et al . , 2010 ) , RGS6 ablation had no effect on any GIRK response parameters in hippocampal pyramidal neurons .\",\n \"The function of R7 RGS proteins is regulated by the membrane anchor R7BP , which augments the ability of R7 RGS proteins to deactivate G protein signaling in reconstituted systems ( Drenan et al . , 2006; Masuho et al . , 2013 ) .\",\n \"Consistent with these observations , we show that knockout of R7BP reduces plasma membrane localization of RGS7 and slows GABABR-GIRK current deactivation in hippocampal pyramidal neurons .\",\n \"Our results also agree well with a recent study reporting similar deceleration of GIRK deactivation kinetics in R7bp−/− hippocampal neurons ( Zhou et al . , 2012 ) .\",\n \"However , the consequences of RGS7 and R7BP elimination are different in two important ways .\",\n \"First , relative to the loss of RGS7 or Gβ5 , elimination of R7BP had a very modest effect on GABABR-GIRK deactivation kinetics , and was seen only at high GABABR agonist concentrations .\",\n \"This suggests that the RGS7/Gβ5 complex can regulate GABABR-GIRK signaling without R7BP .\",\n \"Secondly , loss of R7BP and RGS7 has different effects on GABABR-GIRK coupling sensitivity .\",\n \"While elimination of RGS7 ( or Gβ5 ) results in a parallel leftward shift in the concentration-response relationship , the concentration-response measured in R7bp−/− neurons shows markedly greater cooperativity .\",\n \"Thus , at low levels of GABABR stimulation , R7BP is dispensable and its elimination fails to affect response amplitude .\",\n \"As the response reaches saturation , R7BP becomes indispensable and its elimination has the same effect on response amplitude as loss of RGS7 .\",\n \"These observations are inconsistent with the proposed role for R7BP as a critical factor in the assembly of RGS7-GIRK complexes ( Zhou et al . , 2012 ) , which postulates the equivalence of R7BP effects across agonist concentrations and a similar impact of R7BP and RGS7 elimination on GIRK channel kinetics , and certainly not an increase in cooperativity of GIRK channel activation .\",\n \"Based on our findings , we propose an alternative model , where R7BP sets GABABR-GIRK coupling efficiency .\",\n \"In this model , at low levels of GIRK activation , when released Gβγ subunits do not saturate the GIRK channel , deactivation kinetics are primarily mediated by RGS7/Gβ5 directly associated with the GIRK subunits .\",\n \"Under these conditions , there are virtually no genotype-dependent differences in the lag time of GIRK deactivation upon agonist removal .\",\n \"With higher agonist concentrations , as GABABR produces more Gβγ subunits , the amount of available free Gβγ exceeds that of activatable GIRK channels , evidenced by a delay that precedes GIRK current deactivation upon agonist removal .\",\n \"Under these conditions , R7BP elimination affects response sensitivity as much as elimination of RGS7 .\",\n \"While response deactivation kinetics reflect Gβγ inactivation after it had a chance to interact with the channel , response sensitivity likely relates to the efficiency of the Gβγ reaching the GIRK channel .\",\n \"Considering that R7BP affects both of these parameters only when free Gβγ is produced in excess , we think that the RGS7/Gβ5 complex exists in two states: ( 1 ) anchored to the GIRK complex where it affects deactivation kinetics , and ( 2 ) anchored via R7BP to the plasma membrane outside of GIRK complex where it primarily determines response sensitivity .\",\n \"In agreement with this model , we observe an increase in cooperativity of GIRK channel activation upon R7BP loss that suggests that RGS7/Gβ5 alone , but not in complex with R7BP , promotes more productive association of Gβγ with the channel .\",\n \"On an intuitive level , this may reflect larger proportion of Gβγ subunits that reach GIRK while being deactivated in its vicinity by RGS7/Gβ5 .\",\n \"In contrast , complexes of RGS7/Gβ5 with R7BP may predominantly act elsewhere on the plasma membrane , thereby decreasing the number of Gβγ subunits capable of reaching the channel .\",\n \"By physically binding to the GIRK channel , RGS7/Gβ5 may further act to promote productive interactions of Gβγ ( and/or Gα ) with the channel , thus increasing cooperativity of its activation .\",\n \"This model is also consistent with the observed decrease in RGS7 on the plasma membrane but largely preserved localization at specific postsynaptic sites where it might be anchored via complex formation with GIRK channels .\",\n \"Thus , the main role of R7BP appears to be in tuning the sensitivity of the response by endowing RGS7/Gβ5 complexes with an ability to deactivate G proteins before they reach the GIRK channel .\",\n \"RGS proteins are potent negative regulators of both the extent and duration of neurotransmitter signaling via G protein-coupled receptors ( Sjogren , 2011; Xie and Martemyanov , 2011 ) .\",\n \"As a result , knockout of individual RGS genes in mice is frequently associated with augmented GPCR signaling .\",\n \"This makes the analysis of changes in sensitivity of behavioral or cellular reactions to neurotransmitter actions associated with elimination of individual RGS proteins a powerful strategy that allows for establishing physiological receptor-RGS pairings .\",\n \"Using this strategy , two RGS proteins have been previously implicated in controlling GABABR signaling in vivo: RGS6 in cerebellar neurons ( Maity et al . , 2012 ) and RGS2 in the ventral tegmental area ( Labouebe et al . , 2007 ) .\",\n \"Now , our findings establish that in hippocampal neurons , GABABR signaling is negatively regulated by RGS7 .\",\n \"These observations complement earlier findings with RGS14 , the only other RGS protein implicated in hippocampal synaptic plasticity and spatial learning ( Lee et al . , 2010 ) .\",\n \"However , in contrast to RGS7 that acts in the CA1 region , RGS14 functions in CA2 synapses and its ablation augments LTP and improves spatial learning in mice .\",\n \"The major forms of synaptic plasticity ( LTP , LTD and depotentiation ) in the hippocampus have been implicated in learning and memory ( Martin et al . , 2000 ) .\",\n \"Recent models propose that all three forms cooperate to affect distinct aspects of spatial information storage ( Kemp and Manahan-Vaughan , 2007 ) .\",\n \"In relevance to this thinking , we found a selective disruption of LTD and depotentiation in RGS7−/− mice , but normal LTP .\",\n \"There is a mounting evidence suggesting an active and selective role of LTD in object recognition and creation of spatial representation of memory ( Kemp and Manahan-Vaughan , 2007 ) .\",\n \"For example , LTD but not LTP was found to be important for spatial memory consolidation and memory enhancement for novelty acquisition ( Ge et al . , 2010; Dong et al . , 2012 ) .\",\n \"Consistent with this , electrophysiological recordings in behaving animals show facilitation of LTD during novelty exploration ( Xu et al . , 1997 , 1998; Manahan-Vaughan and Braunewell , 1999; Dong et al . , 2012 ) .\",\n \"Disruption of LTD via genetic mutations often results in memory deficits , particularly affecting behavioral flexibility ( Nicholls et al . , 2008 ) , while LTD enhancement can lead to improvement in spatial reversal learning ( Duffy et al . , 2008 ) .\",\n \"In particular , our findings resemble the phenotype of SRF−/− mice , which exhibited a selective disruption of LTD in hippocampal CA1 neurons paralleled by an inability to learn hippocampus-dependent tasks ( Etkin et al . , 2006 ) .\",\n \"While specific knowledge on how depotentiation contributes to memory is still lacking , it has been observed that exposure to novel environment and spatial exploration depotentiates previously-induced LTP ( Xu et al . , 1998 ) .\",\n \"Together , our observations in mice lacking RGS7 reinforce the idea that normal inhibitory synaptic plasticity ( LTD and depotentiation ) is required for hippocampal-dependent learning and memory .\",\n \"Although , it has never been systematically addressed in a single study , GIRK channel elimination was reported to enhance LTP ( Cramer et al . , 2010 ) .\",\n \"Given that the Girk2−/− neurons are relatively depolarized , the enhanced LTP likely results from a general increase in excitability .\",\n \"Conversely , GIRK2 over-expression in transgenic models of Down syndrome leads to reduced LTP ( Kleschevnikov et al . , 2004; Siarey et al . , 2005 ) , but enhanced LTD ( Siarey et al . , 2005; Cooper et al . , 2012 ) .\",\n \"Interestingly , GIRK2 over-expression and ablation both resulted in decreased depotentiation ( Chung et al . , 2009; Cooper et al . , 2012 ) .\",\n \"Similar to Down syndrome mouse models , Rgs7−/− mice also exhibited augmented GABABR-GIRK signaling .\",\n \"However , in the case of Rgs7−/− mice , this augmentation resulted in yet another distinct phenotype: selective deficits in LTD and depotentiation , with normal LTP .\",\n \"Several lines of evidence argue that these changes in synaptic plasticity are driven by a postsynaptic mechanism whereby RGS7 controls inhibitory GIRK-dependent signaling in the CA1 pyramidal neurons .\",\n \"First , CA1 pyramidal neurons lacking RGS7 are hyperpolarized and less excitable relative to wild-type neurons , a property that is significantly shaped by the GIRK channels .\",\n \"Second , basal excitatory transmission is unaltered in Rgs7−/− slices .\",\n \"Third , direct measurements of events that reflect excitatory presynaptic function ( e . g . , FV and PPR ) reveal no changes caused by the elimination of RGS7 .\",\n \"Given these observations , we propose that GIRK signaling sets the general excitability of postsynaptic pyramidal neurons , which in turn determines a sliding scale window for the induction of different forms of synaptic plasticity .\",\n \"By adjusting signaling strength in the hippocampal GABABR-GIRK pathway , the RGS7/Gβ5/R7BP complex influences the range of neuronal responses necessary for memory formation .\",\n \"While dysregulation of GABABR-GIRK signaling in hippocampus may be sufficient for explaining the effects of RGS7 on learning an memory , at this point we cannot rule out a contribution of other brain regions and/or signaling pathways in the process .\",\n \"In any event , we believe that the results of this study establish RGS7 complex as an important molecule for understanding and/or correcting the pathology of neuropsychiatric disorders associated with disruptions in synaptic plasticity and imbalances in inhibitory signaling .\"\n ],\n [\n \"All studies were carried out in accordance with the National Institute of Health guidelines and were granted formal approval by the Institutional Animal Care and Use Committee of the Scripps Research Institute .\",\n \"The generation of Rgs6−/− ( Posokhova et al . , 2010 ) , Rgs7−/− ( Cao et al . , 2012 ) , R7bp−/− ( Anderson et al . , 2007 ) mice were described earlier .\",\n \"All animals used for comparing genotypes were littermates derived from heterozygous breeding pairs .\",\n \"Mice were housed in groups on a 12 hr light–dark cycle with food and water available ad libitum .\",\n \"Males and females ( 2–5 months ) were used for all experiments .\",\n \"Lysates were prepared by homogenizing hippocampal tissue from age-matched littermates by sonication in the lysis buffer ( 1 × PBS , 150 mm NaCl , 1% Triton X-100 , protease inhibitors ) followed centrifugation at 14 , 000×g for 10 min .\",\n \"The resulting extract was used for protein concentration determination by the BCA protein assay ( Pierce , Rockford , IL ) .\",\n \"The lysates were adjusted to equalize total protein content by adding lysis buffer and 2x SDS sample buffer .\",\n \"Samples were boiled for 5 min , resolved on SDS-PAGE gels , transferred onto PVDF membrane and subjected to western blot analysis using HRP conjugated secondary antibodies and ECL West Pico ( Pierce ) detection system .\",\n \"Signals were captured on film and scanned by densitometer , and band intensities were determined using NIH ImageJ software .\",\n \"Rabbit anti-R7BP ( TRS ) and rabbit Gβ5 ( ADTG ) were generous gifts from Dr William Simonds ( NIDDK/NIH ) .\",\n \"Anti-RGS6 was generated and used previously ( Posokhova et al . , 2010 ) .\",\n \"Chicken IgY anti-RGS7 antibody was from Thermo Scientific ( Waltham , MA ) , anti-GIRK2 was purchased from Alomone labs ( Jerusalem , Israel ) and anti-β-actin was from Sigma ( St . Lois , MO ) .\",\n \"Primary cultures of hippocampal neurons were prepared using a modified version of a published protocol ( Xie et al . , 2010 ) .\",\n \"Briefly , hippocampi were extracted from neonatal ( P1-3 ) pups and placed into an ice-cold HBSS/FBS solution: Hank’s Balanced Salt Solution ( Sigma; St . Louis , MO ) , 4 . 2 mM NaHCO3 , 1 mM HEPES , and 20% FBS .\",\n \"The tissue was washed twice with HBSS/FBS , and then three times with HBSS alone .\",\n \"Hippocampi were digested at room temperature for 5 min with 10 mg/ml Trypsin Type XI ( Sigma; St . Louis , MO ) in a solution that contained ( in mM ) : 137 NaCl , 5 KCl , 7 Na2HPO4 , and 25 HEPES ( pH 7 . 2 ) .\",\n \"The tissue was washed twice with HBSS/FBS and three times with HBSS alone , and then hippocampi were mechanically-dissociated in HBSS ( supplemented with 12 mM MgSO4 ) using Pasteur pipettes of decreasing diameter .\",\n \"The neurons were pelleted by centrifugation ( 600×g for 10 min at 4°C ) and plated onto 8-mm glass coverslips pre-treated with Matrigel ( BD Biosciences; San Jose , CA ) in 48-well plate .\",\n \"Neurons were allowed to adhere for 30 min prior to adding 0 . 3 ml of pre-warmed culture medium consisting of Neurobasal A ( Life Technologies; Carlsbad , CA ) , 2 mM GlutaMAX-I ( Life Technologies , Carlsbad , CA ) , 2% B-27 supplement , and 5% FBS .\",\n \"After 4–12 hr , culture media was completely replaced with the same media without FBS .\",\n \"Neurons were incubated at 37°C/5% CO2 , and half of the medium was replaced with fresh medium on each of the first 3 days of culture .\",\n \"Neurons were kept in culture for 10–14 days prior to experiments .\",\n \"Coverslips containing neurons were transferred to a chamber containing a low-K+ bath solution ( in mM ) : 145 NaCl , 4 KCl , 1 . 8 CaCl2 , 1 MgCl2 , 5 . 5 D-glucose , 5 HEPES/NaOH ( pH 7 . 4 ) .\",\n \"Borosilicate patch pipettes ( 3–5 MΩ ) were filled with ( in mM ) : 130 KCl , 10 NaCl , 1 EGTA/KOH ( pH 7 . 2 ) , 0 . 5 MgCl2 , 10 HEPES/KOH ( pH 7 . 2 ) , 2 Na2ATP , 5 phosphocreatine , 0 . 3 GTP .\",\n \"Baclofen ( R- ( + ) -b- ( aminomethyl ) -4-chlorobenzenepropanoic acid hydrochloride ) was purchased from Sigma ( St . Louis , MO ) .\",\n \"Baclofen-induced currents were measured at room temperature using a high-K+ bath solution ( in mM ) : 120 NaCl , 25 KCl , 1 . 8 CaCl2 , 1 MgCl2 , 5 . 5 D-glucose , 5 HEPES/NaOH ( pH 7 . 4 ) .\",\n \"The high-K+ bath solution ( +/− baclofen ) was applied directly to the soma and proximal dendrites with an SF-77B rapid perfusion system ( Warner Instruments , Inc . ; Hamden , CT ) .\",\n \"Current responses to the application of the high-K+ solution ( +/− baclofen ) were measured at a holding potential of −80 mV .\",\n \"Membrane potentials and whole-cell currents were measured in large neurons ( >75 pF ) with hardware ( Axopatch-700B amplifier , Digidata 1440A ) and software ( pCLAMP v . 10 . 3 ) from Molecular Devices ( Sunnyvale , CA ) .\",\n \"All currents were low-pass filtered at 2 kHz , sampled at 5 kHz , and stored on computer hard disk for subsequent analysis .\",\n \"Peak and steady-state current amplitudes were measured for each experiment .\",\n \"Current activation rates were extracted from a standard exponential fit of the current trace corresponding to the onset of drug effect and the peak evoked current , while deactivation rates were extracted from an exponential fit of the trace corresponding to the return of current to baseline following removal of drug ( Clampfit v . 10 . 3 software ) .\",\n \"Current desensitization was defined as % change in steady state current from the maximal baclofen-evoked response amplitude during 10 s of continuous drug application .\",\n \"Only experiments where access resistances were stable and low ( <20 MΩ ) were included in the analysis .\",\n \"Immunohistochemical reactions were carried out using the pre-embedding immunogold method as described earlier ( Lujan et al . , 1996 ) .\",\n \"Briefly , after blocking with 10% serum for 1 hr at room temperature free-floating sections were incubated for 48 hr with anti-RGS7 antibodies ( 1–2 mg/ml ) .\",\n \"Sections were washed and incubated for 3 hr with goat anti-rabbit IgG coupled to 1 . 4 nm gold ( Nanoprobes Inc ) at 1:100 dilution .\",\n \"Sections were washed , postfixed in 1% glutaraldehyde and processed for silver enhancement of the gold particles with an HQ Silver kit ( Nanoprobes Inc . ) .\",\n \"The reacted sections were treated with osmium tetraoxide ( 1% in 0 . 1 M PB ) , block-stained with uranyl acetate , dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan ( Fluka ) resin .\",\n \"Regions of interest were cut at 70–90 nm on an ultramicrotome ( Reichert Ultracut E; Leica ) .\",\n \"Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds’s lead citrate .\",\n \"Ultrastructural analyses were performed in a Jeol-1010 electron microscope .\",\n \"To establish the relative the abundance of RGS7 immunoreactivity along the plasma membrane of pyramidal cells , we used 60-µm coronal slices processed for pre-embedding immunogold immunohistochemistry .\",\n \"The procedure was similar to that used previously ( Lujan et al . , 1996 ) .\",\n \"Briefly , for each of three animals from different postnatal ages and adult , three samples of tissue were obtained for preparation of embedding blocks ( totalling nine blocks for each age ) .\",\n \"To minimize false negatives , electron microscopic serial ultrathin sections were cut close to the surface of each block , as immunoreactivity decreased with depth .\",\n \"We estimated the quality of immunolabelling by always selecting areas with optimal gold labelling at approximately the same distance from the cutting surface .\",\n \"Randomly selected areas were then photographed from the selected ultrathin sections and printed with a final magnification of 45 000X .\",\n \"Quantification of immunogold labelling was carried out in reference areas totalling approx .\",\n \"1 , 800 µm2 for each age .\",\n \"Immunoparticles identified in each reference area and present in different subcellular compartments ( dendritic spines , dendritic shafts and somata ) were counted .\",\n \"We measured the radial distance of each immunoparticle to the plasma membrane , being 0 for those just located in the plasma membrane .\",\n \"The data was expressed as percentage of immunoparticles along the radial distance from the plasma membrane expressed in nanometers .\",\n \"Mice were sacrificed under isoflurane anesthesia , and brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid ( aCSF ) without CaCl2 , composed of ( mM ) : 124 NaCl , 3 KCl , 24 NaHCO3 , 1 . 25 NaH2PO4 , 1 MgSO4 , and 10 D-Glucose , equilibrated with 95% O2 and 5% CO2 .\",\n \"The tissue was cut in 350–400 µm thick sections with a Vibrating microtome ( Leica VT1200S , Germany ) .\",\n \"The slices were warmed to 35°C for 25–45 min in aCSF supplemented with 2 mM CaCl2 , and equilibrated with 95% O2 and 5% CO2 .\",\n \"Then slices were maintained in gassed aCSF at room temperature until being transferred to submerged-type recording chambers of volume ∼1 . 5 ml .\",\n \"Here , the slices were constantly superfused ( 1–2 ml/min ) with warmed ( 30–31°C ) , gassed aCSF .\",\n \"All measurements were performed by an experimenter blind to genotype .\",\n \"CA1 neurons were visually identified in the hippocampal transverse slices of 350 µM thickness using Scientifica SliceScope system .\",\n \"Glass microelectrodes with an open-tip resistance of 3 . 5–6 . 5 MΩ were used .\",\n \"The following internal solution was used ( mM ) : 130 K-Gluconate , 20 KCl , 10 K-HEPES , 0 . 2 EGTA , 0 . 3 Na-GTP and 4 Mg-ATP ( pH 7 . 3 ) .\",\n \"To determine intrinsic cellular properties such as resting membrane potential , input resistance and spike numbers , 500 ms , 50 pA , 10-step hyperpolarizing and depolarizing current injections were delivered every 10 s .\",\n \"Cells with series resistance >20 MΩ or resting membrane potentials > −55 mV were excluded from analysis .\",\n \"Liquid junction potential was −14 mV .\",\n \"Spontaneous EPSCs ( sEPSCs ) were measured by holding the cells at −70 mV in normal aCSF .\",\n \"At least 100 events , which are above 4 pA , were obtained during sEPSC measurements in each cell .\",\n \"Field excitatory postsynaptic potentials ( fEPSPs ) were elicited by a concentric bipolar stimulating electrode ( inner diameter [ID]: 25 µm; outer diameter [OD]: 125 µm , FHC Inc . , Bowdoin , ME ) connected to a constant current isolated stimulator unit ( A-M Systems; Carlsborg , WA ) and recorded with low resistance ( 3–5 MΩ ) glass pipettes ( ID: 1 . 16 mm , OD: 1 . 5 mm , Harvard Apparatus , Holliston , MA ) filled with aCSF .\",\n \"The electrodes were placed in the stratum radiatum of the CA1 area of the dorsal hippocampus slices .\",\n \"Stimulation frequency was set to 0 . 05 Hz .\",\n \"Input-output curves were generated by adjusting the stimulus intensity in increments of 10 µA , from 0 to 100 µA .\",\n \"Paired pulse ratio ( PPR ) was assessed using a succession of paired pulses separated by time intervals ranging from 25 to 1000 ms , delivered every 20 s .\",\n \"The degree of facilitation was determined by taking the ratio of the initial slope of the second fEPSP to the initial slope of the first fEPSP .\",\n \"A PPR >1 was considered to reflect facilitation .\",\n \"For synaptic plasticity experiments , a stable baseline for at least 30 min was achieved prior to induction .\",\n \"LTP and LTD were recorded for 1 hr after HFS ( 2 tetanized stimuli ( TS ) of 100 Hz for 1s each ) or LFS ( 2 Hz for 10 min , 1200 pulses ) was applied .\",\n \"Depotentiation was achieved by applying HFS followed by LFS after 1-2 min interval .\",\n \"Spatial learning and memory were evaluated in the Morris water maze using a video tracking systems ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .\",\n \"Mice first received 4 cued trials ( visible but variable platform location ) on the first day to determine if non-associative impairment in other behavioral responses affecting performance in this task , such as exploratory activity , motor coordination , vision and motivation .\",\n \"After completing the cued trials , spatial learning acquisition was evaluated during the place condition ( hidden platform , constant location; 4 trials/day , 6 consecutive days ) .\",\n \"Escape latency and success rates in finding hidden platform were calculated for all place trials .\",\n \"Retention performance was evaluated during probe trials ( platform removed ) , which were conducted 24 hr after the last cued trial on the sixth day .\",\n \"Latency to the first target platform area crossing and the number of crossings in the probe trial ( 1 min ) served as the dependent variables .\",\n \"For fear conditioning experiments , mice were habituated in individual conditioning chambers to obtain freezing baseline on the first day .\",\n \"On the second day , mice were trained in individual conditioning chambers ( Med Associates , St . Albans , VT ) .\",\n \"Video images were recorded via video tracking systems ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .\",\n \"In context A , visible light was turned on , a stainless steel grid floor inserted , and the chambers were cleaned with 70% ethanol prior to conditioning .\",\n \"In context B , white plastic inserts were placed inside the chamber to change the shape , size and texture of the wall and the floor .\",\n \"A small weight boat with orange extract is placed behind the wall inserts to provide a novel smell for the chambers .\",\n \"The chambers were cleaned with 70% isopropanol .\",\n \"During the training sessions , mice were placed in context A and allowed to explore for 3 min prior to the delivery shocks ( 0 . 75 mA , 1 s ) .\",\n \"We used auditory tone ( 85 dB , 30 s duration ) as CS and an electric foot shock ( delivered through the grid floor , 0 . 75 mA AC current , 1 s duration ) as US .\",\n \"Mice received 3 tone-shock stimuli ( with 1–2 min interval ) during the training day .\",\n \"In every session , tone always co-terminated with electric shock .\",\n \"Mice were removed from the conditioning chamber 30 s after the last shock .\",\n \"Contextual test ( 5 min ) was done in context A 24 hr after training .\",\n \"Cue test was performed 1 hr after contextual test in context B . The animals were brought into the test room in a different covered mover with different bedding inside to avoid contextual reminders .\",\n \"The animal was immediately placed in the contextual B chamber and allowed to explore for 3 min prior to the delivery the auditory tone ( 85 dB ) for another 3 min .\",\n \"The freezing response was measured with the automated tracking and analyzed offline .\",\n \"For the novel object recognition test , mice were habituated to the arena for two consecutive days , where they were placed in a white plastic open-box of dimensions 43 . 2 cm2 by 30 . 5 cm height for 10 min each .\",\n \"The acquisition phase of the object recognition assay involved placing each individual mouse in the test arena for 8 min in the presence of two identical objects ( either silver or black objects in different shape , size and texture , the order of which alternated between mice ) .\",\n \"The video was recorded via a camera .\",\n \"The time spent investigating both identical objects was recorded by automatic tracking system ( EthoVision XT , Noldus Information Technology , Wageningen , Netherlands ) .\",\n \"24 hr after the acquisition phase , each mouse was reintroduced to the test arena for 8 min .\",\n \"During this phase ( retention testing phase ) the mouse was presented with one copy of the object that it was exposed to during the acquisition phase ( familiar object ) and one novel object ( either silver or black objects , depending on which was presented during the acquisition phase ) .\",\n \"The time spent investigating each object was recorded .\",\n \"All objects and the testing box were thoroughly cleaned with 70% methanol between mice to remove any odor cues .\",\n \"Agonist-dependent cellular measurements of bioluminescence resonance energy transfer ( BRET ) between Venus-Gβ1γ2 and its effector fragment masGRK3ct-Nluc were performed upon reconstitution in living cells as previously described with slight modification ( Hollins et al . , 2009; Masuho et al . , 2013 ) .\",\n \"The masGRK3ct-Nluc construct contained amino acids G495-L688 of bovine GRK3 ( NP_776925 ) , preceded by a myristic acid attachment peptide ( mas; MGSSKSKTSNS ) .\",\n \"The stop codon of GRK3 was replaced with a GGGS linker , which was followed by the NanoLuc ( Nluc ) ( Hall et al . , 2012 ) .\",\n \"Briefly , GABAB1R , GABAB2R , GαoA , Venus156-239-Gβ1 , and Venus1-155-Gγ2 constructs were transfected into HEK293T/17 cells at a 1:1:2:1:1 ratio with increasing masGRK3ct-Nluc from 0 . 125 to 6 in ratio .\",\n \"5 µg total DNA was delivered per 4 × 106 cells in a 6-cm-dish .\",\n \"16–24 hr post transfection cells were stimulated with 100 µM GABA followed by treatment with 100 µM CGP 54626 .\",\n \"The BRET signal is determined by calculating the ration of the light emitted by the Venus-Gβ1γ2 ( 535 nm ) over the light emitted by the masGRKct-Rluc8 ( 475 nm ) .\",\n \"The average baseline value recorded prior to agonist stimulation was subtracted from BRET signal values , and the resulting difference ( ΔBRET ) was obtained .\",\n \"Statistical analyses were performed using Prism ( GraphPad Software , Inc . ; La Jolla , CA ) .\",\n \"Data are presented throughout as the mean ± SEM .\",\n \"Student t test , one-way or two-way ANOVA , followed by Bonferroni’s post hoc test were used as appropriate .\",\n \"The minimal level of significance was set at p<0 . 05 .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["In the hippocampus , the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition .","Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ ( GIRK ) channels .","Here , we show that RGS7 , in cooperation with its binding partner R7BP , regulates GABABR-GIRK signaling in hippocampal pyramidal neurons .","Deletion of RGS7 in mice dramatically sensitizes GIRK responses to GABAB receptor stimulation and markedly slows channel deactivation kinetics .","Enhanced activity of this signaling pathway leads to decreased neuronal excitability and selective disruption of inhibitory forms of synaptic plasticity .","As a result , mice lacking RGS7 exhibit deficits in learning and memory .","We further report that RGS7 is selectively modulated by its membrane anchoring subunit R7BP , which sets the dynamic range of GIRK responses .","Together , these results demonstrate a novel role of RGS7 in hippocampal synaptic plasticity and memory formation ."],"string":"[\n \"In the hippocampus , the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition .\",\n \"Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ ( GIRK ) channels .\",\n \"Here , we show that RGS7 , in cooperation with its binding partner R7BP , regulates GABABR-GIRK signaling in hippocampal pyramidal neurons .\",\n \"Deletion of RGS7 in mice dramatically sensitizes GIRK responses to GABAB receptor stimulation and markedly slows channel deactivation kinetics .\",\n \"Enhanced activity of this signaling pathway leads to decreased neuronal excitability and selective disruption of inhibitory forms of synaptic plasticity .\",\n \"As a result , mice lacking RGS7 exhibit deficits in learning and memory .\",\n \"We further report that RGS7 is selectively modulated by its membrane anchoring subunit R7BP , which sets the dynamic range of GIRK responses .\",\n \"Together , these results demonstrate a novel role of RGS7 in hippocampal synaptic plasticity and memory formation .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Neurons communicate with one another at junctions called synapses .","The arrival of an electrical signal known as an action potential at the first cell causes molecules known as neurotransmitters to be released into the synapse .","These molecules diffuse across the gap between the neurons and bind to receptors on the receiving cell .","Some neurotransmitters , such as glutamate , activate cells when they bind to receptors , thus making it easier for the second neuron to ‘fire’ ( i . e . , to generate an action potential ) .","By contrast , other neurotransmitters , such as GABA , usually make it harder for the second neuron to fire .","Many of the effects of GABA involve a type of receptor called GABAB .","When GABA binds to one of these receptors , a molecule called a G-protein is recruited to the receptor .","This activates the G-protein , triggering a cascade of events inside the cell that lead ultimately to the opening of potassium ion channels , which as known as GIRKs , in the cell membrane .","Positively charged potassium ions then leave the cell through these channels , and this makes it more difficult for the cell to fire .","Now , Ostrovskaya et al . have revealed that a complex of three proteins regulates the interaction between GABAB receptors and GIRK channels .","In neurons that lack either of these proteins , the receptors have less influence on GIRKs than in normal cells .","Moreover , mice that lack one of the proteins ( called RGS7 ) perform less well in various learning and memory tests: for example , they take longer than normal animals to learn the location of an escape platform in a water maze , or to retain a memory of a fearful event .","By identifying the proteins that regulate the interaction between GABAB receptors and GIRKs , Ostrovskaya et al . have helped to unravel a key signaling cascade relevant to cognition .","Given that GIRK channels have recently been implicated in Down’s syndrome , these insights may also increase understanding of cognitive impairments in neuropsychiatric disorders ."],"string":"[\n \"Neurons communicate with one another at junctions called synapses .\",\n \"The arrival of an electrical signal known as an action potential at the first cell causes molecules known as neurotransmitters to be released into the synapse .\",\n \"These molecules diffuse across the gap between the neurons and bind to receptors on the receiving cell .\",\n \"Some neurotransmitters , such as glutamate , activate cells when they bind to receptors , thus making it easier for the second neuron to ‘fire’ ( i . e . , to generate an action potential ) .\",\n \"By contrast , other neurotransmitters , such as GABA , usually make it harder for the second neuron to fire .\",\n \"Many of the effects of GABA involve a type of receptor called GABAB .\",\n \"When GABA binds to one of these receptors , a molecule called a G-protein is recruited to the receptor .\",\n \"This activates the G-protein , triggering a cascade of events inside the cell that lead ultimately to the opening of potassium ion channels , which as known as GIRKs , in the cell membrane .\",\n \"Positively charged potassium ions then leave the cell through these channels , and this makes it more difficult for the cell to fire .\",\n \"Now , Ostrovskaya et al . have revealed that a complex of three proteins regulates the interaction between GABAB receptors and GIRK channels .\",\n \"In neurons that lack either of these proteins , the receptors have less influence on GIRKs than in normal cells .\",\n \"Moreover , mice that lack one of the proteins ( called RGS7 ) perform less well in various learning and memory tests: for example , they take longer than normal animals to learn the location of an escape platform in a water maze , or to retain a memory of a fearful event .\",\n \"By identifying the proteins that regulate the interaction between GABAB receptors and GIRKs , Ostrovskaya et al . have helped to unravel a key signaling cascade relevant to cognition .\",\n \"Given that GIRK channels have recently been implicated in Down’s syndrome , these insights may also increase understanding of cognitive impairments in neuropsychiatric disorders .\"\n]","isConversational":false},"year":{"kind":"string","value":"2014"}}},{"rowIdx":287,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["cell biology","computational and systems biology"],"string":"[\n \"cell biology\",\n \"computational and systems biology\"\n]","isConversational":false},"title":{"kind":"string","value":"Self-establishing communities enable cooperative metabolite exchange in a eukaryote"},"id":{"kind":"string","value":"elife-09943-v2"},"sections":{"kind":"list like","value":[["All living cells possess a system for biochemical reactions , the metabolic network , which supplies cells with their necessary molecular constituents .","The reactions participating in this network are highly conserved , so much so that all life is made up of a markedly similar set of metabolites ( Braakman and Smith , 2013; Caetano-Anolles et al . , 2009 ) .","The functionality of the metabolic system is bound to a series of transport reactions that facilitate the uptake of metabolites from the environment , as well as metabolite export .","Metabolite export primarily occurs for the purpose of maintaining balance of the metabolic system ( 'overflow metabolism' ) and to maintain chemical and physical integrity of the metabolic network .","This includes indiscriminate metabolite export through non-specific multi-drug transporters required in removal of toxic metabolites for cells ( Paczia et al . , 2012; Piedrafita et al . , 2015 ) .","Co-growing cells can uptake the released metabolites and exploit their presence .","Indeed , to a lower extent , metabolite export of metabolites can specifically occur for the purpose of establishing inter-cellular metabolic interactions in biosynthetic metabolism ( Nigam , 2015; Paczia et al . , 2012; Yazaki , 2005 ) and can lead to mutualistic situations in which cells profit from coexistence ( Foster and Bell , 2012; Oliveira et al . , 2014 ) .","In between species , mutually positive interactions can readily establish when exchange concerns an overflow metabolite , exemplified by yeast–algae interactions that can form on the basis of a CO2 and sugar exchange ( Hom and Murray , 2014 ) , or between different cells of the same species or tissue , exemplified in tumours , when lactate produced in excess by one cell type is metabolised by another ( Bonuccelli et al . , 2010 ) , or between neurons and glial cells that exchange sugar metabolites ( Bélanger et al . , 2011; Volkenhoff et al . , 2015 ) .","It is more difficult to assess whether metabolite exchange is indicative of non-cell-autonomous metabolism , when exchange concerns metabolites that are needed by both exchange partners , amino acids , and nucleobases for instance .","Exchange of costly intermediates is associated with a significant risk , as exported metabolites can be lost through diffusion , chemical damage , or cheating ( Dobay et al . , 2014; Oliveira et al . , 2014; Wintermute and Silver , 2010 ) .","Despite these constraints , exchange of intermediates is frequently observed within bacterial microbial communities .","Many bacterial species lose essential biosynthetic pathways , disabling them from living autonomously , which may explain why more than 90% of bacteria cannot be cultivated in the absence of a community environment ( Costerton et al . , 1994; Johnson et al . , 2012 ) .","The energetic benefit and selective advantage associated with non-autonomous cellular metabolism is often not clear but might involve , for example , the ability to reduce genome size which would in turn facilitate faster proliferation .","This may explain why bacteria frequently appear to cooperate in the biosynthesis of more costly and biosynthetically complex metabolites , such as aromatics ( Mee et al . , 2014 ) .","While metagenomics has boosted the knowledge of metabolite exchange strategies in bacteria ( Blaser et al . , 2013; Manor et al . , 2014; Zelezniak et al . , 2015 ) , relatively little is known about eukaryotic species .","This includes yeast , a popular single cellular eukaryotic model organism , whose metabolic capacities are regularly exploited in biotechnology .","Yeast cells are known to participate in multi-species communities ( e . g . on human skin ( Findley et al . , 2013 ) ) , but as wild yeast isolates usually maintain similar prototrophic genomes ( Jeffares et al . , 2015; Liti et al . , 2009 ) , metagenomic data are not conclusive about yeast's metabolite exchange strategies .","In laboratory experiments , yeast cultures were , however , not effective in supporting co-growth of auxotrophs that have complementary defects in amino acid and nucleotide metabolism , unless they were genetically modified to increase metabolite export ( Müller et al . , 2014; Shou et al . , 2007 ) .","This contrasts with analogous studies in bacterial species , in which such growth experiments regularly show that co-cultured cells can overcome complementary metabolic deficiencies ( Foster and Bell , 2012; Oliveira et al . , 2014; Pande et al . , 2014; Vetsigian et al . , 2011 ) .","In the absence of quantitative metabolite data , this observation has triggered the conclusion that co-growing prototrophic yeast cells produce amino acid and nucleotide metabolites predominantly for themselves and export them at insufficient quantities to support growth of co-growing cells ( Momeni et al . , 2013; Shou et al . , 2007 ) .","Conflicting with this interpretation , we here report that yeast colonies maintain a rich exometabolome and that cells exploit this metabolic pool preferentially over their own biosynthetic capacities , which implies that metabolite exchange establishes as a natural property of yeast growth .","To test whether yeast indeed possesses the capacity for metabolite exchange at growth relevant quantities , we established an alternative method to co-culture experiments .","We exploited the stochastic segregation of episomes to randomly and progressively introduce metabolic auxotrophies into a yeast population which self-establishes from an initially prototrophic cell .","This strategy enabled co-growing auxotrophs to enter an efficient state of metabolic cooperation , named self-establishing metabolically cooperating communities ( SeMeCos ) .","Despite an auxotrophic cell composition of up to 97% , SeMeCos achieve metabolic efficiency , growth parameters , and cell viability similar to that of genetically prototrophic cells , revealing a natural capacity of yeast to exchange metabolites at growth relevant quantities .","In a SeMeCo that possesses auxotrophies in histidine , leucine , uracil and methionine metabolism , we distinguish up to eight cell types , each of which is unable to survive on its own , or in co-culture studies , however , could adapt effectively and cooperatively to overcoming metabolic deficiencies , once self-established in a community structure .","Communities have a stable population composition as well as distinct spatial heterogeneity , which was , however , not essential for metabolite exchange , as SeMeCos maintain growth in liquid suspension .","Self-establishing , complex communities thus demonstrate that yeast is not only exchanging metabolites , but is also able to do so at growth relevant quantities , to facilitate growth on the basis of a non-cell-autonomous metabolism ."],["Histidine , leucine , uracil , and methionine biosynthetic pathways were chosen for our study as","( i ) they can be interrupted by deletion of a single , non-redundant gene , which has been reported not to cause compensatory mutations and","( ii ) because cells possess efficient uptake mechanisms for these nutrients ( Mülleder et al . , 2012; Pronk , 2002; Teng et al . , 2013 ) .","Paired combinations of histidine ( his3Δ ) , leucine ( leu2Δ ) , uracil ( ura3Δ ) , or methionine ( met15Δ ) auxotrophs were unable to sustain growth in the absence of supplementation required for both individual cell types ( Figure 1A ) .","A similar result was obtained by co-culturing flocculating yeast cells ( Figure 1—figure supplement 1 ) , which are able to maintain biofilm-like physical contact ( Smukalla et al . , 2008 ) , and in Schizosaccharomyces pombe ( Figure 1B ) , indicating evolutionary conservation of this observation in yeast species . 10 . 7554/eLife . 09943 . 003Figure 1 . Yeast auxotrophs do not compensate for metabolic deficiencies upon co-culturing , yet export the relevant metabolites and prefer metabolite uptake over self-synthesis .","( A ) Complementary pairs of Saccharomyces cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .","his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts were combined in complementary pairs and spotted on corresponding selective media .","No pairs exhibited co-growth together .","( B ) A complementary pair of Schizosaccharomyces pombe auxotrophs does not overcome metabolic deficiencies upon co-culturing .","leu1Δ and ura4Δ yeasts were combined in a complementary pair and spotted on corresponding selective media .","No co-growth occurred .","( C ) The concentration of metabolites in the S . cerevisiae colony exometabolome obtained from 1 . 3e08 YSBN5 cells grown in a colony on synthetic minimal agar media ( SM ) and quantified by LC-MS/MS .","Abbreviations: single letter IUPAC amino acid codes , O = ornithine , CIT = citrulline .","n = 3 , error bars = ± SD .","( D )","( i ) Metabolites quantified as in ( C ) , comparing intracellular ( total cell extracts ) and extracellular metabolite concentrations in YSBN5 .","n = 3 , error bars = ± SD .","Dashed line: linear regression fit , grey band shows 95% confidence region .","( ii ) Metabolites quantified as in ( C ) , comparing extracellular metabolite concentrations of YSBN5 and BY4741-pHLUM yeast colonies grown on minimal media .","H , L , U , and M are highlighted in red circles .","n = 3 , error bars = ± SD .","Dashed line: linear regression fit , grey band shows 95% confidence region .","Abbreviations IUPAC codes; H = histidine , L = leucine , U = uracil , M = methionine .","( E ) Consumption of uracil , histidine , leucine , and methionine in yeast batch cultures in synthetic complete ( SC ) media as measured by LC-MS/MS .","Uracil , histidine , leucine , and methionine prototrophic cells consume these metabolites at rates and quantities comparable to the corresponding auxotrophic strains .","( F )","( i ) Deletion of URA3 ( Orotidine-5'-phosphate decarboxylase ) causes accumulation of the Ura3p substrate orotidine-5'-phosphate ( OMP ) , when cells are supplemented with 20 mg/L uracil ( fold change of OMP abundance , relative to URA3 without uracil supplementation ) , as determined by LC-MS/MS .","Error bars = ± SD .","( ii ) Uracil supplementation of wild-type cells alters their metabolite profile to resemble ura3∆ cells , which obtain uracil solely from the growth media .","Heatmap scaling ( [0 , 1] and min , max per metabolite ) was based on median concentration .","The dendrogram was constructed by comparing euclidean distance ( dissimilarity ) between samples . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00310 . 7554/eLife . 09943 . 004Figure 1—figure supplement 1 . Flocculation does not enable Saccharomyces cerevisiae cells to establish viable co-cultures .","( i ) ( left ) The FLO+ phenotype in yeast cells transforms their typical cell suspension ( right ) into a physiological state reminiscent of biofilms ( left ) ( Smukalla et al . , 2008 ) .","Cultures were grown to stationary phase in rich media ( YPD ) and flocculation was detected via an inability of cells to re-suspend following repeated tube inversion .","( ii ) Complementary pairs of flocculating S . cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .","his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts were combined in complementary pairs and spotted on corresponding selective media .","No pairs exhibited co-growth together . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00410 . 7554/eLife . 09943 . 005Figure 1—figure supplement 2 . Uracil biosynthetic genes in the uracil prototroph ( URA3 ) and auxotroph ( ura3Δ ) remain expressed in ( uracil supplemented ) SC media , as determined by RNA sequencing . Abbreviations: RPKM = reads per kilobase per million .","n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 005 In two previous studies , leucine/tryptophan and adenine/lysine auxotrophic cell pairs , respectively ( Müller et al . , 2014; Shou et al . , 2007 ) , could co-grow upon removing metabolic feedback control .","Feedback resistance renders cells metabolite over-exporters , leading to the conclusion that wild-type yeast cells produce intermediates primarily for themselves , at quantities that are not sufficient for growth relevant metabolite exchange ( Momeni et al . , 2013; Shou et al . , 2007 ) .","In a detailed analysis of the intra-colony exometabolome , using an ultra-sensitive mass spectrometry method , the intra-colony fluid showed however to contain a plethora of metabolites , with the amino acids glutamine , glutamate , and alanine being the most highly concentrated ( Figure 1C ) .","Furthermore , histidine , leucine , methionine , and uracil all showed to be part of this exometabolome ( Figure 1C ) .","These measurements were obtained from cells in exponential growth phase , where apoptosis and necrosis are negligible .","Comparing extracellular metabolite concentrations to intracellular levels ( the endometabolome ) we observed a general trend of correlation between the highest and lowest concentrated metabolites ( r2 = 0 . 517; Figure 1Di ) , but overall extracellular metabolite concentrations do not replicate the corresponding endometabolome .","Tryptophan , phenylalanine , proline , and valine , for instance , were over-proportionally more concentrated inside the cell , whereas uracil , serine , tyrosine , and glycine were relatively over-represented in the extracellular fluid ( Figure 1Di ) .","Instead , highly similar exometabolome concentration values ( r2 = 0 . 971 ) were observed in the related yeast strain BY4741 upon complementing its auxotrophies with the centromere-containing single-copy vector ( a minichromosome ) , 'pHLUM' , which contains all four marker genes ( Mülleder et al . , 2012 ) ( Figure 1Dii ) .","Metabolite concentrations in the exometabolome between these two related yeast strains are hence substantially more similar than the endo- versus exometabolome in the same strain , implying that the intra-colony exometabolome is a distinct metabolite pool .","A second requirement to establish metabolite exchange is that cells need to be able to sense extracellular metabolites and to exploit them as a nutrient source .","Yeast is known to uptake amino acids when they are available extracellularly ( Stahl and James , 2014 ) .","We tested how extensive this uptake was by comparing the uptake rates between auxotrophs and prototrophs .","Remarkably , prototrophic cells consumed histidine , leucine , methionine , and uracil at a comparable rate to the genetic auxotrophs , who depend 100% on external metabolite pools ( Figure 1E ) .","This demonstrated that yeast cells completely shift from de novo synthesis to uptake in the presence of each of the four metabolites .","Studying the URA3 genotype in greater detail confirmed the preference of uptake over self-synthesis .","Enzymes involved in uracil biosynthesis remained expressed in both the URA3 and the ura3Δ strains under fully supplemented conditions ( Figure 1—figure supplement 2 ) , but uracil biosynthesis-related intermediates shifted to similar concentrations both in the wild-type strain and in the ura3Δ strain once uracil was supplemented ( Figure 1F ) .","The only exception was the direct substrate of the URA3 enzyme ( orotidine-5'-phosphate decarboxylase ) , orotidine-5'-phosphate ( OMP ) , which accumulated upon uracil supplementation once its metabolising enzyme ( URA3 ) was deleted ( Figure 1Fi ) .","In summary , yeast cells do not compensate for metabolic deficiencies in co-culture experiments consistently as others reported previously ( Müller et al . , 2014; Shou et al . , 2007 ) , but they","( i ) export the relevant metabolites even when grown on minimal media and","( ii ) take up histidine , leucine , uracil , and methionine at similar rates to auxotrophs if supplementation is available .","At least for uracil ,","( iii ) the biosynthetic enzymes and majority of biosynthetic intermediates in the supplemented wild-type cell resemble those of the corresponding auxotroph .","In light of these results , we speculated that the inability to cooperate could be found in the nature of the co-culturing experiment .","To establish an alternative method , we made use of a , in other circumstances disadvantageous , property of yeast plasmids , their occasional , stochastic loss from cells ( segregation ) .","Segregation is observed for both popular replication types , centromeric 'cen' and 2µ , at a rate of 2–4% expressed per cell division ( Christianson et al . , 1992 ) .","This property allowed us to randomly and progressively introduce auxotrophies into a developing yeast community starting from a single , initially prototrophic , cell: when a plasmid carries a gene that complements for an auxotrophy , a newly budded cell re-gains the metabolic deficiency according to the segregation of its plasmid .","We transformed plasmids from the classic pRS and p400 series which express HIS3 , LEU2 , MET15 , or URA3 genes under the respective S . cerevisiae promoters ( Christianson et al . , 1992; Mumberg et al . , 1995; Sikorski and Hieter , 1989 ) into the standard laboratory strain BY4741 , deficient in these markers ( Brachmann et al . , 1998 ) ( Figure 2Ai ) .","As expected , the transformed cells grew competently in the absence of histidine , leucine , uracil , and methionine supplementations .","We then quantified plasmid segregation and confirmed earlier literature values ( Figure 2Aii and Figure 2—source data 1 ) ( Christianson et al . , 1992; Ghosh et al . , 2007 ) . 10 . 7554/eLife . 09943 . 006Figure 2 . A self-establishing yeast community can cooperatively compensate for progressive loss of prototrophy on minimal media .","( A )","( i ) Schematic illustration of BY4741 carrying four plasmids to complement its auxotrophies in histidine ( his3Δ1 ) , leucine ( leu2Δ0 ) , methionine ( met15Δ0 ) , and uracil ( ura3Δ0 ) .","( ii ) Plasmid segregation rates ( probability of plasmid loss per cell division ) of BY4741 carrying four plasmids encoding HIS3 ( p423 ) , LEU2 ( pRS425 ) , URA3 ( p426 ) , and MET15 ( pRS411 ) ( y-axis ) compared to BY4741 carrying one plasmid at a time ( x-axis ) .","n = 3 , error bars = ± SD .","Dashed line: linear regression fit .","( B ) Schematic illustration of the segregate strain composition over time on rich or complete media where no cooperation is necessary for cells to survive .","Sequential plasmid loss leads to an increase in auxotrophy , with loss of up to four plasmids leading to the formation of 16 cell types with varying metabolic capacity ( metabotypes ) .","( C ) Three possible outcomes for BY4741 carrying four segregating plasmids , when establishing a colony on minimal media;","( i ) no cooperation , only cells carrying four plasmids grow ,","( ii ) no cooperation but plasmid segregation is faster than the growth rate of cells carrying four plasmids leading to no growth capacity .","Finally","( iii ) , cells cooperate , wherein cells that have obtained auxotrophy continue growth by sharing metabolites with neighbouring cells in the colony .","( D ) Auxotrophy of BY4741 colonies carrying single plasmids encoding HIS3 ( p423 ) , LEU2 ( pRS425 ) , URA3 ( p426 ) , and MET15 ( pRS411 ) on selective media after approximately 33 doublings .","The number of plasmid-free cells ( % auxotrophy abundance ) was measured by replica plating .","n = 3 , error bars = ± SD .","( E ) Mathematical simulation of segregation over time , starting from 100% cells carrying four plasmids , based on the experimentally measured segregation rates .","Highlighted is the situation after 57 doublings ( achieved in dashed line ) where >99 . 9% of cells have segregated >1 plasmid .","( F ) Segregation over time in a colony on rich media ( no selection to maintain the plasmids ) ; starting from a micro-colony of four-plasmid prototrophic cells on minimal media , cells were transferred to rich ( YPD ) media and established as a giant colony , segregation was followed by replica plating .","Biomass gain is counted from the single cell .","( G ) Giant colonies established for 57 biomass doublings on minimal media are composed of ( left ) 73 . 3% auxotrophic cells , ( centre ) contain a mixed number of auxotrophies and ( right ) a non 1:1 ratio of auxotrophy types .","( n = 542 genotyped cells ) .","Colony growth is achieved , despite the majority of cells possessing one or more auxotrophies . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00610 . 7554/eLife . 09943 . 007Figure 2—source data 1 . Plasmid segregation rates . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00710 . 7554/eLife . 09943 . 008Figure 2—figure supplement 1 . Experimentally obtained colony composition , compared to the composition expected if segregation continued without selective pressure to maintain cells able to synthesise leucine , uracil , methionine and histidine .","( i ) ( left ) Colonies established for 57 biomass doublings on minimal media ( SM ) are composed of 73 . 3% auxotrophic cells ( n = 542 genotyped cells ) .","( right )","Uninterrupted segregation would lead to 99 . 9% auxotrophic cells .","( ii ) ( left ) Auxotrophy number ( 0 to 4 ) for cells within the synthetic metabolically cooperating colony ( SeMeCo ) .","( right )","Result when there is uninterrupted segregation ( theoretical composition ) .","( iii ) ( left ) Composition of SeMeCo in terms of auxotrophy type ( histidine , leucine , uracil and methionine ) .","( right )","Result when there is uninterrupted segregation ( theoretical composition ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00810 . 7554/eLife . 09943 . 009Figure 2—figure supplement 2 . Schizosaccharomyces pombe , like Saccharomyces cerevisiae , are also able to establish SeMeCo colonies . The four possible metabotypes resulting from a combination of uracil and leucine auxotrophies are found within S . pombe SeMeCos , despite growing colonies on selective media ( n = 3 ) .","Separately established populations ( >90 cells ) were genotyped per replicate . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00910 . 7554/eLife . 09943 . 010Figure 2—figure supplement 3 . Complementary pairs of auxotrophs , re-isolated from established SeMeCo colonies , do not overcome metabolic deficiencies upon co-culturing , similar to the original strains .","his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts isolated from SeMeCo colonies were combined in complementary pairs and spotted on corresponding selective media .","No pairs exhibited co-growth together , indicating that SeMeCo metabotypes did not acquire secondary mutations to overcome metabolic deficiencies , while establishing a cooperating community . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01010 . 7554/eLife . 09943 . 011Figure 2—figure supplement 4 .","Different auxotrophy combinations do not enable metabolic cooperation .","( left )","Quadruple mixed cultures of Saccharomyces cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .","his3Δ , leu2Δ , met15Δ , and ura3Δ yeasts were combined together in mixed ratios and spotted as a co-culture on corresponding selective media .","( right )","A similar outcome upon co-culturing the four genotypes over night in rich media prior to transferring to minimal media . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 011 Cells having lost prototrophy can only continue growth if they obtain the relevant nutrient from the environment ( Figure 2B ) .","Transferred to minimal media , the lack of nutrient supplementation leads to three possible outcomes ( Figure 2C ) : First , if the cooperative potential would not suffice to overcome the increasing content of metabolically deficient cells , colony growth would only be explained by cells maintaining all four plasmids ( Figure 2C left ) .","Alternatively , if the segregation is faster than the growth rate of cells carrying four plasmids , the colonies would not be able to grow ( Figure 2C centre ) .","Finally , the third outcome is that colony growth continues , despite an increasing auxotrophic composition , facilitated by cells exchanging histidine , leucine , uracil , and methionine at growth relevant quantities ( Figure 2C right ) .","First , we observed that upon approximately 33 biomass doublings , segregation for HIS3 and URA3 had continued until less than 50% of cells were prototrophs ( Figure 2D ) , even though a 1:1 co-culture of the same auxotrophs was not able to co-grow ( Figure 1A ) .","Then , we assayed for the formation of a heterogeneous yeast community , starting from the four-plasmid ( 4P ) strain , that can give rise to the emergence of 16 complementary auxotrophic genotypes ( Figure 2B ) .","In the 4P strain , individual plasmid segregation rates were similar but not identical to yeast carrying one plasmid at a time and were in linear correlation , indicating that no specific interaction between the plasmids occurred ( Figure 2Aii ) .","With a total segregation rate of 11% , 4P cells regain auxotrophy rapidly so that only 21 cell divisions ( doublings ) would result in >90% of cells losing prototrophy ( Figure 2E ) .","The continuous loss of prototrophy from the 4P strain was experimentally confirmed on rich ( YPD ) media; In the presence of rich supplementation , only 45 biomass doublings resulted in 96% of cells losing prototrophy ( Figure 2F ) .","Testing whether cells can maintain growth by cooperating in the biosynthesis of histidine , leucine , uracil , and methionine , colonies were grown over 7 days on minimal media agar through dilution and re-spotting once giant colonies had formed ( every 48 hr ) , so that the continuous gain in biomass necessitates constant de novo synthesis of intermediate metabolites .","The experiment yielded viable colonies and from the obtained biomass , we calculated that 57 doublings had occurred .","Fifty-seven doublings would have been sufficient for >99% of cells to segregate ( Figure 2E ) .","Replica plating revealed a predominantly auxotrophic composition of the obtained colonies .","These were composed of 73 . 3 ± 3 . 7% auxotrophic cells ( Figure 2G left ) , of which 39 . 9% had lost one plasmid , 20 . 6% two , 6 . 6% three , and 6 . 1% had lost all four markers ( Figure 2G centre ) .","No auxotrophies were in a 1:1 ratio with each other ( 36 . 9% for uracil , 27 . 7% leucine , 23 . 7% histidine , and 11 . 7% methionine ( Figure 2G right ) , despite the segregation rates predicting a relatively equal distribution , implying that selection pressure for certain metabotypes affected colony composition ( Figure 2G right and Figure 2—figure supplement 1 ) .","We also confirmed that S . pombe is capable of forming similar communities , indicating conservation in these evolutionary distant yeast species ( Figure 2—figure supplement 2 ) .","As additional controls , we","( i ) re-isolated the auxotrophs from the established colonies , and repeated the co-culture experiment , after having grown the cells for 48 hr in supplemented media , as with the original strains ( Figure 1A ) .","Even when isolated from a functional cooperating colony , complementary auxotrophic cells did not complement each other's deficiencies upon co-culturing ( Figure 2—figure supplement 3 ) , ruling out the possibility that new mutations altering metabolite exchange capacities could explain the formation of the cooperating community .","We also mixed all four auxotrophs together , both in a 1:1 mixture , as well as in the ratio observed from the community and performed co-culturing both with and without co-cultivation before spotting; These attempts did not result in successful co-growth either ( Figure 2–figure supplement 4 ) .","Hence , by exploiting plasmid segregation to overcome culturing and allowing the community to self-establish , heterogeneous yeast colonies were formed , which could sustain exponential growth under nutrient limitation , despite the majority of cells being auxotrophic for at least one metabolite .","These co-growing cells could therefore overcome metabolic deficiencies through cooperative metabolism , demonstrating that yeast possesses metabolite exchange capacities at a growth relevant quantity .","The obtained colonies were viable on minimal media and showed no apparent growth defects , despite containing a content of 73% auxotrophs , each of which were non-viable in co-culture studies ( Figure 1A , B , Figure 1—figure supplement 1 , Figure 2—figure supplement 3 , 4 ) .","To characterise the properties of this community , we started with LC-MS/MS to compare its exometabolome against prototrophic yeast strains ( YSBN5 , BY4741-pHLUM ) , and the unpassaged strain carrying the four plasmids ( 4P ) ; ( Figure 3A , B and Figure 3—source data 1 ) .","SeMeCo colonies possessed similar extracellular metabolite concentrations to prototrophic controls ( Figure 3Bi ) .","Of particular note are the extracellular concentrations of H , L , U , and M . Aside from a statistically non-significant trend towards a lower leucine concentration , only uracil ( U ) was significantly affected .","To our surprise , however , the concentration of this metabolite was increased , indicating that SeMeCo had adapted by maintaining a higher level of uracil in its exometabolome ( Figure 3Bii ) . 10 . 7554/eLife . 09943 . 012Figure 3 . Growth and physiological parameters of the self-established metabolically cooperating yeast community 'SeMeCo' .","( A ) Schematic illustration of colonies derived from the genomically prototrophic yeast strain YSBN5 , the single-vector complemented BY4741-pHLUM ( 'pHLUM' ) , BY4741 complemented with four plasmids ( 'FourP' ) , and the self established yeast population ( SeMeCo; self-established metabolically cooperating yeast community ) ; ( from left to right ) .","( B )","( i ) Extracellular concentrations of metabolites in colonies of YSBN5 , pHLUM and SeMeCo growing exponentially on minimal media as determined by LC-MS/MS , n = 3 .","Histidine ( H ) , leucine ( L ) , methionine ( M ) , and uracil ( U ) are highlighted in red circles .","( ii ) Detailed extracellular concentration values of uracil , leucine , methionine , and histidine as determined by LC-MS/MS .","n = 3 , error bars = ± SD .","( C ) ( left ) Growth curve of YSBN5 , pHLUM , FourP , and SeMeCo as determined by measuring optical density ( OD595 ) .","n = 3 , error area = ± SD .","( centre )","Dry biomass collected from 100 mL batch cultures after three days growth in minimal media , 30°C , n = 3 , error bars = ± SD .","( right )","Maximum specific growth rate ( µmax ) as determined from OD595 growth curves using a model-richards fit ( Kahm et al . , 2010 ) .","n = 3 , error bars = ± SD .","( D ) The ratio of colony-forming units ( CFUs ) to number of cells used for plating , for YSBN5 and SeMeCo .","n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01210 . 7554/eLife . 09943 . 013Figure 3—source data 1 . Absolute quantification of amino acids and uracil in yeast strains YSBN5 , pHLUM and SeMeCo , absolute concentration values . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 013 SeMeCo also maintained wild-type like growth efficiency and biomass-forming capacities under nutrient limitation ( Figure 3C ) .","Comparing SeMeCo against prototrophic yeast strains ( Figure 3A ) , dry biomass formation did not vary significantly ( Figure 3C centre; p-values = 0 . 27 , 0 . 70 , and 0 . 09 for FourP , pHLUM , and YSBN5 , respectively ) .","In liquid media , lag phase was prolonged , and the maximum specific growth rate ( µmax ) was slightly reduced compared to the genetically prototrophic YSBN5 or pHLUM cells ( 0 . 17 OD595/hr vs 0 . 20 and 0 . 21 OD595/hr , respectively ) ( Figure 3C right ) .","However , this difference appeared more as a cost of plasmid segregation , as both lag phase and µmax did not vary significantly between SeMeCo and the FourP strain ( 0 . 17 and 0 . 16 OD595/hr , respectively ) ( Figure 3C right ) .","Finally , we tested to what extent cell death occurs in the cooperating community .","Both a wild-type ( YSBN5 ) and a SeMeCo culture were grown to exponential phase and cells were counted .","Then , the cultures were plated on SC media and the number of colony-forming units ( CFUs ) determined .","The CFU count was nearly equal between SeMeCo and YSBN5 , and similar to a 1:1 relationship to the cell count measured prior to spotting ( Figure 3D ) .","This indicates that cells in SeMeCo have a comparable colony-forming capacity to that of exponentially growing wild-type cells , and in both populations , virtually every cell can form a new colony .","To establish if cells cooperating in SeMeCo are distributed in a random or organised manner , we analysed colony spatial structure using confocal fluorescence microscopy ( Figure 4A ) .","For this , the community was re-established with alternative plasmids that express the fluorescent protein markers CFP ( cyan fluorescent protein ) , Venus ( yellow fluorescent protein ) , Sapphire ( a UV-excitable green fluorescent protein [Sheff and Thorn , 2004] ) , and mCherry ( red fluorescent protein ) coupled to the auxotrophic markers HIS3 , URA3 , LEU2 , and MET15 , respectively ( Bilsland et al . , 2013 ) .","Segregation of the labelled plasmids were within the same range , although not identical to the original pRS and p400 plasmids ( Figure 4—figure supplement 1 ) .","Images were acquired with a SP5 on a DMI6000 inverted microscope ( Leica , Wetzlar , Germany ) and show the underside of a live two day micro-colony which had , prior to imaging , been growing on minimal media ( SM ) .","In our hands , the Sapphire-LEU2 fluorescence was also visible under the imaging conditions used to visualise Venus-URA3 .","For this reason , the Venus-URA3 channel was removed from the colony image .","The spatial heterogeneity of fluorescent markers in the micro-colony revealed that cells in SeMeCo unequally distribute over the macroscopic structure , and form regions where the biosynthesis of a particular metabolite dominates ( Figure 4A ) .","A prediction in truly cooperating communities is , however , whether complementary cells maintain physical proximity to each other , to oppose diffusion of exchanged metabolites ( Müller et al . , 2014 ) .","Using computational image analyses of colony micrographs , we find that even when the most stringent cut-off was applied , complementary metabotypes across the community maintained an average distance ( 6 . 86 μm ) of less than two cell diameters ( Figure 4B ) .","Cells are hence most likely to exchange the majority of metabolites with those maintaining close proximity .","Despite these results , SeMeCo could , however , continue growth after disruption of this spatial structure in liquid media ( Figure 3C ) .","To verify this assumption , SeMeCo was replicated for 7 days in liquid minimal media , as previously , with re-dilution every 2 days .","Indeed , the liquid culture maintained a similar content of auxotrophs as obtained with colony grown SeMeCos ( Figure 4—figure supplement 2 ) .","The capacity of SeMeCos to overcome metabolic deficiencies through metabolic cooperation is hence not in essence bound to colonial growth . 10 . 7554/eLife . 09943 . 014Figure 4 . Spatial organisation of SeMeCo .","( A ) Spatial organisation of metabolically cooperating yeast micro-colony on minimal agar media ( SM ) .","SeMeCo was established with plasmids expressing fluorescent protein coupled to each auxotrophic marker; LEU2 , MET15 , and HIS3 cells are coloured green , red , and blue , respectively .","Cells containing more than one marker are coloured as a product of the additive RGB colour model .","Two–day-old live and growing micro-colony is visualised from underneath .","( B ) Minimum , mean and maximum distances between leucine , histidine , and methionine auxotrophs and their corresponding prototrophs in a SeMeCo colony .","Maximum distance between auxotroph and prototroph for 90% of cells shows an average distance of 6 . 86 μm , using the highest cut-off .","Despite the heterogeneous macroscopic colony composition , complementary auxotrophs are maintained in physical proximity to each other . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01410 . 7554/eLife . 09943 . 015Figure 4—source data 1 . Segregation rates of fluorescent protein plasmids from the yEp , pRS and p400 series . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01510 . 7554/eLife . 09943 . 016Figure 4—figure supplement 1 . Plasmid segregation rates of fluorescent protein plasmids ( %; probability of plasmid loss per cell division ) of BY4741 carrying plasmids encoding HIS3 ( yEpCFP_HIS ) , LEU2 ( yEpSapphire_LEU ) , URA3 ( yEpVenus_URA ) , and MET15 ( pRS411-GPDpr-mCherry ) respectively , compared to BY4741 carrying all four at the same time .","n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01610 . 7554/eLife . 09943 . 017Figure 4—figure supplement 2 . SeMeCos continue growth in minimal ( SM ) liquid culture . As in the colony growth experiment , cells were transferred from a micro-colony on SM agar , and then grown for 7 days with re-diluting every 2 days , however , here cells were in shaking batch liquid culture ( 25 mL ) .","Auxotrophy abundance is 70 . 3 ± 2 . 5% despite cells growing in minimal media . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 017 To determine not only the spatial but also the population structure , we switched back to the non-fluorescent SeMeCo to avoid confounding effects of fluorescent protein expression , and quantified by replica plating the colony contribution of all 16 possible metabotypes , resulting from all possible combinations of the four auxotrophies ( Figure 2B ) .","These experiments revealed that within SeMeCo , 95 . 6% of cells belonged only to 8 of the 16 possible metabolic combinations ( Figure 5A ) .","We questioned whether this composition was the result of a stochastic event , however , the dominance of the same metabotypes establish three times independently .","Moreover , the eight successful metabotypes contributed to SeMeCo at comparable percentages ( Figure 5A inset ) .","Using our segregation rate model as well as growth rate data , we could rule out this colony composition being a result of","( i ) varying plasmid segregation rate ,","( ii ) the number or type of auxotrophy , or","( iii ) differences in growth rates .","First , a community composition calculated on the basis of the experimentally determined plasmid segregation values ( Figure 2E , Figure 2—figure supplement 1 ) showed zero correlation with the actual population composition ( r² = 0 . 051 ) ( Figure 5B ) .","Second , all histidine , leucine , uracil , or methionine auxotrophies , as well as all plasmid numbers ( 1 to 4 ) were found amongst both the frequent and rare metabotypes .","For instance , while single uracil ( HIS3 , LEU2 , MET15 , ura3∆; 19 . 7% ) or leucine ( HIS3 , URA3 , MET15 , leu2∆; 11 . 1% ) auxotrophs were amongst the most frequent cells , their methionine-deficient counterparts ( HIS3 , URA3 , LEU2 , met15∆; 0 . 4% ) were among the most rare ( Figure 5A ) .","Also , the high frequency of the dual auxotrophs LEU2 , MET15 , his3∆ , ura3∆ ( 8 . 1% ) and HIS3 , MET15 , leu2∆ , ura3∆ ( 10 . 1% ) , contrasts with the rareness of the other dual auxotrophs ( HIS3 , URA3 , leu2∆ , met15∆ ( 1 . 1% ) , MET15 , URA3 , leu2∆ , his3∆ ( 0 . 2% ) , URA3 , LEU2 , his3∆ , met15∆ ( 0 . 5% ) , and LEU2 , HIS3 , ura3∆ , met15∆ ( 0 . 6% ) ) .","Thus , the number of plasmids or type of auxotrophy a cell had did not indicate whether a cell-type would be rare or frequent ( Figure 5A ) .","Finally , the growth rate of 16 strains , carrying the same marker and supplement combination that replicates the 16 metabotypes ( Mülleder et al . , 2012 ) did not distinguish the depleted from the selected metabotypes either ( Figure 5C ) . 10 . 7554/eLife . 09943 . 018Figure 5 . The community composition is distinct and dynamic .","( A ) Frequency of the 16 metabotypes that result from combination of histidine , leucine , methionine , and uracil auxotrophies as found within SeMeCo colonies .","Separately established populations ( n=3 ) were genotyped ( >180 cells per colony ) and eight metabotypes showed to dominate in the populations ( inset ) .","Frequency of the 16 metabotypes in independently established cell populations .","The eight metabotypes of low frequency , which were depleted in all experiments , are highlighted with a red circle .","( B ) No correlation shown between the frequency of the 16 metabotypes in SeMeCo versus a segregation rate-predicted colony composition .","Coloured points correspond to the experimentally observed eight frequent metabotypes .","Dashed line: linear regression fit .","( C ) Maximum specific growth rate ( µmax ) in supplemented minimal media ( red dots ) , of the 16 strains carrying HIS3 , LEU2 , URA3 , and MET15 plasmids in all combinations obtained from ( Mülleder et al . , 2012 ) , relative to the frequency of the specific metabotype in SeMeCo ( light blue ) .","Dashed line indicates average µmax for the eight most and least frequent metabotypes within SeMeCo colonies .","( D ) SeMeCo re-established on minimal media supplemented with uracil .","After 7 days of growth ( with re-spotting every two days ) , SeMeCo adapted with an entirely different composition of metabotypes ( green box plot ) compared to original SeMeCo colony composition ( grey bars ) .","Uracil producing cells decline , including the FourP genotype , so that 97% of cells are cooperating auxotrophs . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01810 . 7554/eLife . 09943 . 019Figure 5—figure supplement 1 . Abundance over time of a fluorescent labelled frequent ( HIS3 , LEU2 , MET15 , ura3∆ ) and rare ( his3∆ , leu2∆ , URA3 , MET15 ) genotype spiked into SeMeCo , as measured by FACS .","( Metabotype frequency determined from SeMeCo colony , Figure 5A ) .","( left ) % Fluorescence of the frequent and rare metabotype established individually as a colony shows frequent and rare abundance respectively ( unlabelled prototroph control is BY4741-pHLUM ) .","( right )","Frequent and rare metabotypes spiked into pre-established SeMeCo shows depletion of both cell types after approx .","48 hr . n = 3 , error bars = ± SD .","FACS: Fluorescence-activated cell sortingDOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 019 As growth potential and segregation parameters did not explain the population architecture of SeMeCo , we conclude that this community was selected for on its ability to cooperate effectively .","If this interpretation is correct , it would imply that a different pressure to cooperate would result in a different SeMeCo composition .","To test this hypothesis , we focussed on uracil , as mass spectrometry had detected an increase in uracil concentration in the SeMeCo colony exometabolome , indicating that uracil is the most limiting metabolite ( Figures 2G right and 3Bii ) .","SeMeCos established on uracil adapted a different composition , resulting from a dramatic decline in cells needed to produce uracil .","Importantly , this included the prototroph with its total content in the community decreasing from 26 . 7% in the original SeMeCo to solely 3 . 0% , so that 97 . 0% of cells were cooperating auxotrophs ( Figure 5D ) .","Hence , SeMeCo colonies establish a population that is dynamic to changes in the external metabolite pool , and can persist in a state with virtually all cells being genetically auxotrophic for at least one essential metabolite ."],["Metabolic exchange interactions occur frequently among cells that grow in proximity to one another , as metabolites are constantly released from cells for different reasons , such as overflow metabolism , metabolite repair , as well as export to facilitate metabolite exchange .","In bacteria , a subset of such metabolite exchanges are of a cooperative nature in the sense that all exchange partners profit from this situation ( Oliveira et al . , 2014 ) , whereas for the majority of eukaryotic organisms , metabolite exchange strategies remain unclear .","Despite yeast auxotrophs being viable in supplemented and rich media ( Mülleder et al . , 2012 ) , in the absence of amino acid supplementation , they fail to complement metabolic deficiencies in several pairs or higher order co-culture experiments ( Figure 1A , B , [Müller et al . , 2014; Shou et al . , 2007] ) , a clear difference to bacterial studies , where similar experiments were effective ( Foster and Bell , 2012; Freilich et al . , 2011; Harcombe , 2010; Pande et al . , 2014; Ramsey et al . , 2011; Vetsigian et al . , 2011 ) .","This led to speculations that yeast might , in contrast to many bacterial species , lack the required export capacities to enable growth relevant exchange of intermediary metabolites such as amino acids and nucleobases ( Shou et al . , 2007 ) .","Analysing the intra-colony exometabolome we could , however , detect the required metabolites; in fact , we found that cells within a colony are surrounded by a rich exometabolome .","We also found that yeast would efficiently exploit the nutrients when available , to the extent that they solely rely on these extracellular metabolites .","This result implied that metabolite exchange among co-growing yeast cells is frequent by nature; the lack of complementation in the co-culture experiments could thus reflect a limit of the experiment itself , and not represent the metabolite exchange capacities of yeast cells .","To circumvent combining two or more cultures , we chose an approach of synthetic biology and exploited the stochastic loss of plasmids to progressively introduce the metabolic deficiencies in random combination from an initially single cell .","The progressive loss of prototrophy allowed cells to maintain cell growth on the basis of metabolite exchange , resulting in a community with 73% auxotrophy , which increased to 97% upon supplementation with the most limiting metabolite , uracil .","Despite its dominant auxotrophic composition , the SeMeCo community could maintain a wild-type like exometabolome , metabolic efficiency , as well as cell viability , implying that this type of cooperation is a robust physiological property .","Hence yeast's natural metabolite export and import capacities are wholly sufficient to support co-growth on the basis of metabolite exchange .","The establishment of SeMeCos was not facilitated by mixing the auxotrophs in a higher order combination either .","This is consistent with the notion that losing more metabolic genes reduces biochemical capabilities and does not add new ones .","The key of the SeMeCo system is instead to allow the progressive self-establishment of the community starting from the single cell ( Figure 2 ) .","Metabolic feedback regulatory systems therefore do not inhibit metabolite export in general but prevent cooperative co-growth when already pre-established co-cultures are mixed ( Müller et al . , 2014; Shou et al . , 2007 ) .","A possible role of these mechanisms could perhaps prevent the spread of foreign , potentially cheating , cells that derive from a competing yeast colony .","We could replicate behaviour which is in favour of such an assumption; By spiking into SeMeCo a cell culture possessing the same genotype as a frequent ( HIS3 LEU2 ura3Δ MET15 ) and rare ( his3Δ leu2Δ URA3 MET15 ) genotype ( Figure 5A ) , we observed that both genotypes were rapidly depleted from the pre-established SeMeCo , irrespective of the frequency of the respective genotype in SeMeCo ( Figure 5—figure supplement 1 ) .","Studying the genotypic composition of SeMeCo implied that there are a defined set of interactions underlying the properties of the cooperating community , which maintained a similar population composition involving eight reproducibly concentrated metabotypes when established independently .","This indicates that this quantitatively defined community composition was most effective in metabolic cooperation .","This finding may close an important gap in the understanding of the evolution of multicellularity; If a defined composition is most effective in cooperative growth , a selection advantage could be provided by any sort of physical bonding which can maintain cooperation partners in the defined equilibria , and would in addition , provide additional protection against the invasion of cheating cell types .","Indeed , the exometabolome data implies that the number of metabolite exchange interactions among co-growing cells could be significant .","The colony exometabolome contained a vast array of biomolecules , including the majority of amino acids ( Figure 1C ) ( Castrillo et al . , 2007; Paczia et al . , 2012; Silva and Northen , 2015 ) .","The finding that yeast cells prefer uptake over synthesis of amino acids and uracil , even when they are genetically prototrophic , shows that exchange interactions will readily establish once the cellular environment has acquired a critical concentration of metabolites .","This has implications for the interpretation of metagenomic studies and cheating/benefactor experiments , as for this reason , it cannot be concluded from the genetic presence or absence of a single metabolic pathway , or from following the synthesis of a single metabolite , how many other metabolites are being exchanged as well .","Even without selective pressure , both wild-type yeasts and SeMeCo established an amino-acid-rich exometabolome on minimal media ( Figure 1C ) and engaged in metabolite uptake when nutrients were available ( Figure 1E ) .","This implies that these features are a natural property of yeast and raises the question of why natural yeast communities are not composed of co-growing , genetic , auxotrophs .","To answer this question , one needs to keep in mind that possessing metabolic genes in the genome is not equal to the pathway being constantly active; Indeed , prototrophs can flexibly switch from self-synthesis to amino acid uptake ( Figure 1 ) .","Being genetically prototrophic hence gives a higher level of metabolic flexibility , as prototrophic cells can re-activate a synthetic pathway when required .","Unlike in SeMeCo , the genotype of a cell in a natural community is not in essence reflecting its metabotype or its metabolic role in the community .","Additionally , the natural life cycle of yeast involves the formation of endospores , which are important for enduring starvation , and to spread between habitats .","Without a prototrophic genotype , a single spore can no longer establish a colony on its own as genetic auxotrophy would interrupt the yeast life cycle .","Second , only a fraction of the natural yeast life cycle occurs under exponential growth that requires abundant carbohydrate and nitrogen supply .","The maintenance of a prototrophic genotype both in S . pombe and in S . cerevisiae wild isolates ( Jeffares et al . , 2015; Liti et al . , 2009 ) is hence fully compatible with the presence of elaborate amino acid and nucleotide exchange mechanisms .","The finding that these cells fully shift from self-synthesis to uptake for histidine , leucine , methionine , and uracil , once these metabolites are provided , has direct implications on research using yeast , a primary eukaryotic model organism in genome-scale studies .","A majority of yeast genetic experiments are conducted in auxotrophic strains , requiring amino-acid supplemented or rich media compositions .","Important parts of biosynthetic metabolism ( amino acid biosynthesis can account for up to 50% of metabolic flux towards biomass ) may have thus stayed silent in a significant amount of functional genomics experiments .","The effects of metabolic–genetic interactions on cellular physiology could thus substantially exceed our current knowledge and could be discovered upon switching to minimal nutrient supplementations .","In this context , SeMeCos are simple to handle , establish rapidly and are easy to analyse , and therefore represent an effective and broadly applicable eukaryotic model system to study both cooperativity and effects of metabolism in the laboratory .","In summary , using histidine , leucine , methionine , and uracil as model metabolic pathways for exchangeable metabolites , we found that S . cerevisiae cells prefer these nutrients' uptake over their self-synthesis and maintain an amino-acid-rich exometabolome in the extracellular colony space , indicators of ongoing inter-cellular metabolite exchange .","Although yeast is known to fail in compensating for auxotrophy in pairwise and higher order co-culture experiments , the cells did successfully enter a state of metabolic cooperative growth upon exploiting stochastic plasmid segregation so that a single cell could progressively develop into a complex heterogeneous community .","Composed of auxotrophic cell types that are non-viable on their own , SeMCo communities were able to overcome metabolic deficiencies and maintain metabolite concentrations and robustness similar to wild-type cells .","Additionally , cooperation had imposed different metabolic roles on contributing cells .","Progressive community formation thus reveals that yeast possesses full capacity to exchange anabolic metabolites at growth relevant quantities and readily establishes a non-cell-autonomous metabolism within complex but defined community structures ."],["Yeast cells were grown under standard conditions on synthetic minimal ( SM or EMM ) , SC and rich ( YPD or YES ) media .","Plasmid segregation was calculated according to Christianson et al . ( 1992 ) , by monitoring plasmid retention after cells are shifted from non-selective to selective media , and by expressing the number of cells that have lost the marker as a function of generation time .","Metabolites were quantified after quenching using an online UPLC-coupled 6460 ( Agilent Technologies , Waldbronn , Germany ) triple quadrupole mass spectrometer .","Confocal fluorescence microscopy was conducted with a SP5 confocal on a DMI6000 inverted microscope ( Leica ) using a 10x/0 . 3 HC PL Fluotar Air objective .","All experiments involved , unless otherwise indicated , used BY4741 yeast strain ( his3Δ1 , leu2Δ0 , ura3Δ0 , met15Δ0 ) ( Brachmann et al . , 1998 ) with prototrophy restored by complementation either with vectors p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) ( Christianson et al . , 1992; Mumberg et al . , 1995; Sikorski and Hieter , 1989 ) , with the centromeric vector ( minichromosome ) pHLUM ( Mülleder et al . , 2012 ) ; Addgene number: 40276 ) , or with the fluorescent protein vectors yEpCFP_HIS ( HIS3 ) , yEpSapphire_LEU ( LEU2 ) , yEpVenus_URA ( URA3 ) ( Bilsland et al . , 2013 ) , and pRS411-GPDpr-mCherry ( MET15 ) ( Table 1 ) .","Cloning was conducted according to standard procedures; oligonucleotides are listed in Table 2 . 10 . 7554/eLife . 09943 . 020Table 1 . Strains and plasmids used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 020NameDescriptionReferenceStrainsBY4741MATa , his3∆1 leu2∆0 met15∆0 ura3∆0 ( ATCC 201388 ) ( Brachmann et al . , 1998 ) BY4741 FLO+ Derived from tetrad dissection after crossing and sporulating a flocculating BY4741 strain derived from the knock out collection ( ∆tpo1 ) with BY4742 and isolating a FLO+ TPO1 wild-type progeny This studyYSBN5MATa , FY3 ho::Ble ( Canelas et al . , 2010 ) ED666h+ ade6-M210 ura4-D18 leu1-32Bioneer Cat .","No .","M-3030HPlasmids p423GPD2 µ vector with HIS3 marker ( Mumberg et al . , 1995 ) pRS4252 µ vector with LEU2 marker ( Christianson et al . , 1992 ) p426GPD2 µ vector with URA3 marker ( Mumberg et al . , 1995 ) pRS411Yeast centromeric vector with MET15 marker ( Brachmann et al . , 1998 ) pHLUMYeast centromeric vector with HIS3 , URA3 , LEU2 and MET15 markers ( minichromosome ) .","( Addgene number: 40276 ) ( Mülleder et al . , 2012 ) pFS118Yeast high-copy vector with endogenous promoter forura4+ ( Addgene number: 12378 ) ( Sivakumar et al . , 2004 ) pREP41-MCS+Yeast high-copy vector with endogenous promoter for LEU2 .","( Addgene number: 52690 ) A gift from Michael Nick Boddyp416GPDYeast centromeric vector with endogenous promoter for URA3 ( Mumberg et al . , 1995 ) pHS12-mCherryYeast vector with mCherry fluorescent tag and LEU2 marker .","( Addgene number: 25444 ) A gift from Benjamin Glickp426-GPDpr-mCherryYeast 2 µ vector with endogenous promoter for URA3 marker and a GPD promoter for mCherry fluorescent tagThis study .","Derived from p416GPD , pHS12-mCherry and p426GPDpRS411-GPDpr-mCherryYeast 2 µ vector with endogenous promoter for MET15 marker and a GPD promoter for mCherry fluorescent tagThis study .","Derived from p426-GPDpr-mCherry and pRS411yEpVenus_URAYeast 2 µ vector with TDH3-promoter-driven Venus ( YFP ) and URA3 marker ( Bilsland et al . , 2013 ) yEpCFP_HISYeast 2 µ vector with TDH3-promoter-driven CFP and HIS3 marker ( Bilsland et al . , 2013 ) yEpSapphire_LEUYeast 2 µ vector with TDH3-promoter-driven Sapphire ( a UV-excitable GFP ) and LEU2 marker ( Bilsland et al . , 2013 ) pHLM-GPDpr-mCherryYeast centromeric vector with a GPD promoter for mCherry fluorescent tagThis study .","Derived from pHLUMpUM-GPDpr-mCherryYeast centromeric vector with a GPD promoter for mCherry fluorescent tagThis study .","Derived from pHLUM10 . 7554/eLife . 09943 . 021Table 2 . Oligonucleotides used to create expression plasmid p426-GPDpr-mCherry . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 021NameSequencemCherry_Bam_Sac_fwAAGAAGAGCTCAAAAGGATCCGGGATGGTGAGCAAGGGCGAGGmCherry_Xho_rvCCTTTTCTCGAGCTTGTACAGCTCGTCCATGC All experiments involving wild-type yeast strains were carried out using as indicated , and using YSBN5 , a prototrophic haploid variant of S . cerevisiae S288c ( Canelas et al . , 2010 ) .","For microscopy analyses of colony spatial organisation , BY4741 had prototrophy restored by complementation with the above fluorescent protein vectors ( Table 1 ) .","For fluorescence-activated cell sorting ( FACS ) , BY4741 was used with mCherry labelled derivatives of pHLUM , pHLM-GPDpr-mCherry , and pUM-GPDpr-mCherry ( Table 1 ) .","For S . pombe experiments , ED666 yeast strain ( ade6-M210 ura4-D18 leu1-32 ) was used that had uracil and leucine prototrophy restored by complementation with vectors pFS118 ( ura4+ ) and pREP41-MCS+ ( LEU2 ) ( Table 1 ) .","Yeast was cultivated if not otherwise indicated at 30ºC , in rich ( YPD; 1% yeast extract [Bacto] , 2% peptone [Bacto] or YES; [Formedium; 35 . 25 g/L] ) , complete supplemented synthetic media ( SC; CSM complete supplement mixture [MP Biomedicals; 0 . 56 g/L] , YNB , yeast nitrogen base [Sigma; 6 . 8 g/L] ) , or minimal supplemented synthetic media ( SM; YNB [Sigma; 6 . 8 g/L] or EMM; [Formedium; 32 . 3 g/L] ) , with 2% glucose ( Sigma ) as the carbon source .","Media recipes and amino acid compositions for S . cerevisiae were used as previously published ( Mülleder et al . , 2012 ) .","For S . cerevisiae , auxotrophic derivatives of prototrophic BY4741 ( Mülleder et al . , 2012 ) were cultured alone or mixed in combination with other auxotrophs , and 1 . 1e05 cells of individual or mixed auxotrophs were spotted on respective selective media .","Growth was then documented after 2 days incubation at 30°C .","For the flocculation experiments , a FLO+ derivative of BY4741 was obtained by back-crossing and tetrad dissection of a tpo1Δ ( YLL028W ) strain obtained from Euroscarf ( Frankfurt , Germany ) .","For S . pombe , 1 . 9e04 cells of auxotrophic derivatives of ED666 h+ , prototrophic for leucine and uracil , were spotted alone or mixed together on corresponding selective media .","Growth was then documented after 2 days incubation at 30°C .","All proteogenic amino acids ( except for cysteine ) and uracil , citrulline , and ornithine were analysed by selective reaction monitoring ( SRM ) using an online coupled UPLC ( 1290 Infinity , ( Agilent ) ) / triple quadrupole mass spectrometer ( 6460 , ( Agilent ) ) system .","The compounds were separated by hydrophilic interaction chromatography on an ACQUITY UPLC BEH amide column ( 2 . 1 mm × 100 mm , 1 . 7 µm ) by gradient elution .","Solvent A consisted of 95:5:5 acetonitrile:methanol:water , 10 mM ammonium formate , 0 . 176% formic acid , and solvent B of 50:50 acetonitrile:water , 10 mM ammonium formate , 0 . 176% formic acid .","The gradient conditions were 0–07 min 85% B , 0 . 7–27–2 . 55 min 85–585–5% B , 2 . 55–255–2 . 75 5% B , 2 . 75–275–2 . 8 min 5–855–85% B and 2 . 8–38–3 . 25 min 85% B at a constant flow rate of 0 . 9 mL/min and 25°C column temperature .","SRM ( Q1/3 settings ) are given in Table 3 .","Metabolite signals were automatically integrated using Masshunter ( Agilent ) corrected after manual inspection and quantified by external calibration . 10 . 7554/eLife . 09943 . 022Table 3 . SRM transitions for quantification of amino acids and uracil . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 022Compound nameCompound abbreviationSRM transitionFragmentor ( V ) Collision energy ( V ) PolarityUracilU111 . 0 > 42 . 1629-−PhenylalanineF166 . 1 > 1201009+LeucineL132 . 1 > 86808+TryptophanW205 . 1 > 188855+IsoleucineI132 . 1 > 86808+MethionineM150 . 1 > 104408+TaurineTau126 > 44 . 111016+ValineV118 . 1 > 71 . 910010+ProlineP116 . 1 > 70 . 110013+TyrosineY182 > 165905+AlanineA90 > 44 . 1508+ThreonineT120 . 1 > 74809+GlycineG76 > 30 . 1505+GlutamineQ147 . 1 > 845016+GlutamateE148 . 1 > 84 . 17510+SerineS106 > 60409+AsparagineN133 . 1 > 74809+AspartateD134 . 1 > 748010+HistidineH156 . 1 > 110 . 28012+ArginineR175 . 1 > 7010015+LysineK147 . 1 > 845016+CitrullineCit176 > 159604+OrnithineO133 > 709010+ Cells were spotted on SM solid media and incubated at 30°C in FLUOstar OPTIMA plate reader ( BMG LABTECH , Aylesbury , United Kingdom ) to establish giant colony .","Cells were collected at 26 hr ( exponential phase ) and re-suspended in H2O .","Cells were then gently centrifuged , and pellet ( intracellular ) and supernatant ( extracellular ) fractions were separated .","Metabolites were extracted from both fractions using 75% boiling ethanol containing l-taurine as an internal standard .","Here , samples were left to incubate with extraction solvent in water bath ( 80°C ) for 2 min then mixed vigorously .","Incubation and vigorous mixing step was then repeated .","Solvent was evaporated using a Concentrator plus Speed Vac ( Eppendorf , Hamburg , Germany ) and samples were reconstituted in 50 µL 80% ethanol with intracellular fraction diluted 1:5 with 80% ethanol .","All samples were submitted to LC-MS/MS and metabolite identification , and quantification was then performed as in 'LC-MS/MS based quantification of amino acids and uracil' .","Data was illustrated following correction to the internal standard of amino acid abundances from both intra- and extracellular fractions .","S . cerevisiae strains were transferred from cryo-preserved cultures to SC solid media , grown for 2 days and selected on SM solid media , supplemented only with required amino acids/ nucleobases for 1 day .","Pre-cultures were inoculated in 1 . 5 mL SM containing the minimal supplementation and cultured O/N at 30°C .","Main cultures were started at an OD595 of 0 . 15 in 1 . 5 mL of SC media in deep well 96-well plates and cultured in a Titramax ( Heidolph , Schwabach , Germany ) for 30 hr ( 950 rpm , 30°C , 4 mm stirring bead/ well ) .","Samples of 50 µL were harvested every 3 hr , where cells were removed by centrifugation ( 3000 g , 5 min ) and the supernatant diluted 1:20 in absolute ethanol .","Then , 1 µL of supernatant was used for quantification of extracellular metabolites by LC-MS/MS .","‘gcFitModel’ function from ‘grofit’ R package ( Kahm et al , 2010 ) was used to estimate the uptake rate of histidine , leucine , uracil , and methionine in different auxotrophic strains .","After O/N pre-culture S288c MATa yeast without auxotrophies and ura3Δ yeast ( S288c , MATa ) ( Mülleder et al . , 2012 ) were grown in 30 mL SM in shake flasks at 30°C , 300 rpm .","The media contained either","( i ) no additives ,","( ii ) uracil ( 20 mg/L )","( iii ) or uracil ( 20 mg/L ) , leucine ( 60 mg/L ) , methionine ( 20 mg/L ) and histidine ( 20 mg/L ) .","During mid-exponential growth ( OD595 between 0 . 7 and 1 . 2 ) , 1 mL samples of the cultures were quenched in 4 mL -40°C 60% methanol , 10 mM NH4-acetate .","After centrifugation ( -9°C , 4500 g ) , the cell pellet was stored at -80°C until extraction .","Prior to extraction , 13C-yeast internal standard was spiked into the cell pellets , which were subsequently extracted with 1 mL 80°C 75% ethanol , 10 mM NH4-acetate for 3 min .","During extraction , the suspension was vortexed on a 0 . 5–15–1 min time interval .","After extraction , the suspension was centrifuged ( -9°C , 4500 g ) and the supernatant , hence the extract , was dried in a vacuum centrifuge before being stored at -80°C until measurement .","For LC-MS measurements , the dried extracts were dissolved in 50–10050–100 µL H2O .","The metabolites were separated with reversed phase ion-pairing chromatography on a Acquity UPLC ( Waters , Cheshire , United Kingdom ) with a Waters Acquity T3-endcapped column ( 150 mm , 2 . 1 mm , 18 µm ) as described in ( Buescher et al . , 2010 ) .","Subsequently , the metabolites were analysed with a TSQ quantum ultra triple quadrupole mass spectrometer ( Thermo Fisher Scientific , Waltham , MA ) ( Buescher et al . , 2010 ) .","Specifically , the metabolites were ionised with an electro spray ( ESI ) and the mass spectrometer was run in negative mode with SRM .","The SRM transitions used are described in ( Buescher et al . , 2010 ) and for orotidine-monophosphate , where no standard was available , we used the phosphate fragment ( m/z 367 → 79 ) trajectory ( Horai et al . , 2010 ) .","The obtained data was integrated with an in-house software and normalised to 13C-internal standard and OD595 , hence biomass .","The median value of different replicates were then scaled and used to illustrate data .","Plasmid stability ( segregation ) of vectors p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) was determined according to Christianson et al . ( 1992 ) .","BY4741 ( his3Δ1 , leu2Δ0 , ura3Δ0 , met15Δ0 ) either transformed with one or all four plasmids , respectively , for either the four non-fluorescent or fluorescent vectors , were cultured in 25 mL of YPD media for 48 hr then plated at 1:100 , 000 dilution on YPD solid media .","Plasmid retention was then calculated by replica plating CFUs from YPD solid media onto selective solid media .","Number of doublings ( g ) and segregation rate ( m ) were calculated as in ( Christianson et al . , 1992 ) .","Segregation rates of p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) were simulated over generation time in R by iterative cycling ( looping ) .","The script is given in Supplementary file 1 .","Plasmid abundances were binned by plasmid number ( 0 to 4 ) and illustrated with R package 'ggplot2' in terms of auxotrophy .","Unless otherwise indicated , cells were first spotted and grown for 2 nights on SM solid media to establish a giant colony .","The colony was then re-suspended in H2O and diluted to 3 . 4e03 cells in 200 μL SM , and their optical density ( OD595 ) was recorded in a FLUOstar OPTIMA plate reader ( BMG LABTECH ) every 20 min for 40 hr at 30ºC .","Both maximum specific growth rate ( μmax ) and lag phase were determined from growth curves using a model-richards fit from the R ‘grofit’ package ( Kahm et al , 2010 ) .","For determining dry biomass , colony was re-suspended in H2O and normalised to 1 . 1e07 cells in 100 mL SM , then incubated for 72 hr at 30°C and pelleted .","Pellets were dried for 5 days at 50°C and then weighed to obtain dry biomass .","μmax for individual metabotypes was determined in batch SM culture ( 50 mL ) and supplemented accordingly for the different auxotrophic requirements .","Cells from giant colonies of SeMeCo and YSBN5 were grown to exponential growth phase in SC media , and cell number was then measured with a CASY Model TTC ( Roche Innovatis , Switzerland ) cell counter .","Cells were then diluted and plated on solid SC media to establish individual CFUs and the number of CFUs with initial cell number were compared .","A micro-colony of BY4741 with prototrophy restored by complementation with the fluorescent protein vectors yEpCFP_HIS ( HIS3 ) , yEpSapphire_LEU ( LEU2 ) , yEpVenus_URA ( URA3 ) ( Bilsland et al . , 2013 ) , and pRS411-GPDpr-mCherry ( MET15 ) ( Table 1 ) was grown for 2 nights on SM .","Prior to imaging , colony was embedded in 2% agarose ( Type I-B; Sigma ) and gently transferred to a μ-slide glass bottom ( ibidi ) .","Cells were imaged with a DMI6000 inverted Leica SP5 confocal microscope , using a 10×/0 . 3 HC PL Fluotar Air objective , running LAS AF software ( version 2 . 7 . 3 . 9723 ) .","Fluorescence for each marker was separated by excitation ( CFP: 458 nm , Sapphire: 405 nm , Venus: 514 nm and mCherry: 561 nm ) .","In our hands , the Sapphire-LEU2 was also visible under the imaging conditions used to visualise the Venus-URA3 .","For this reason , we removed Venus-URA3 channel from the colony image .","A look-up table was applied to each channel post-acquisition to allow visualisation of the different channels together using ImageJ software .","For the microscopy image showing fluorescent-labelled cells in a colony , prototrophs and auxotrophs were identified as being present or absent using several cut-offs for fluorescent signal intensity .","Based on these different cut-off values ( 0 . 1 , 0 . 2 , 0 . 3 , and 0 . 4 ) , the monochrome fluorescence microscopy images of each individual marker ( HIS3 , LEU2 and MET15 ) were recognised as metabolite producing ( prototrophic ) or requiring ( auxotrophic ) pixels .","The auxotrophic pixels were marked black , prototrophic pixels white , and the background was illustrated grey to separate it from the colony pixels .","For each auxotrophic pixel , the distance to the next prototrophic pixel was calculated and distance values for the minimum , mean , overall maximum , and maximum for 90% of cells were taken for each marker .","Genotypes depicting SeMeCo frequent ( HIS3 , LEU2 , MET15 , ura3Δ ) and rare ( his3Δ , leu2Δ , URA3 , MET15 ) metabotypes were reconstructed by transforming mCherry labelled derivatives of pHLUM ( pHLM-GPDpr-mCherry and pUM-GPDpr-mCherry , respectively ) into BY4741 .","Strains were spiked into established SeMeCo in 1:10 ( frequent or rare metabotype: SeMeCo ) ratio taken from their established giant colonies on selective media ( where plasmid segregation is ongoing ) .","Abundance of fluorescent cells was monitored immediately after mixing and approximately 48 hr after re-establishment of giant colony on minimal solid media with a BD LSRFortessa cell analyser .","Data analysis was performed with FlowJo ."]],"string":"[\n [\n \"All living cells possess a system for biochemical reactions , the metabolic network , which supplies cells with their necessary molecular constituents .\",\n \"The reactions participating in this network are highly conserved , so much so that all life is made up of a markedly similar set of metabolites ( Braakman and Smith , 2013; Caetano-Anolles et al . , 2009 ) .\",\n \"The functionality of the metabolic system is bound to a series of transport reactions that facilitate the uptake of metabolites from the environment , as well as metabolite export .\",\n \"Metabolite export primarily occurs for the purpose of maintaining balance of the metabolic system ( 'overflow metabolism' ) and to maintain chemical and physical integrity of the metabolic network .\",\n \"This includes indiscriminate metabolite export through non-specific multi-drug transporters required in removal of toxic metabolites for cells ( Paczia et al . , 2012; Piedrafita et al . , 2015 ) .\",\n \"Co-growing cells can uptake the released metabolites and exploit their presence .\",\n \"Indeed , to a lower extent , metabolite export of metabolites can specifically occur for the purpose of establishing inter-cellular metabolic interactions in biosynthetic metabolism ( Nigam , 2015; Paczia et al . , 2012; Yazaki , 2005 ) and can lead to mutualistic situations in which cells profit from coexistence ( Foster and Bell , 2012; Oliveira et al . , 2014 ) .\",\n \"In between species , mutually positive interactions can readily establish when exchange concerns an overflow metabolite , exemplified by yeast–algae interactions that can form on the basis of a CO2 and sugar exchange ( Hom and Murray , 2014 ) , or between different cells of the same species or tissue , exemplified in tumours , when lactate produced in excess by one cell type is metabolised by another ( Bonuccelli et al . , 2010 ) , or between neurons and glial cells that exchange sugar metabolites ( Bélanger et al . , 2011; Volkenhoff et al . , 2015 ) .\",\n \"It is more difficult to assess whether metabolite exchange is indicative of non-cell-autonomous metabolism , when exchange concerns metabolites that are needed by both exchange partners , amino acids , and nucleobases for instance .\",\n \"Exchange of costly intermediates is associated with a significant risk , as exported metabolites can be lost through diffusion , chemical damage , or cheating ( Dobay et al . , 2014; Oliveira et al . , 2014; Wintermute and Silver , 2010 ) .\",\n \"Despite these constraints , exchange of intermediates is frequently observed within bacterial microbial communities .\",\n \"Many bacterial species lose essential biosynthetic pathways , disabling them from living autonomously , which may explain why more than 90% of bacteria cannot be cultivated in the absence of a community environment ( Costerton et al . , 1994; Johnson et al . , 2012 ) .\",\n \"The energetic benefit and selective advantage associated with non-autonomous cellular metabolism is often not clear but might involve , for example , the ability to reduce genome size which would in turn facilitate faster proliferation .\",\n \"This may explain why bacteria frequently appear to cooperate in the biosynthesis of more costly and biosynthetically complex metabolites , such as aromatics ( Mee et al . , 2014 ) .\",\n \"While metagenomics has boosted the knowledge of metabolite exchange strategies in bacteria ( Blaser et al . , 2013; Manor et al . , 2014; Zelezniak et al . , 2015 ) , relatively little is known about eukaryotic species .\",\n \"This includes yeast , a popular single cellular eukaryotic model organism , whose metabolic capacities are regularly exploited in biotechnology .\",\n \"Yeast cells are known to participate in multi-species communities ( e . g . on human skin ( Findley et al . , 2013 ) ) , but as wild yeast isolates usually maintain similar prototrophic genomes ( Jeffares et al . , 2015; Liti et al . , 2009 ) , metagenomic data are not conclusive about yeast's metabolite exchange strategies .\",\n \"In laboratory experiments , yeast cultures were , however , not effective in supporting co-growth of auxotrophs that have complementary defects in amino acid and nucleotide metabolism , unless they were genetically modified to increase metabolite export ( Müller et al . , 2014; Shou et al . , 2007 ) .\",\n \"This contrasts with analogous studies in bacterial species , in which such growth experiments regularly show that co-cultured cells can overcome complementary metabolic deficiencies ( Foster and Bell , 2012; Oliveira et al . , 2014; Pande et al . , 2014; Vetsigian et al . , 2011 ) .\",\n \"In the absence of quantitative metabolite data , this observation has triggered the conclusion that co-growing prototrophic yeast cells produce amino acid and nucleotide metabolites predominantly for themselves and export them at insufficient quantities to support growth of co-growing cells ( Momeni et al . , 2013; Shou et al . , 2007 ) .\",\n \"Conflicting with this interpretation , we here report that yeast colonies maintain a rich exometabolome and that cells exploit this metabolic pool preferentially over their own biosynthetic capacities , which implies that metabolite exchange establishes as a natural property of yeast growth .\",\n \"To test whether yeast indeed possesses the capacity for metabolite exchange at growth relevant quantities , we established an alternative method to co-culture experiments .\",\n \"We exploited the stochastic segregation of episomes to randomly and progressively introduce metabolic auxotrophies into a yeast population which self-establishes from an initially prototrophic cell .\",\n \"This strategy enabled co-growing auxotrophs to enter an efficient state of metabolic cooperation , named self-establishing metabolically cooperating communities ( SeMeCos ) .\",\n \"Despite an auxotrophic cell composition of up to 97% , SeMeCos achieve metabolic efficiency , growth parameters , and cell viability similar to that of genetically prototrophic cells , revealing a natural capacity of yeast to exchange metabolites at growth relevant quantities .\",\n \"In a SeMeCo that possesses auxotrophies in histidine , leucine , uracil and methionine metabolism , we distinguish up to eight cell types , each of which is unable to survive on its own , or in co-culture studies , however , could adapt effectively and cooperatively to overcoming metabolic deficiencies , once self-established in a community structure .\",\n \"Communities have a stable population composition as well as distinct spatial heterogeneity , which was , however , not essential for metabolite exchange , as SeMeCos maintain growth in liquid suspension .\",\n \"Self-establishing , complex communities thus demonstrate that yeast is not only exchanging metabolites , but is also able to do so at growth relevant quantities , to facilitate growth on the basis of a non-cell-autonomous metabolism .\"\n ],\n [\n \"Histidine , leucine , uracil , and methionine biosynthetic pathways were chosen for our study as\",\n \"( i ) they can be interrupted by deletion of a single , non-redundant gene , which has been reported not to cause compensatory mutations and\",\n \"( ii ) because cells possess efficient uptake mechanisms for these nutrients ( Mülleder et al . , 2012; Pronk , 2002; Teng et al . , 2013 ) .\",\n \"Paired combinations of histidine ( his3Δ ) , leucine ( leu2Δ ) , uracil ( ura3Δ ) , or methionine ( met15Δ ) auxotrophs were unable to sustain growth in the absence of supplementation required for both individual cell types ( Figure 1A ) .\",\n \"A similar result was obtained by co-culturing flocculating yeast cells ( Figure 1—figure supplement 1 ) , which are able to maintain biofilm-like physical contact ( Smukalla et al . , 2008 ) , and in Schizosaccharomyces pombe ( Figure 1B ) , indicating evolutionary conservation of this observation in yeast species . 10 . 7554/eLife . 09943 . 003Figure 1 . Yeast auxotrophs do not compensate for metabolic deficiencies upon co-culturing , yet export the relevant metabolites and prefer metabolite uptake over self-synthesis .\",\n \"( A ) Complementary pairs of Saccharomyces cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .\",\n \"his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts were combined in complementary pairs and spotted on corresponding selective media .\",\n \"No pairs exhibited co-growth together .\",\n \"( B ) A complementary pair of Schizosaccharomyces pombe auxotrophs does not overcome metabolic deficiencies upon co-culturing .\",\n \"leu1Δ and ura4Δ yeasts were combined in a complementary pair and spotted on corresponding selective media .\",\n \"No co-growth occurred .\",\n \"( C ) The concentration of metabolites in the S . cerevisiae colony exometabolome obtained from 1 . 3e08 YSBN5 cells grown in a colony on synthetic minimal agar media ( SM ) and quantified by LC-MS/MS .\",\n \"Abbreviations: single letter IUPAC amino acid codes , O = ornithine , CIT = citrulline .\",\n \"n = 3 , error bars = ± SD .\",\n \"( D )\",\n \"( i ) Metabolites quantified as in ( C ) , comparing intracellular ( total cell extracts ) and extracellular metabolite concentrations in YSBN5 .\",\n \"n = 3 , error bars = ± SD .\",\n \"Dashed line: linear regression fit , grey band shows 95% confidence region .\",\n \"( ii ) Metabolites quantified as in ( C ) , comparing extracellular metabolite concentrations of YSBN5 and BY4741-pHLUM yeast colonies grown on minimal media .\",\n \"H , L , U , and M are highlighted in red circles .\",\n \"n = 3 , error bars = ± SD .\",\n \"Dashed line: linear regression fit , grey band shows 95% confidence region .\",\n \"Abbreviations IUPAC codes; H = histidine , L = leucine , U = uracil , M = methionine .\",\n \"( E ) Consumption of uracil , histidine , leucine , and methionine in yeast batch cultures in synthetic complete ( SC ) media as measured by LC-MS/MS .\",\n \"Uracil , histidine , leucine , and methionine prototrophic cells consume these metabolites at rates and quantities comparable to the corresponding auxotrophic strains .\",\n \"( F )\",\n \"( i ) Deletion of URA3 ( Orotidine-5'-phosphate decarboxylase ) causes accumulation of the Ura3p substrate orotidine-5'-phosphate ( OMP ) , when cells are supplemented with 20 mg/L uracil ( fold change of OMP abundance , relative to URA3 without uracil supplementation ) , as determined by LC-MS/MS .\",\n \"Error bars = ± SD .\",\n \"( ii ) Uracil supplementation of wild-type cells alters their metabolite profile to resemble ura3∆ cells , which obtain uracil solely from the growth media .\",\n \"Heatmap scaling ( [0 , 1] and min , max per metabolite ) was based on median concentration .\",\n \"The dendrogram was constructed by comparing euclidean distance ( dissimilarity ) between samples . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00310 . 7554/eLife . 09943 . 004Figure 1—figure supplement 1 . Flocculation does not enable Saccharomyces cerevisiae cells to establish viable co-cultures .\",\n \"( i ) ( left ) The FLO+ phenotype in yeast cells transforms their typical cell suspension ( right ) into a physiological state reminiscent of biofilms ( left ) ( Smukalla et al . , 2008 ) .\",\n \"Cultures were grown to stationary phase in rich media ( YPD ) and flocculation was detected via an inability of cells to re-suspend following repeated tube inversion .\",\n \"( ii ) Complementary pairs of flocculating S . cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .\",\n \"his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts were combined in complementary pairs and spotted on corresponding selective media .\",\n \"No pairs exhibited co-growth together . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00410 . 7554/eLife . 09943 . 005Figure 1—figure supplement 2 . Uracil biosynthetic genes in the uracil prototroph ( URA3 ) and auxotroph ( ura3Δ ) remain expressed in ( uracil supplemented ) SC media , as determined by RNA sequencing . Abbreviations: RPKM = reads per kilobase per million .\",\n \"n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 005 In two previous studies , leucine/tryptophan and adenine/lysine auxotrophic cell pairs , respectively ( Müller et al . , 2014; Shou et al . , 2007 ) , could co-grow upon removing metabolic feedback control .\",\n \"Feedback resistance renders cells metabolite over-exporters , leading to the conclusion that wild-type yeast cells produce intermediates primarily for themselves , at quantities that are not sufficient for growth relevant metabolite exchange ( Momeni et al . , 2013; Shou et al . , 2007 ) .\",\n \"In a detailed analysis of the intra-colony exometabolome , using an ultra-sensitive mass spectrometry method , the intra-colony fluid showed however to contain a plethora of metabolites , with the amino acids glutamine , glutamate , and alanine being the most highly concentrated ( Figure 1C ) .\",\n \"Furthermore , histidine , leucine , methionine , and uracil all showed to be part of this exometabolome ( Figure 1C ) .\",\n \"These measurements were obtained from cells in exponential growth phase , where apoptosis and necrosis are negligible .\",\n \"Comparing extracellular metabolite concentrations to intracellular levels ( the endometabolome ) we observed a general trend of correlation between the highest and lowest concentrated metabolites ( r2 = 0 . 517; Figure 1Di ) , but overall extracellular metabolite concentrations do not replicate the corresponding endometabolome .\",\n \"Tryptophan , phenylalanine , proline , and valine , for instance , were over-proportionally more concentrated inside the cell , whereas uracil , serine , tyrosine , and glycine were relatively over-represented in the extracellular fluid ( Figure 1Di ) .\",\n \"Instead , highly similar exometabolome concentration values ( r2 = 0 . 971 ) were observed in the related yeast strain BY4741 upon complementing its auxotrophies with the centromere-containing single-copy vector ( a minichromosome ) , 'pHLUM' , which contains all four marker genes ( Mülleder et al . , 2012 ) ( Figure 1Dii ) .\",\n \"Metabolite concentrations in the exometabolome between these two related yeast strains are hence substantially more similar than the endo- versus exometabolome in the same strain , implying that the intra-colony exometabolome is a distinct metabolite pool .\",\n \"A second requirement to establish metabolite exchange is that cells need to be able to sense extracellular metabolites and to exploit them as a nutrient source .\",\n \"Yeast is known to uptake amino acids when they are available extracellularly ( Stahl and James , 2014 ) .\",\n \"We tested how extensive this uptake was by comparing the uptake rates between auxotrophs and prototrophs .\",\n \"Remarkably , prototrophic cells consumed histidine , leucine , methionine , and uracil at a comparable rate to the genetic auxotrophs , who depend 100% on external metabolite pools ( Figure 1E ) .\",\n \"This demonstrated that yeast cells completely shift from de novo synthesis to uptake in the presence of each of the four metabolites .\",\n \"Studying the URA3 genotype in greater detail confirmed the preference of uptake over self-synthesis .\",\n \"Enzymes involved in uracil biosynthesis remained expressed in both the URA3 and the ura3Δ strains under fully supplemented conditions ( Figure 1—figure supplement 2 ) , but uracil biosynthesis-related intermediates shifted to similar concentrations both in the wild-type strain and in the ura3Δ strain once uracil was supplemented ( Figure 1F ) .\",\n \"The only exception was the direct substrate of the URA3 enzyme ( orotidine-5'-phosphate decarboxylase ) , orotidine-5'-phosphate ( OMP ) , which accumulated upon uracil supplementation once its metabolising enzyme ( URA3 ) was deleted ( Figure 1Fi ) .\",\n \"In summary , yeast cells do not compensate for metabolic deficiencies in co-culture experiments consistently as others reported previously ( Müller et al . , 2014; Shou et al . , 2007 ) , but they\",\n \"( i ) export the relevant metabolites even when grown on minimal media and\",\n \"( ii ) take up histidine , leucine , uracil , and methionine at similar rates to auxotrophs if supplementation is available .\",\n \"At least for uracil ,\",\n \"( iii ) the biosynthetic enzymes and majority of biosynthetic intermediates in the supplemented wild-type cell resemble those of the corresponding auxotroph .\",\n \"In light of these results , we speculated that the inability to cooperate could be found in the nature of the co-culturing experiment .\",\n \"To establish an alternative method , we made use of a , in other circumstances disadvantageous , property of yeast plasmids , their occasional , stochastic loss from cells ( segregation ) .\",\n \"Segregation is observed for both popular replication types , centromeric 'cen' and 2µ , at a rate of 2–4% expressed per cell division ( Christianson et al . , 1992 ) .\",\n \"This property allowed us to randomly and progressively introduce auxotrophies into a developing yeast community starting from a single , initially prototrophic , cell: when a plasmid carries a gene that complements for an auxotrophy , a newly budded cell re-gains the metabolic deficiency according to the segregation of its plasmid .\",\n \"We transformed plasmids from the classic pRS and p400 series which express HIS3 , LEU2 , MET15 , or URA3 genes under the respective S . cerevisiae promoters ( Christianson et al . , 1992; Mumberg et al . , 1995; Sikorski and Hieter , 1989 ) into the standard laboratory strain BY4741 , deficient in these markers ( Brachmann et al . , 1998 ) ( Figure 2Ai ) .\",\n \"As expected , the transformed cells grew competently in the absence of histidine , leucine , uracil , and methionine supplementations .\",\n \"We then quantified plasmid segregation and confirmed earlier literature values ( Figure 2Aii and Figure 2—source data 1 ) ( Christianson et al . , 1992; Ghosh et al . , 2007 ) . 10 . 7554/eLife . 09943 . 006Figure 2 . A self-establishing yeast community can cooperatively compensate for progressive loss of prototrophy on minimal media .\",\n \"( A )\",\n \"( i ) Schematic illustration of BY4741 carrying four plasmids to complement its auxotrophies in histidine ( his3Δ1 ) , leucine ( leu2Δ0 ) , methionine ( met15Δ0 ) , and uracil ( ura3Δ0 ) .\",\n \"( ii ) Plasmid segregation rates ( probability of plasmid loss per cell division ) of BY4741 carrying four plasmids encoding HIS3 ( p423 ) , LEU2 ( pRS425 ) , URA3 ( p426 ) , and MET15 ( pRS411 ) ( y-axis ) compared to BY4741 carrying one plasmid at a time ( x-axis ) .\",\n \"n = 3 , error bars = ± SD .\",\n \"Dashed line: linear regression fit .\",\n \"( B ) Schematic illustration of the segregate strain composition over time on rich or complete media where no cooperation is necessary for cells to survive .\",\n \"Sequential plasmid loss leads to an increase in auxotrophy , with loss of up to four plasmids leading to the formation of 16 cell types with varying metabolic capacity ( metabotypes ) .\",\n \"( C ) Three possible outcomes for BY4741 carrying four segregating plasmids , when establishing a colony on minimal media;\",\n \"( i ) no cooperation , only cells carrying four plasmids grow ,\",\n \"( ii ) no cooperation but plasmid segregation is faster than the growth rate of cells carrying four plasmids leading to no growth capacity .\",\n \"Finally\",\n \"( iii ) , cells cooperate , wherein cells that have obtained auxotrophy continue growth by sharing metabolites with neighbouring cells in the colony .\",\n \"( D ) Auxotrophy of BY4741 colonies carrying single plasmids encoding HIS3 ( p423 ) , LEU2 ( pRS425 ) , URA3 ( p426 ) , and MET15 ( pRS411 ) on selective media after approximately 33 doublings .\",\n \"The number of plasmid-free cells ( % auxotrophy abundance ) was measured by replica plating .\",\n \"n = 3 , error bars = ± SD .\",\n \"( E ) Mathematical simulation of segregation over time , starting from 100% cells carrying four plasmids , based on the experimentally measured segregation rates .\",\n \"Highlighted is the situation after 57 doublings ( achieved in dashed line ) where >99 . 9% of cells have segregated >1 plasmid .\",\n \"( F ) Segregation over time in a colony on rich media ( no selection to maintain the plasmids ) ; starting from a micro-colony of four-plasmid prototrophic cells on minimal media , cells were transferred to rich ( YPD ) media and established as a giant colony , segregation was followed by replica plating .\",\n \"Biomass gain is counted from the single cell .\",\n \"( G ) Giant colonies established for 57 biomass doublings on minimal media are composed of ( left ) 73 . 3% auxotrophic cells , ( centre ) contain a mixed number of auxotrophies and ( right ) a non 1:1 ratio of auxotrophy types .\",\n \"( n = 542 genotyped cells ) .\",\n \"Colony growth is achieved , despite the majority of cells possessing one or more auxotrophies . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00610 . 7554/eLife . 09943 . 007Figure 2—source data 1 . Plasmid segregation rates . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00710 . 7554/eLife . 09943 . 008Figure 2—figure supplement 1 . Experimentally obtained colony composition , compared to the composition expected if segregation continued without selective pressure to maintain cells able to synthesise leucine , uracil , methionine and histidine .\",\n \"( i ) ( left ) Colonies established for 57 biomass doublings on minimal media ( SM ) are composed of 73 . 3% auxotrophic cells ( n = 542 genotyped cells ) .\",\n \"( right )\",\n \"Uninterrupted segregation would lead to 99 . 9% auxotrophic cells .\",\n \"( ii ) ( left ) Auxotrophy number ( 0 to 4 ) for cells within the synthetic metabolically cooperating colony ( SeMeCo ) .\",\n \"( right )\",\n \"Result when there is uninterrupted segregation ( theoretical composition ) .\",\n \"( iii ) ( left ) Composition of SeMeCo in terms of auxotrophy type ( histidine , leucine , uracil and methionine ) .\",\n \"( right )\",\n \"Result when there is uninterrupted segregation ( theoretical composition ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00810 . 7554/eLife . 09943 . 009Figure 2—figure supplement 2 . Schizosaccharomyces pombe , like Saccharomyces cerevisiae , are also able to establish SeMeCo colonies . The four possible metabotypes resulting from a combination of uracil and leucine auxotrophies are found within S . pombe SeMeCos , despite growing colonies on selective media ( n = 3 ) .\",\n \"Separately established populations ( >90 cells ) were genotyped per replicate . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 00910 . 7554/eLife . 09943 . 010Figure 2—figure supplement 3 . Complementary pairs of auxotrophs , re-isolated from established SeMeCo colonies , do not overcome metabolic deficiencies upon co-culturing , similar to the original strains .\",\n \"his3∆ , leu2∆ , met15∆ , and ura3∆ yeasts isolated from SeMeCo colonies were combined in complementary pairs and spotted on corresponding selective media .\",\n \"No pairs exhibited co-growth together , indicating that SeMeCo metabotypes did not acquire secondary mutations to overcome metabolic deficiencies , while establishing a cooperating community . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01010 . 7554/eLife . 09943 . 011Figure 2—figure supplement 4 .\",\n \"Different auxotrophy combinations do not enable metabolic cooperation .\",\n \"( left )\",\n \"Quadruple mixed cultures of Saccharomyces cerevisiae auxotrophs do not overcome metabolic deficiencies upon co-culturing .\",\n \"his3Δ , leu2Δ , met15Δ , and ura3Δ yeasts were combined together in mixed ratios and spotted as a co-culture on corresponding selective media .\",\n \"( right )\",\n \"A similar outcome upon co-culturing the four genotypes over night in rich media prior to transferring to minimal media . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 011 Cells having lost prototrophy can only continue growth if they obtain the relevant nutrient from the environment ( Figure 2B ) .\",\n \"Transferred to minimal media , the lack of nutrient supplementation leads to three possible outcomes ( Figure 2C ) : First , if the cooperative potential would not suffice to overcome the increasing content of metabolically deficient cells , colony growth would only be explained by cells maintaining all four plasmids ( Figure 2C left ) .\",\n \"Alternatively , if the segregation is faster than the growth rate of cells carrying four plasmids , the colonies would not be able to grow ( Figure 2C centre ) .\",\n \"Finally , the third outcome is that colony growth continues , despite an increasing auxotrophic composition , facilitated by cells exchanging histidine , leucine , uracil , and methionine at growth relevant quantities ( Figure 2C right ) .\",\n \"First , we observed that upon approximately 33 biomass doublings , segregation for HIS3 and URA3 had continued until less than 50% of cells were prototrophs ( Figure 2D ) , even though a 1:1 co-culture of the same auxotrophs was not able to co-grow ( Figure 1A ) .\",\n \"Then , we assayed for the formation of a heterogeneous yeast community , starting from the four-plasmid ( 4P ) strain , that can give rise to the emergence of 16 complementary auxotrophic genotypes ( Figure 2B ) .\",\n \"In the 4P strain , individual plasmid segregation rates were similar but not identical to yeast carrying one plasmid at a time and were in linear correlation , indicating that no specific interaction between the plasmids occurred ( Figure 2Aii ) .\",\n \"With a total segregation rate of 11% , 4P cells regain auxotrophy rapidly so that only 21 cell divisions ( doublings ) would result in >90% of cells losing prototrophy ( Figure 2E ) .\",\n \"The continuous loss of prototrophy from the 4P strain was experimentally confirmed on rich ( YPD ) media; In the presence of rich supplementation , only 45 biomass doublings resulted in 96% of cells losing prototrophy ( Figure 2F ) .\",\n \"Testing whether cells can maintain growth by cooperating in the biosynthesis of histidine , leucine , uracil , and methionine , colonies were grown over 7 days on minimal media agar through dilution and re-spotting once giant colonies had formed ( every 48 hr ) , so that the continuous gain in biomass necessitates constant de novo synthesis of intermediate metabolites .\",\n \"The experiment yielded viable colonies and from the obtained biomass , we calculated that 57 doublings had occurred .\",\n \"Fifty-seven doublings would have been sufficient for >99% of cells to segregate ( Figure 2E ) .\",\n \"Replica plating revealed a predominantly auxotrophic composition of the obtained colonies .\",\n \"These were composed of 73 . 3 ± 3 . 7% auxotrophic cells ( Figure 2G left ) , of which 39 . 9% had lost one plasmid , 20 . 6% two , 6 . 6% three , and 6 . 1% had lost all four markers ( Figure 2G centre ) .\",\n \"No auxotrophies were in a 1:1 ratio with each other ( 36 . 9% for uracil , 27 . 7% leucine , 23 . 7% histidine , and 11 . 7% methionine ( Figure 2G right ) , despite the segregation rates predicting a relatively equal distribution , implying that selection pressure for certain metabotypes affected colony composition ( Figure 2G right and Figure 2—figure supplement 1 ) .\",\n \"We also confirmed that S . pombe is capable of forming similar communities , indicating conservation in these evolutionary distant yeast species ( Figure 2—figure supplement 2 ) .\",\n \"As additional controls , we\",\n \"( i ) re-isolated the auxotrophs from the established colonies , and repeated the co-culture experiment , after having grown the cells for 48 hr in supplemented media , as with the original strains ( Figure 1A ) .\",\n \"Even when isolated from a functional cooperating colony , complementary auxotrophic cells did not complement each other's deficiencies upon co-culturing ( Figure 2—figure supplement 3 ) , ruling out the possibility that new mutations altering metabolite exchange capacities could explain the formation of the cooperating community .\",\n \"We also mixed all four auxotrophs together , both in a 1:1 mixture , as well as in the ratio observed from the community and performed co-culturing both with and without co-cultivation before spotting; These attempts did not result in successful co-growth either ( Figure 2–figure supplement 4 ) .\",\n \"Hence , by exploiting plasmid segregation to overcome culturing and allowing the community to self-establish , heterogeneous yeast colonies were formed , which could sustain exponential growth under nutrient limitation , despite the majority of cells being auxotrophic for at least one metabolite .\",\n \"These co-growing cells could therefore overcome metabolic deficiencies through cooperative metabolism , demonstrating that yeast possesses metabolite exchange capacities at a growth relevant quantity .\",\n \"The obtained colonies were viable on minimal media and showed no apparent growth defects , despite containing a content of 73% auxotrophs , each of which were non-viable in co-culture studies ( Figure 1A , B , Figure 1—figure supplement 1 , Figure 2—figure supplement 3 , 4 ) .\",\n \"To characterise the properties of this community , we started with LC-MS/MS to compare its exometabolome against prototrophic yeast strains ( YSBN5 , BY4741-pHLUM ) , and the unpassaged strain carrying the four plasmids ( 4P ) ; ( Figure 3A , B and Figure 3—source data 1 ) .\",\n \"SeMeCo colonies possessed similar extracellular metabolite concentrations to prototrophic controls ( Figure 3Bi ) .\",\n \"Of particular note are the extracellular concentrations of H , L , U , and M . Aside from a statistically non-significant trend towards a lower leucine concentration , only uracil ( U ) was significantly affected .\",\n \"To our surprise , however , the concentration of this metabolite was increased , indicating that SeMeCo had adapted by maintaining a higher level of uracil in its exometabolome ( Figure 3Bii ) . 10 . 7554/eLife . 09943 . 012Figure 3 . Growth and physiological parameters of the self-established metabolically cooperating yeast community 'SeMeCo' .\",\n \"( A ) Schematic illustration of colonies derived from the genomically prototrophic yeast strain YSBN5 , the single-vector complemented BY4741-pHLUM ( 'pHLUM' ) , BY4741 complemented with four plasmids ( 'FourP' ) , and the self established yeast population ( SeMeCo; self-established metabolically cooperating yeast community ) ; ( from left to right ) .\",\n \"( B )\",\n \"( i ) Extracellular concentrations of metabolites in colonies of YSBN5 , pHLUM and SeMeCo growing exponentially on minimal media as determined by LC-MS/MS , n = 3 .\",\n \"Histidine ( H ) , leucine ( L ) , methionine ( M ) , and uracil ( U ) are highlighted in red circles .\",\n \"( ii ) Detailed extracellular concentration values of uracil , leucine , methionine , and histidine as determined by LC-MS/MS .\",\n \"n = 3 , error bars = ± SD .\",\n \"( C ) ( left ) Growth curve of YSBN5 , pHLUM , FourP , and SeMeCo as determined by measuring optical density ( OD595 ) .\",\n \"n = 3 , error area = ± SD .\",\n \"( centre )\",\n \"Dry biomass collected from 100 mL batch cultures after three days growth in minimal media , 30°C , n = 3 , error bars = ± SD .\",\n \"( right )\",\n \"Maximum specific growth rate ( µmax ) as determined from OD595 growth curves using a model-richards fit ( Kahm et al . , 2010 ) .\",\n \"n = 3 , error bars = ± SD .\",\n \"( D ) The ratio of colony-forming units ( CFUs ) to number of cells used for plating , for YSBN5 and SeMeCo .\",\n \"n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01210 . 7554/eLife . 09943 . 013Figure 3—source data 1 . Absolute quantification of amino acids and uracil in yeast strains YSBN5 , pHLUM and SeMeCo , absolute concentration values . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 013 SeMeCo also maintained wild-type like growth efficiency and biomass-forming capacities under nutrient limitation ( Figure 3C ) .\",\n \"Comparing SeMeCo against prototrophic yeast strains ( Figure 3A ) , dry biomass formation did not vary significantly ( Figure 3C centre; p-values = 0 . 27 , 0 . 70 , and 0 . 09 for FourP , pHLUM , and YSBN5 , respectively ) .\",\n \"In liquid media , lag phase was prolonged , and the maximum specific growth rate ( µmax ) was slightly reduced compared to the genetically prototrophic YSBN5 or pHLUM cells ( 0 . 17 OD595/hr vs 0 . 20 and 0 . 21 OD595/hr , respectively ) ( Figure 3C right ) .\",\n \"However , this difference appeared more as a cost of plasmid segregation , as both lag phase and µmax did not vary significantly between SeMeCo and the FourP strain ( 0 . 17 and 0 . 16 OD595/hr , respectively ) ( Figure 3C right ) .\",\n \"Finally , we tested to what extent cell death occurs in the cooperating community .\",\n \"Both a wild-type ( YSBN5 ) and a SeMeCo culture were grown to exponential phase and cells were counted .\",\n \"Then , the cultures were plated on SC media and the number of colony-forming units ( CFUs ) determined .\",\n \"The CFU count was nearly equal between SeMeCo and YSBN5 , and similar to a 1:1 relationship to the cell count measured prior to spotting ( Figure 3D ) .\",\n \"This indicates that cells in SeMeCo have a comparable colony-forming capacity to that of exponentially growing wild-type cells , and in both populations , virtually every cell can form a new colony .\",\n \"To establish if cells cooperating in SeMeCo are distributed in a random or organised manner , we analysed colony spatial structure using confocal fluorescence microscopy ( Figure 4A ) .\",\n \"For this , the community was re-established with alternative plasmids that express the fluorescent protein markers CFP ( cyan fluorescent protein ) , Venus ( yellow fluorescent protein ) , Sapphire ( a UV-excitable green fluorescent protein [Sheff and Thorn , 2004] ) , and mCherry ( red fluorescent protein ) coupled to the auxotrophic markers HIS3 , URA3 , LEU2 , and MET15 , respectively ( Bilsland et al . , 2013 ) .\",\n \"Segregation of the labelled plasmids were within the same range , although not identical to the original pRS and p400 plasmids ( Figure 4—figure supplement 1 ) .\",\n \"Images were acquired with a SP5 on a DMI6000 inverted microscope ( Leica , Wetzlar , Germany ) and show the underside of a live two day micro-colony which had , prior to imaging , been growing on minimal media ( SM ) .\",\n \"In our hands , the Sapphire-LEU2 fluorescence was also visible under the imaging conditions used to visualise Venus-URA3 .\",\n \"For this reason , the Venus-URA3 channel was removed from the colony image .\",\n \"The spatial heterogeneity of fluorescent markers in the micro-colony revealed that cells in SeMeCo unequally distribute over the macroscopic structure , and form regions where the biosynthesis of a particular metabolite dominates ( Figure 4A ) .\",\n \"A prediction in truly cooperating communities is , however , whether complementary cells maintain physical proximity to each other , to oppose diffusion of exchanged metabolites ( Müller et al . , 2014 ) .\",\n \"Using computational image analyses of colony micrographs , we find that even when the most stringent cut-off was applied , complementary metabotypes across the community maintained an average distance ( 6 . 86 μm ) of less than two cell diameters ( Figure 4B ) .\",\n \"Cells are hence most likely to exchange the majority of metabolites with those maintaining close proximity .\",\n \"Despite these results , SeMeCo could , however , continue growth after disruption of this spatial structure in liquid media ( Figure 3C ) .\",\n \"To verify this assumption , SeMeCo was replicated for 7 days in liquid minimal media , as previously , with re-dilution every 2 days .\",\n \"Indeed , the liquid culture maintained a similar content of auxotrophs as obtained with colony grown SeMeCos ( Figure 4—figure supplement 2 ) .\",\n \"The capacity of SeMeCos to overcome metabolic deficiencies through metabolic cooperation is hence not in essence bound to colonial growth . 10 . 7554/eLife . 09943 . 014Figure 4 . Spatial organisation of SeMeCo .\",\n \"( A ) Spatial organisation of metabolically cooperating yeast micro-colony on minimal agar media ( SM ) .\",\n \"SeMeCo was established with plasmids expressing fluorescent protein coupled to each auxotrophic marker; LEU2 , MET15 , and HIS3 cells are coloured green , red , and blue , respectively .\",\n \"Cells containing more than one marker are coloured as a product of the additive RGB colour model .\",\n \"Two–day-old live and growing micro-colony is visualised from underneath .\",\n \"( B ) Minimum , mean and maximum distances between leucine , histidine , and methionine auxotrophs and their corresponding prototrophs in a SeMeCo colony .\",\n \"Maximum distance between auxotroph and prototroph for 90% of cells shows an average distance of 6 . 86 μm , using the highest cut-off .\",\n \"Despite the heterogeneous macroscopic colony composition , complementary auxotrophs are maintained in physical proximity to each other . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01410 . 7554/eLife . 09943 . 015Figure 4—source data 1 . Segregation rates of fluorescent protein plasmids from the yEp , pRS and p400 series . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01510 . 7554/eLife . 09943 . 016Figure 4—figure supplement 1 . Plasmid segregation rates of fluorescent protein plasmids ( %; probability of plasmid loss per cell division ) of BY4741 carrying plasmids encoding HIS3 ( yEpCFP_HIS ) , LEU2 ( yEpSapphire_LEU ) , URA3 ( yEpVenus_URA ) , and MET15 ( pRS411-GPDpr-mCherry ) respectively , compared to BY4741 carrying all four at the same time .\",\n \"n = 3 , error bars = ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01610 . 7554/eLife . 09943 . 017Figure 4—figure supplement 2 . SeMeCos continue growth in minimal ( SM ) liquid culture . As in the colony growth experiment , cells were transferred from a micro-colony on SM agar , and then grown for 7 days with re-diluting every 2 days , however , here cells were in shaking batch liquid culture ( 25 mL ) .\",\n \"Auxotrophy abundance is 70 . 3 ± 2 . 5% despite cells growing in minimal media . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 017 To determine not only the spatial but also the population structure , we switched back to the non-fluorescent SeMeCo to avoid confounding effects of fluorescent protein expression , and quantified by replica plating the colony contribution of all 16 possible metabotypes , resulting from all possible combinations of the four auxotrophies ( Figure 2B ) .\",\n \"These experiments revealed that within SeMeCo , 95 . 6% of cells belonged only to 8 of the 16 possible metabolic combinations ( Figure 5A ) .\",\n \"We questioned whether this composition was the result of a stochastic event , however , the dominance of the same metabotypes establish three times independently .\",\n \"Moreover , the eight successful metabotypes contributed to SeMeCo at comparable percentages ( Figure 5A inset ) .\",\n \"Using our segregation rate model as well as growth rate data , we could rule out this colony composition being a result of\",\n \"( i ) varying plasmid segregation rate ,\",\n \"( ii ) the number or type of auxotrophy , or\",\n \"( iii ) differences in growth rates .\",\n \"First , a community composition calculated on the basis of the experimentally determined plasmid segregation values ( Figure 2E , Figure 2—figure supplement 1 ) showed zero correlation with the actual population composition ( r² = 0 . 051 ) ( Figure 5B ) .\",\n \"Second , all histidine , leucine , uracil , or methionine auxotrophies , as well as all plasmid numbers ( 1 to 4 ) were found amongst both the frequent and rare metabotypes .\",\n \"For instance , while single uracil ( HIS3 , LEU2 , MET15 , ura3∆; 19 . 7% ) or leucine ( HIS3 , URA3 , MET15 , leu2∆; 11 . 1% ) auxotrophs were amongst the most frequent cells , their methionine-deficient counterparts ( HIS3 , URA3 , LEU2 , met15∆; 0 . 4% ) were among the most rare ( Figure 5A ) .\",\n \"Also , the high frequency of the dual auxotrophs LEU2 , MET15 , his3∆ , ura3∆ ( 8 . 1% ) and HIS3 , MET15 , leu2∆ , ura3∆ ( 10 . 1% ) , contrasts with the rareness of the other dual auxotrophs ( HIS3 , URA3 , leu2∆ , met15∆ ( 1 . 1% ) , MET15 , URA3 , leu2∆ , his3∆ ( 0 . 2% ) , URA3 , LEU2 , his3∆ , met15∆ ( 0 . 5% ) , and LEU2 , HIS3 , ura3∆ , met15∆ ( 0 . 6% ) ) .\",\n \"Thus , the number of plasmids or type of auxotrophy a cell had did not indicate whether a cell-type would be rare or frequent ( Figure 5A ) .\",\n \"Finally , the growth rate of 16 strains , carrying the same marker and supplement combination that replicates the 16 metabotypes ( Mülleder et al . , 2012 ) did not distinguish the depleted from the selected metabotypes either ( Figure 5C ) . 10 . 7554/eLife . 09943 . 018Figure 5 . The community composition is distinct and dynamic .\",\n \"( A ) Frequency of the 16 metabotypes that result from combination of histidine , leucine , methionine , and uracil auxotrophies as found within SeMeCo colonies .\",\n \"Separately established populations ( n=3 ) were genotyped ( >180 cells per colony ) and eight metabotypes showed to dominate in the populations ( inset ) .\",\n \"Frequency of the 16 metabotypes in independently established cell populations .\",\n \"The eight metabotypes of low frequency , which were depleted in all experiments , are highlighted with a red circle .\",\n \"( B ) No correlation shown between the frequency of the 16 metabotypes in SeMeCo versus a segregation rate-predicted colony composition .\",\n \"Coloured points correspond to the experimentally observed eight frequent metabotypes .\",\n \"Dashed line: linear regression fit .\",\n \"( C ) Maximum specific growth rate ( µmax ) in supplemented minimal media ( red dots ) , of the 16 strains carrying HIS3 , LEU2 , URA3 , and MET15 plasmids in all combinations obtained from ( Mülleder et al . , 2012 ) , relative to the frequency of the specific metabotype in SeMeCo ( light blue ) .\",\n \"Dashed line indicates average µmax for the eight most and least frequent metabotypes within SeMeCo colonies .\",\n \"( D ) SeMeCo re-established on minimal media supplemented with uracil .\",\n \"After 7 days of growth ( with re-spotting every two days ) , SeMeCo adapted with an entirely different composition of metabotypes ( green box plot ) compared to original SeMeCo colony composition ( grey bars ) .\",\n \"Uracil producing cells decline , including the FourP genotype , so that 97% of cells are cooperating auxotrophs . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 01810 . 7554/eLife . 09943 . 019Figure 5—figure supplement 1 . Abundance over time of a fluorescent labelled frequent ( HIS3 , LEU2 , MET15 , ura3∆ ) and rare ( his3∆ , leu2∆ , URA3 , MET15 ) genotype spiked into SeMeCo , as measured by FACS .\",\n \"( Metabotype frequency determined from SeMeCo colony , Figure 5A ) .\",\n \"( left ) % Fluorescence of the frequent and rare metabotype established individually as a colony shows frequent and rare abundance respectively ( unlabelled prototroph control is BY4741-pHLUM ) .\",\n \"( right )\",\n \"Frequent and rare metabotypes spiked into pre-established SeMeCo shows depletion of both cell types after approx .\",\n \"48 hr . n = 3 , error bars = ± SD .\",\n \"FACS: Fluorescence-activated cell sortingDOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 019 As growth potential and segregation parameters did not explain the population architecture of SeMeCo , we conclude that this community was selected for on its ability to cooperate effectively .\",\n \"If this interpretation is correct , it would imply that a different pressure to cooperate would result in a different SeMeCo composition .\",\n \"To test this hypothesis , we focussed on uracil , as mass spectrometry had detected an increase in uracil concentration in the SeMeCo colony exometabolome , indicating that uracil is the most limiting metabolite ( Figures 2G right and 3Bii ) .\",\n \"SeMeCos established on uracil adapted a different composition , resulting from a dramatic decline in cells needed to produce uracil .\",\n \"Importantly , this included the prototroph with its total content in the community decreasing from 26 . 7% in the original SeMeCo to solely 3 . 0% , so that 97 . 0% of cells were cooperating auxotrophs ( Figure 5D ) .\",\n \"Hence , SeMeCo colonies establish a population that is dynamic to changes in the external metabolite pool , and can persist in a state with virtually all cells being genetically auxotrophic for at least one essential metabolite .\"\n ],\n [\n \"Metabolic exchange interactions occur frequently among cells that grow in proximity to one another , as metabolites are constantly released from cells for different reasons , such as overflow metabolism , metabolite repair , as well as export to facilitate metabolite exchange .\",\n \"In bacteria , a subset of such metabolite exchanges are of a cooperative nature in the sense that all exchange partners profit from this situation ( Oliveira et al . , 2014 ) , whereas for the majority of eukaryotic organisms , metabolite exchange strategies remain unclear .\",\n \"Despite yeast auxotrophs being viable in supplemented and rich media ( Mülleder et al . , 2012 ) , in the absence of amino acid supplementation , they fail to complement metabolic deficiencies in several pairs or higher order co-culture experiments ( Figure 1A , B , [Müller et al . , 2014; Shou et al . , 2007] ) , a clear difference to bacterial studies , where similar experiments were effective ( Foster and Bell , 2012; Freilich et al . , 2011; Harcombe , 2010; Pande et al . , 2014; Ramsey et al . , 2011; Vetsigian et al . , 2011 ) .\",\n \"This led to speculations that yeast might , in contrast to many bacterial species , lack the required export capacities to enable growth relevant exchange of intermediary metabolites such as amino acids and nucleobases ( Shou et al . , 2007 ) .\",\n \"Analysing the intra-colony exometabolome we could , however , detect the required metabolites; in fact , we found that cells within a colony are surrounded by a rich exometabolome .\",\n \"We also found that yeast would efficiently exploit the nutrients when available , to the extent that they solely rely on these extracellular metabolites .\",\n \"This result implied that metabolite exchange among co-growing yeast cells is frequent by nature; the lack of complementation in the co-culture experiments could thus reflect a limit of the experiment itself , and not represent the metabolite exchange capacities of yeast cells .\",\n \"To circumvent combining two or more cultures , we chose an approach of synthetic biology and exploited the stochastic loss of plasmids to progressively introduce the metabolic deficiencies in random combination from an initially single cell .\",\n \"The progressive loss of prototrophy allowed cells to maintain cell growth on the basis of metabolite exchange , resulting in a community with 73% auxotrophy , which increased to 97% upon supplementation with the most limiting metabolite , uracil .\",\n \"Despite its dominant auxotrophic composition , the SeMeCo community could maintain a wild-type like exometabolome , metabolic efficiency , as well as cell viability , implying that this type of cooperation is a robust physiological property .\",\n \"Hence yeast's natural metabolite export and import capacities are wholly sufficient to support co-growth on the basis of metabolite exchange .\",\n \"The establishment of SeMeCos was not facilitated by mixing the auxotrophs in a higher order combination either .\",\n \"This is consistent with the notion that losing more metabolic genes reduces biochemical capabilities and does not add new ones .\",\n \"The key of the SeMeCo system is instead to allow the progressive self-establishment of the community starting from the single cell ( Figure 2 ) .\",\n \"Metabolic feedback regulatory systems therefore do not inhibit metabolite export in general but prevent cooperative co-growth when already pre-established co-cultures are mixed ( Müller et al . , 2014; Shou et al . , 2007 ) .\",\n \"A possible role of these mechanisms could perhaps prevent the spread of foreign , potentially cheating , cells that derive from a competing yeast colony .\",\n \"We could replicate behaviour which is in favour of such an assumption; By spiking into SeMeCo a cell culture possessing the same genotype as a frequent ( HIS3 LEU2 ura3Δ MET15 ) and rare ( his3Δ leu2Δ URA3 MET15 ) genotype ( Figure 5A ) , we observed that both genotypes were rapidly depleted from the pre-established SeMeCo , irrespective of the frequency of the respective genotype in SeMeCo ( Figure 5—figure supplement 1 ) .\",\n \"Studying the genotypic composition of SeMeCo implied that there are a defined set of interactions underlying the properties of the cooperating community , which maintained a similar population composition involving eight reproducibly concentrated metabotypes when established independently .\",\n \"This indicates that this quantitatively defined community composition was most effective in metabolic cooperation .\",\n \"This finding may close an important gap in the understanding of the evolution of multicellularity; If a defined composition is most effective in cooperative growth , a selection advantage could be provided by any sort of physical bonding which can maintain cooperation partners in the defined equilibria , and would in addition , provide additional protection against the invasion of cheating cell types .\",\n \"Indeed , the exometabolome data implies that the number of metabolite exchange interactions among co-growing cells could be significant .\",\n \"The colony exometabolome contained a vast array of biomolecules , including the majority of amino acids ( Figure 1C ) ( Castrillo et al . , 2007; Paczia et al . , 2012; Silva and Northen , 2015 ) .\",\n \"The finding that yeast cells prefer uptake over synthesis of amino acids and uracil , even when they are genetically prototrophic , shows that exchange interactions will readily establish once the cellular environment has acquired a critical concentration of metabolites .\",\n \"This has implications for the interpretation of metagenomic studies and cheating/benefactor experiments , as for this reason , it cannot be concluded from the genetic presence or absence of a single metabolic pathway , or from following the synthesis of a single metabolite , how many other metabolites are being exchanged as well .\",\n \"Even without selective pressure , both wild-type yeasts and SeMeCo established an amino-acid-rich exometabolome on minimal media ( Figure 1C ) and engaged in metabolite uptake when nutrients were available ( Figure 1E ) .\",\n \"This implies that these features are a natural property of yeast and raises the question of why natural yeast communities are not composed of co-growing , genetic , auxotrophs .\",\n \"To answer this question , one needs to keep in mind that possessing metabolic genes in the genome is not equal to the pathway being constantly active; Indeed , prototrophs can flexibly switch from self-synthesis to amino acid uptake ( Figure 1 ) .\",\n \"Being genetically prototrophic hence gives a higher level of metabolic flexibility , as prototrophic cells can re-activate a synthetic pathway when required .\",\n \"Unlike in SeMeCo , the genotype of a cell in a natural community is not in essence reflecting its metabotype or its metabolic role in the community .\",\n \"Additionally , the natural life cycle of yeast involves the formation of endospores , which are important for enduring starvation , and to spread between habitats .\",\n \"Without a prototrophic genotype , a single spore can no longer establish a colony on its own as genetic auxotrophy would interrupt the yeast life cycle .\",\n \"Second , only a fraction of the natural yeast life cycle occurs under exponential growth that requires abundant carbohydrate and nitrogen supply .\",\n \"The maintenance of a prototrophic genotype both in S . pombe and in S . cerevisiae wild isolates ( Jeffares et al . , 2015; Liti et al . , 2009 ) is hence fully compatible with the presence of elaborate amino acid and nucleotide exchange mechanisms .\",\n \"The finding that these cells fully shift from self-synthesis to uptake for histidine , leucine , methionine , and uracil , once these metabolites are provided , has direct implications on research using yeast , a primary eukaryotic model organism in genome-scale studies .\",\n \"A majority of yeast genetic experiments are conducted in auxotrophic strains , requiring amino-acid supplemented or rich media compositions .\",\n \"Important parts of biosynthetic metabolism ( amino acid biosynthesis can account for up to 50% of metabolic flux towards biomass ) may have thus stayed silent in a significant amount of functional genomics experiments .\",\n \"The effects of metabolic–genetic interactions on cellular physiology could thus substantially exceed our current knowledge and could be discovered upon switching to minimal nutrient supplementations .\",\n \"In this context , SeMeCos are simple to handle , establish rapidly and are easy to analyse , and therefore represent an effective and broadly applicable eukaryotic model system to study both cooperativity and effects of metabolism in the laboratory .\",\n \"In summary , using histidine , leucine , methionine , and uracil as model metabolic pathways for exchangeable metabolites , we found that S . cerevisiae cells prefer these nutrients' uptake over their self-synthesis and maintain an amino-acid-rich exometabolome in the extracellular colony space , indicators of ongoing inter-cellular metabolite exchange .\",\n \"Although yeast is known to fail in compensating for auxotrophy in pairwise and higher order co-culture experiments , the cells did successfully enter a state of metabolic cooperative growth upon exploiting stochastic plasmid segregation so that a single cell could progressively develop into a complex heterogeneous community .\",\n \"Composed of auxotrophic cell types that are non-viable on their own , SeMCo communities were able to overcome metabolic deficiencies and maintain metabolite concentrations and robustness similar to wild-type cells .\",\n \"Additionally , cooperation had imposed different metabolic roles on contributing cells .\",\n \"Progressive community formation thus reveals that yeast possesses full capacity to exchange anabolic metabolites at growth relevant quantities and readily establishes a non-cell-autonomous metabolism within complex but defined community structures .\"\n ],\n [\n \"Yeast cells were grown under standard conditions on synthetic minimal ( SM or EMM ) , SC and rich ( YPD or YES ) media .\",\n \"Plasmid segregation was calculated according to Christianson et al . ( 1992 ) , by monitoring plasmid retention after cells are shifted from non-selective to selective media , and by expressing the number of cells that have lost the marker as a function of generation time .\",\n \"Metabolites were quantified after quenching using an online UPLC-coupled 6460 ( Agilent Technologies , Waldbronn , Germany ) triple quadrupole mass spectrometer .\",\n \"Confocal fluorescence microscopy was conducted with a SP5 confocal on a DMI6000 inverted microscope ( Leica ) using a 10x/0 . 3 HC PL Fluotar Air objective .\",\n \"All experiments involved , unless otherwise indicated , used BY4741 yeast strain ( his3Δ1 , leu2Δ0 , ura3Δ0 , met15Δ0 ) ( Brachmann et al . , 1998 ) with prototrophy restored by complementation either with vectors p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) ( Christianson et al . , 1992; Mumberg et al . , 1995; Sikorski and Hieter , 1989 ) , with the centromeric vector ( minichromosome ) pHLUM ( Mülleder et al . , 2012 ) ; Addgene number: 40276 ) , or with the fluorescent protein vectors yEpCFP_HIS ( HIS3 ) , yEpSapphire_LEU ( LEU2 ) , yEpVenus_URA ( URA3 ) ( Bilsland et al . , 2013 ) , and pRS411-GPDpr-mCherry ( MET15 ) ( Table 1 ) .\",\n \"Cloning was conducted according to standard procedures; oligonucleotides are listed in Table 2 . 10 . 7554/eLife . 09943 . 020Table 1 . Strains and plasmids used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 020NameDescriptionReferenceStrainsBY4741MATa , his3∆1 leu2∆0 met15∆0 ura3∆0 ( ATCC 201388 ) ( Brachmann et al . , 1998 ) BY4741 FLO+ Derived from tetrad dissection after crossing and sporulating a flocculating BY4741 strain derived from the knock out collection ( ∆tpo1 ) with BY4742 and isolating a FLO+ TPO1 wild-type progeny This studyYSBN5MATa , FY3 ho::Ble ( Canelas et al . , 2010 ) ED666h+ ade6-M210 ura4-D18 leu1-32Bioneer Cat .\",\n \"No .\",\n \"M-3030HPlasmids p423GPD2 µ vector with HIS3 marker ( Mumberg et al . , 1995 ) pRS4252 µ vector with LEU2 marker ( Christianson et al . , 1992 ) p426GPD2 µ vector with URA3 marker ( Mumberg et al . , 1995 ) pRS411Yeast centromeric vector with MET15 marker ( Brachmann et al . , 1998 ) pHLUMYeast centromeric vector with HIS3 , URA3 , LEU2 and MET15 markers ( minichromosome ) .\",\n \"( Addgene number: 40276 ) ( Mülleder et al . , 2012 ) pFS118Yeast high-copy vector with endogenous promoter forura4+ ( Addgene number: 12378 ) ( Sivakumar et al . , 2004 ) pREP41-MCS+Yeast high-copy vector with endogenous promoter for LEU2 .\",\n \"( Addgene number: 52690 ) A gift from Michael Nick Boddyp416GPDYeast centromeric vector with endogenous promoter for URA3 ( Mumberg et al . , 1995 ) pHS12-mCherryYeast vector with mCherry fluorescent tag and LEU2 marker .\",\n \"( Addgene number: 25444 ) A gift from Benjamin Glickp426-GPDpr-mCherryYeast 2 µ vector with endogenous promoter for URA3 marker and a GPD promoter for mCherry fluorescent tagThis study .\",\n \"Derived from p416GPD , pHS12-mCherry and p426GPDpRS411-GPDpr-mCherryYeast 2 µ vector with endogenous promoter for MET15 marker and a GPD promoter for mCherry fluorescent tagThis study .\",\n \"Derived from p426-GPDpr-mCherry and pRS411yEpVenus_URAYeast 2 µ vector with TDH3-promoter-driven Venus ( YFP ) and URA3 marker ( Bilsland et al . , 2013 ) yEpCFP_HISYeast 2 µ vector with TDH3-promoter-driven CFP and HIS3 marker ( Bilsland et al . , 2013 ) yEpSapphire_LEUYeast 2 µ vector with TDH3-promoter-driven Sapphire ( a UV-excitable GFP ) and LEU2 marker ( Bilsland et al . , 2013 ) pHLM-GPDpr-mCherryYeast centromeric vector with a GPD promoter for mCherry fluorescent tagThis study .\",\n \"Derived from pHLUMpUM-GPDpr-mCherryYeast centromeric vector with a GPD promoter for mCherry fluorescent tagThis study .\",\n \"Derived from pHLUM10 . 7554/eLife . 09943 . 021Table 2 . Oligonucleotides used to create expression plasmid p426-GPDpr-mCherry . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 021NameSequencemCherry_Bam_Sac_fwAAGAAGAGCTCAAAAGGATCCGGGATGGTGAGCAAGGGCGAGGmCherry_Xho_rvCCTTTTCTCGAGCTTGTACAGCTCGTCCATGC All experiments involving wild-type yeast strains were carried out using as indicated , and using YSBN5 , a prototrophic haploid variant of S . cerevisiae S288c ( Canelas et al . , 2010 ) .\",\n \"For microscopy analyses of colony spatial organisation , BY4741 had prototrophy restored by complementation with the above fluorescent protein vectors ( Table 1 ) .\",\n \"For fluorescence-activated cell sorting ( FACS ) , BY4741 was used with mCherry labelled derivatives of pHLUM , pHLM-GPDpr-mCherry , and pUM-GPDpr-mCherry ( Table 1 ) .\",\n \"For S . pombe experiments , ED666 yeast strain ( ade6-M210 ura4-D18 leu1-32 ) was used that had uracil and leucine prototrophy restored by complementation with vectors pFS118 ( ura4+ ) and pREP41-MCS+ ( LEU2 ) ( Table 1 ) .\",\n \"Yeast was cultivated if not otherwise indicated at 30ºC , in rich ( YPD; 1% yeast extract [Bacto] , 2% peptone [Bacto] or YES; [Formedium; 35 . 25 g/L] ) , complete supplemented synthetic media ( SC; CSM complete supplement mixture [MP Biomedicals; 0 . 56 g/L] , YNB , yeast nitrogen base [Sigma; 6 . 8 g/L] ) , or minimal supplemented synthetic media ( SM; YNB [Sigma; 6 . 8 g/L] or EMM; [Formedium; 32 . 3 g/L] ) , with 2% glucose ( Sigma ) as the carbon source .\",\n \"Media recipes and amino acid compositions for S . cerevisiae were used as previously published ( Mülleder et al . , 2012 ) .\",\n \"For S . cerevisiae , auxotrophic derivatives of prototrophic BY4741 ( Mülleder et al . , 2012 ) were cultured alone or mixed in combination with other auxotrophs , and 1 . 1e05 cells of individual or mixed auxotrophs were spotted on respective selective media .\",\n \"Growth was then documented after 2 days incubation at 30°C .\",\n \"For the flocculation experiments , a FLO+ derivative of BY4741 was obtained by back-crossing and tetrad dissection of a tpo1Δ ( YLL028W ) strain obtained from Euroscarf ( Frankfurt , Germany ) .\",\n \"For S . pombe , 1 . 9e04 cells of auxotrophic derivatives of ED666 h+ , prototrophic for leucine and uracil , were spotted alone or mixed together on corresponding selective media .\",\n \"Growth was then documented after 2 days incubation at 30°C .\",\n \"All proteogenic amino acids ( except for cysteine ) and uracil , citrulline , and ornithine were analysed by selective reaction monitoring ( SRM ) using an online coupled UPLC ( 1290 Infinity , ( Agilent ) ) / triple quadrupole mass spectrometer ( 6460 , ( Agilent ) ) system .\",\n \"The compounds were separated by hydrophilic interaction chromatography on an ACQUITY UPLC BEH amide column ( 2 . 1 mm × 100 mm , 1 . 7 µm ) by gradient elution .\",\n \"Solvent A consisted of 95:5:5 acetonitrile:methanol:water , 10 mM ammonium formate , 0 . 176% formic acid , and solvent B of 50:50 acetonitrile:water , 10 mM ammonium formate , 0 . 176% formic acid .\",\n \"The gradient conditions were 0–07 min 85% B , 0 . 7–27–2 . 55 min 85–585–5% B , 2 . 55–255–2 . 75 5% B , 2 . 75–275–2 . 8 min 5–855–85% B and 2 . 8–38–3 . 25 min 85% B at a constant flow rate of 0 . 9 mL/min and 25°C column temperature .\",\n \"SRM ( Q1/3 settings ) are given in Table 3 .\",\n \"Metabolite signals were automatically integrated using Masshunter ( Agilent ) corrected after manual inspection and quantified by external calibration . 10 . 7554/eLife . 09943 . 022Table 3 . SRM transitions for quantification of amino acids and uracil . DOI: http://dx . doi . org/10 . 7554/eLife . 09943 . 022Compound nameCompound abbreviationSRM transitionFragmentor ( V ) Collision energy ( V ) PolarityUracilU111 . 0 > 42 . 1629-−PhenylalanineF166 . 1 > 1201009+LeucineL132 . 1 > 86808+TryptophanW205 . 1 > 188855+IsoleucineI132 . 1 > 86808+MethionineM150 . 1 > 104408+TaurineTau126 > 44 . 111016+ValineV118 . 1 > 71 . 910010+ProlineP116 . 1 > 70 . 110013+TyrosineY182 > 165905+AlanineA90 > 44 . 1508+ThreonineT120 . 1 > 74809+GlycineG76 > 30 . 1505+GlutamineQ147 . 1 > 845016+GlutamateE148 . 1 > 84 . 17510+SerineS106 > 60409+AsparagineN133 . 1 > 74809+AspartateD134 . 1 > 748010+HistidineH156 . 1 > 110 . 28012+ArginineR175 . 1 > 7010015+LysineK147 . 1 > 845016+CitrullineCit176 > 159604+OrnithineO133 > 709010+ Cells were spotted on SM solid media and incubated at 30°C in FLUOstar OPTIMA plate reader ( BMG LABTECH , Aylesbury , United Kingdom ) to establish giant colony .\",\n \"Cells were collected at 26 hr ( exponential phase ) and re-suspended in H2O .\",\n \"Cells were then gently centrifuged , and pellet ( intracellular ) and supernatant ( extracellular ) fractions were separated .\",\n \"Metabolites were extracted from both fractions using 75% boiling ethanol containing l-taurine as an internal standard .\",\n \"Here , samples were left to incubate with extraction solvent in water bath ( 80°C ) for 2 min then mixed vigorously .\",\n \"Incubation and vigorous mixing step was then repeated .\",\n \"Solvent was evaporated using a Concentrator plus Speed Vac ( Eppendorf , Hamburg , Germany ) and samples were reconstituted in 50 µL 80% ethanol with intracellular fraction diluted 1:5 with 80% ethanol .\",\n \"All samples were submitted to LC-MS/MS and metabolite identification , and quantification was then performed as in 'LC-MS/MS based quantification of amino acids and uracil' .\",\n \"Data was illustrated following correction to the internal standard of amino acid abundances from both intra- and extracellular fractions .\",\n \"S . cerevisiae strains were transferred from cryo-preserved cultures to SC solid media , grown for 2 days and selected on SM solid media , supplemented only with required amino acids/ nucleobases for 1 day .\",\n \"Pre-cultures were inoculated in 1 . 5 mL SM containing the minimal supplementation and cultured O/N at 30°C .\",\n \"Main cultures were started at an OD595 of 0 . 15 in 1 . 5 mL of SC media in deep well 96-well plates and cultured in a Titramax ( Heidolph , Schwabach , Germany ) for 30 hr ( 950 rpm , 30°C , 4 mm stirring bead/ well ) .\",\n \"Samples of 50 µL were harvested every 3 hr , where cells were removed by centrifugation ( 3000 g , 5 min ) and the supernatant diluted 1:20 in absolute ethanol .\",\n \"Then , 1 µL of supernatant was used for quantification of extracellular metabolites by LC-MS/MS .\",\n \"‘gcFitModel’ function from ‘grofit’ R package ( Kahm et al , 2010 ) was used to estimate the uptake rate of histidine , leucine , uracil , and methionine in different auxotrophic strains .\",\n \"After O/N pre-culture S288c MATa yeast without auxotrophies and ura3Δ yeast ( S288c , MATa ) ( Mülleder et al . , 2012 ) were grown in 30 mL SM in shake flasks at 30°C , 300 rpm .\",\n \"The media contained either\",\n \"( i ) no additives ,\",\n \"( ii ) uracil ( 20 mg/L )\",\n \"( iii ) or uracil ( 20 mg/L ) , leucine ( 60 mg/L ) , methionine ( 20 mg/L ) and histidine ( 20 mg/L ) .\",\n \"During mid-exponential growth ( OD595 between 0 . 7 and 1 . 2 ) , 1 mL samples of the cultures were quenched in 4 mL -40°C 60% methanol , 10 mM NH4-acetate .\",\n \"After centrifugation ( -9°C , 4500 g ) , the cell pellet was stored at -80°C until extraction .\",\n \"Prior to extraction , 13C-yeast internal standard was spiked into the cell pellets , which were subsequently extracted with 1 mL 80°C 75% ethanol , 10 mM NH4-acetate for 3 min .\",\n \"During extraction , the suspension was vortexed on a 0 . 5–15–1 min time interval .\",\n \"After extraction , the suspension was centrifuged ( -9°C , 4500 g ) and the supernatant , hence the extract , was dried in a vacuum centrifuge before being stored at -80°C until measurement .\",\n \"For LC-MS measurements , the dried extracts were dissolved in 50–10050–100 µL H2O .\",\n \"The metabolites were separated with reversed phase ion-pairing chromatography on a Acquity UPLC ( Waters , Cheshire , United Kingdom ) with a Waters Acquity T3-endcapped column ( 150 mm , 2 . 1 mm , 18 µm ) as described in ( Buescher et al . , 2010 ) .\",\n \"Subsequently , the metabolites were analysed with a TSQ quantum ultra triple quadrupole mass spectrometer ( Thermo Fisher Scientific , Waltham , MA ) ( Buescher et al . , 2010 ) .\",\n \"Specifically , the metabolites were ionised with an electro spray ( ESI ) and the mass spectrometer was run in negative mode with SRM .\",\n \"The SRM transitions used are described in ( Buescher et al . , 2010 ) and for orotidine-monophosphate , where no standard was available , we used the phosphate fragment ( m/z 367 → 79 ) trajectory ( Horai et al . , 2010 ) .\",\n \"The obtained data was integrated with an in-house software and normalised to 13C-internal standard and OD595 , hence biomass .\",\n \"The median value of different replicates were then scaled and used to illustrate data .\",\n \"Plasmid stability ( segregation ) of vectors p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) was determined according to Christianson et al . ( 1992 ) .\",\n \"BY4741 ( his3Δ1 , leu2Δ0 , ura3Δ0 , met15Δ0 ) either transformed with one or all four plasmids , respectively , for either the four non-fluorescent or fluorescent vectors , were cultured in 25 mL of YPD media for 48 hr then plated at 1:100 , 000 dilution on YPD solid media .\",\n \"Plasmid retention was then calculated by replica plating CFUs from YPD solid media onto selective solid media .\",\n \"Number of doublings ( g ) and segregation rate ( m ) were calculated as in ( Christianson et al . , 1992 ) .\",\n \"Segregation rates of p423 ( HIS3 ) , pRS425 ( LEU2 ) , p426 ( URA3 ) , and pRS411 ( MET15 ) were simulated over generation time in R by iterative cycling ( looping ) .\",\n \"The script is given in Supplementary file 1 .\",\n \"Plasmid abundances were binned by plasmid number ( 0 to 4 ) and illustrated with R package 'ggplot2' in terms of auxotrophy .\",\n \"Unless otherwise indicated , cells were first spotted and grown for 2 nights on SM solid media to establish a giant colony .\",\n \"The colony was then re-suspended in H2O and diluted to 3 . 4e03 cells in 200 μL SM , and their optical density ( OD595 ) was recorded in a FLUOstar OPTIMA plate reader ( BMG LABTECH ) every 20 min for 40 hr at 30ºC .\",\n \"Both maximum specific growth rate ( μmax ) and lag phase were determined from growth curves using a model-richards fit from the R ‘grofit’ package ( Kahm et al , 2010 ) .\",\n \"For determining dry biomass , colony was re-suspended in H2O and normalised to 1 . 1e07 cells in 100 mL SM , then incubated for 72 hr at 30°C and pelleted .\",\n \"Pellets were dried for 5 days at 50°C and then weighed to obtain dry biomass .\",\n \"μmax for individual metabotypes was determined in batch SM culture ( 50 mL ) and supplemented accordingly for the different auxotrophic requirements .\",\n \"Cells from giant colonies of SeMeCo and YSBN5 were grown to exponential growth phase in SC media , and cell number was then measured with a CASY Model TTC ( Roche Innovatis , Switzerland ) cell counter .\",\n \"Cells were then diluted and plated on solid SC media to establish individual CFUs and the number of CFUs with initial cell number were compared .\",\n \"A micro-colony of BY4741 with prototrophy restored by complementation with the fluorescent protein vectors yEpCFP_HIS ( HIS3 ) , yEpSapphire_LEU ( LEU2 ) , yEpVenus_URA ( URA3 ) ( Bilsland et al . , 2013 ) , and pRS411-GPDpr-mCherry ( MET15 ) ( Table 1 ) was grown for 2 nights on SM .\",\n \"Prior to imaging , colony was embedded in 2% agarose ( Type I-B; Sigma ) and gently transferred to a μ-slide glass bottom ( ibidi ) .\",\n \"Cells were imaged with a DMI6000 inverted Leica SP5 confocal microscope , using a 10×/0 . 3 HC PL Fluotar Air objective , running LAS AF software ( version 2 . 7 . 3 . 9723 ) .\",\n \"Fluorescence for each marker was separated by excitation ( CFP: 458 nm , Sapphire: 405 nm , Venus: 514 nm and mCherry: 561 nm ) .\",\n \"In our hands , the Sapphire-LEU2 was also visible under the imaging conditions used to visualise the Venus-URA3 .\",\n \"For this reason , we removed Venus-URA3 channel from the colony image .\",\n \"A look-up table was applied to each channel post-acquisition to allow visualisation of the different channels together using ImageJ software .\",\n \"For the microscopy image showing fluorescent-labelled cells in a colony , prototrophs and auxotrophs were identified as being present or absent using several cut-offs for fluorescent signal intensity .\",\n \"Based on these different cut-off values ( 0 . 1 , 0 . 2 , 0 . 3 , and 0 . 4 ) , the monochrome fluorescence microscopy images of each individual marker ( HIS3 , LEU2 and MET15 ) were recognised as metabolite producing ( prototrophic ) or requiring ( auxotrophic ) pixels .\",\n \"The auxotrophic pixels were marked black , prototrophic pixels white , and the background was illustrated grey to separate it from the colony pixels .\",\n \"For each auxotrophic pixel , the distance to the next prototrophic pixel was calculated and distance values for the minimum , mean , overall maximum , and maximum for 90% of cells were taken for each marker .\",\n \"Genotypes depicting SeMeCo frequent ( HIS3 , LEU2 , MET15 , ura3Δ ) and rare ( his3Δ , leu2Δ , URA3 , MET15 ) metabotypes were reconstructed by transforming mCherry labelled derivatives of pHLUM ( pHLM-GPDpr-mCherry and pUM-GPDpr-mCherry , respectively ) into BY4741 .\",\n \"Strains were spiked into established SeMeCo in 1:10 ( frequent or rare metabotype: SeMeCo ) ratio taken from their established giant colonies on selective media ( where plasmid segregation is ongoing ) .\",\n \"Abundance of fluorescent cells was monitored immediately after mixing and approximately 48 hr after re-establishment of giant colony on minimal solid media with a BD LSRFortessa cell analyser .\",\n \"Data analysis was performed with FlowJo .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Metabolite exchange among co-growing cells is frequent by nature , however , is not necessarily occurring at growth-relevant quantities indicative of non-cell-autonomous metabolic function .","Complementary auxotrophs of Saccharomyces cerevisiae amino acid and nucleotide metabolism regularly fail to compensate for each other's deficiencies upon co-culturing , a situation which implied the absence of growth-relevant metabolite exchange interactions .","Contrastingly , we find that yeast colonies maintain a rich exometabolome and that cells prefer the uptake of extracellular metabolites over self-synthesis , indicators of ongoing metabolite exchange .","We conceived a system that circumvents co-culturing and begins with a self-supporting cell that grows autonomously into a heterogeneous community , only able to survive by exchanging histidine , leucine , uracil , and methionine .","Compensating for the progressive loss of prototrophy , self-establishing communities successfully obtained an auxotrophic composition in a nutrition-dependent manner , maintaining a wild-type like exometabolome , growth parameters , and cell viability .","Yeast , as a eukaryotic model , thus possesses extensive capacity for growth-relevant metabolite exchange and readily cooperates in metabolism within progressively establishing communities ."],"string":"[\n \"Metabolite exchange among co-growing cells is frequent by nature , however , is not necessarily occurring at growth-relevant quantities indicative of non-cell-autonomous metabolic function .\",\n \"Complementary auxotrophs of Saccharomyces cerevisiae amino acid and nucleotide metabolism regularly fail to compensate for each other's deficiencies upon co-culturing , a situation which implied the absence of growth-relevant metabolite exchange interactions .\",\n \"Contrastingly , we find that yeast colonies maintain a rich exometabolome and that cells prefer the uptake of extracellular metabolites over self-synthesis , indicators of ongoing metabolite exchange .\",\n \"We conceived a system that circumvents co-culturing and begins with a self-supporting cell that grows autonomously into a heterogeneous community , only able to survive by exchanging histidine , leucine , uracil , and methionine .\",\n \"Compensating for the progressive loss of prototrophy , self-establishing communities successfully obtained an auxotrophic composition in a nutrition-dependent manner , maintaining a wild-type like exometabolome , growth parameters , and cell viability .\",\n \"Yeast , as a eukaryotic model , thus possesses extensive capacity for growth-relevant metabolite exchange and readily cooperates in metabolism within progressively establishing communities .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Life is sustained by an array of chemical reactions that is collectively referred to as metabolism .","Some of these reactions break down complex substances to release energy and vital compounds , while others make new molecules from smaller building blocks .","Bacterial communities are regularly composed of heterogeneous species , several of which have lost one or more essential metabolic pathways .","Nevertheless , these cells can still survive by making use of metabolic products released by their neighbouring cells .","Yeast are single-celled fungi that also form colonies and , as eukaryotes , they possess cells that are more similar to our own .","However , in the laboratory , complementary metabolically deficient yeast cells do not survive when mixed together .","It was presumed this is because yeast cells make only enough of each essential metabolite for themself , and so can’t replace those that are missing from their neighbouring cells .","Campbell et al . now challenge this view by finding that yeast cells release a variety of metabolites , they use these released metabolites in preference to making their own , and possess the capacity to grow on the basis of a non-cell-autonomous metabolism .","This discovery came with the design of a new experimental test to study metabolite exchange interactions .","This method uses yeast cells that have one or more of their own metabolic genes disabled , and instead have a copy of these genes on small circular DNA 'mini-chromosomes' ( called plasmids ) .","The gene on the plasmid can compensate for the yeast having its own gene missing , and allows the cell to still make the metabolic product it needs to survive .","However , as a single cell divides to form a colony , cells randomly lose these plasmids , leaving some of the cells deficient for a particular metabolite .","These cells can only survive if the other cells in the colony export the missing metabolite in the quantity needed for growth .","Using this test , Campbell et al . found that yeast cells can export missing metabolites at levels needed to sustain these emerging metabolic mutants .","Additionally , these yeast communities could grow at levels comparable to other yeast without metabolic deficiencies .","The resulting colonies also feature one of several different genetic and metabolic profiles , which change in response to the metabolite that is missing .","These findings demonstrate that yeast cells can exchange high amounts of metabolites , sufficient to form cooperative colonies , and as metabolite concentrations are not altered compared to normal cells , it is likely that exchange of metabolites is ongoing between neighbours in yeast communities .","The additional discovery that yeast stop making metabolites when they can obtain them from neighbouring cells has implications for research .","This is because many yeast genetic studies use metabolically deficient strains that are supplemented in culture with metabolites .","Future work could address whether such supplementation has kept certain functions of metabolism hidden ."],"string":"[\n \"Life is sustained by an array of chemical reactions that is collectively referred to as metabolism .\",\n \"Some of these reactions break down complex substances to release energy and vital compounds , while others make new molecules from smaller building blocks .\",\n \"Bacterial communities are regularly composed of heterogeneous species , several of which have lost one or more essential metabolic pathways .\",\n \"Nevertheless , these cells can still survive by making use of metabolic products released by their neighbouring cells .\",\n \"Yeast are single-celled fungi that also form colonies and , as eukaryotes , they possess cells that are more similar to our own .\",\n \"However , in the laboratory , complementary metabolically deficient yeast cells do not survive when mixed together .\",\n \"It was presumed this is because yeast cells make only enough of each essential metabolite for themself , and so can’t replace those that are missing from their neighbouring cells .\",\n \"Campbell et al . now challenge this view by finding that yeast cells release a variety of metabolites , they use these released metabolites in preference to making their own , and possess the capacity to grow on the basis of a non-cell-autonomous metabolism .\",\n \"This discovery came with the design of a new experimental test to study metabolite exchange interactions .\",\n \"This method uses yeast cells that have one or more of their own metabolic genes disabled , and instead have a copy of these genes on small circular DNA 'mini-chromosomes' ( called plasmids ) .\",\n \"The gene on the plasmid can compensate for the yeast having its own gene missing , and allows the cell to still make the metabolic product it needs to survive .\",\n \"However , as a single cell divides to form a colony , cells randomly lose these plasmids , leaving some of the cells deficient for a particular metabolite .\",\n \"These cells can only survive if the other cells in the colony export the missing metabolite in the quantity needed for growth .\",\n \"Using this test , Campbell et al . found that yeast cells can export missing metabolites at levels needed to sustain these emerging metabolic mutants .\",\n \"Additionally , these yeast communities could grow at levels comparable to other yeast without metabolic deficiencies .\",\n \"The resulting colonies also feature one of several different genetic and metabolic profiles , which change in response to the metabolite that is missing .\",\n \"These findings demonstrate that yeast cells can exchange high amounts of metabolites , sufficient to form cooperative colonies , and as metabolite concentrations are not altered compared to normal cells , it is likely that exchange of metabolites is ongoing between neighbours in yeast communities .\",\n \"The additional discovery that yeast stop making metabolites when they can obtain them from neighbouring cells has implications for research .\",\n \"This is because many yeast genetic studies use metabolically deficient strains that are supplemented in culture with metabolites .\",\n \"Future work could address whether such supplementation has kept certain functions of metabolism hidden .\"\n]","isConversational":false},"year":{"kind":"string","value":"2015"}}},{"rowIdx":288,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["tools and resources","neuroscience"],"string":"[\n \"tools and resources\",\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"An open-source device for measuring food intake and operant behavior in rodent home-cages"},"id":{"kind":"string","value":"elife-66173-v2"},"sections":{"kind":"list like","value":[["Feeding is critical for survival , and dysregulation of food intake underlies medical conditions such as obesity and anorexia nervosa .","Quantifying food intake is necessary for understanding these disorders in animal models .","However , it is challenging to accurately measure food intake in rodents due to the small volume of food that they eat .","Researchers have devised multiple methods for quantifying food intake in rodents , each with advantages and drawbacks ( Ali and Kravitz , 2018 ) .","Manual weighing of a food container is a simple and widely used method for quantifying food intake in a rodent home-cage .","Yet this is time consuming to complete , is subject to error and variability , and does not allow for fine temporal analysis of consumption patterns ( Acosta-Rodríguez et al . , 2017; Reinert et al . , 2019 ) .","Automated tools have been developed for measuring food intake in home-cages with high temporal resolution , although most require modified caging , powdered foods , or connected computers which limit throughput ( Ahloy-Dallaire et al . , 2019; Farley et al . , 2003; Moran , 2003; Yan et al . , 2011 ) .","These include automated weighing ( Hulsey and Martin , 1991; Meguid et al . , 1990; Minematsu et al . , 1991 ) , pellet dispensers ( Aponte et al . , 2011; Gill et al . , 1989; Oh et al . , 2017 ) , or video detection-based systems ( Burnett et al . , 2016; Jhuang et al . , 2010; Salem et al . , 2015 ) .","In addition to measuring total food intake , understanding neural circuits involved in feeding requires exploring why animals seek and consume food .","Has their motivation for a specific nutrient changed ?","Has their feeding gained a compulsive nature that is insensitive to satiety signals ?","These questions can be answered with operant tasks , where rodents receive food contingent on their actions ( Curtis et al . , 2019; Mourra et al . , 2020; O'Connor et al . , 2015; Skinner , 1938; Thorndike , 1898; Wald et al . , 2020 ) .","Typically , operant behavior is tested in dedicated chambers for a few hours each day .","Commercial systems for testing operant behaviors typically comprise a dedicated arena equipped with levers , food or liquid dispensers , tones or spotlights , and video cameras .","Programmable software interfaces allow experimenters to control this equipment to train animals on behavioral tasks such as fixed-ratio or progressive ratio responding .","Some chambers are also equipped with video cameras and tracking software , so the location of the animal can be used as a trigger to control task events ( London et al . , 2018 ) .","Training in dedicated operant chambers has limitations: tasks can take weeks for animals to learn , animals may be tested at different phases of their circadian cycle due to equipment availability , and food restriction can be necessary to get animals to seek food outside of their home-cage , which can confound feeding studies .","To mitigate these issues , several researchers have begun to test operant behavior in rodent home-cages , resulting in both fewer interventions from the researcher and faster rates of learning ( Balzani et al . , 2018; Francis and Kanold , 2017; Lee et al . , 2020 ) .","Here , we present a new solution for monitoring food intake and testing operant behavior in rodent home-cages: the Feeding Experimentation Device version 3 ( FED3 ) .","Our goal was to develop a device for measuring food intake in rodent home-cages with high temporal resolution , while also measuring food motivation via operant behavior .","FED3 is a stand-alone device that contains a pellet dispenser , two ‘nose-poke’ sensors for operant behavior , visual and auditory stimuli , and a screen for experimenter feedback .","FED3 is compact and battery powered , fitting in most commercial vivarium home-cages without any connected computers or external wiring .","FED3 also has a programmable output that can control other equipment , for example to trigger optogenetic stimulation after a nose-poke or pellet removal , or to synchronize feeding behavior with electrophysiological or fiber-photometry recordings , or with in vivo calcium imaging .","Finally , FED3 is open source and was designed to be customized and re-programmed to perform novel tasks to help researchers understand food intake and food motivation .","To this end , we have written a user-friendly Arduino library to facilitate custom behavioral programming of FED3 .","Here , we describe the design and construction of FED3 and present several experiments that demonstrate its functionality .","These include measuring circadian patterns of food intake over multiple days , performing meal pattern analysis , automated operant training , closed-economy motivational testing , and optogenetic self-stimulation .","FED3 extends existing methods for quantifying food intake and operant behavior in rodents to help researchers achieve a deeper understanding of feeding and feeding disorders ."],["FED3 is controlled by a 48 mHz ATSAMD21G18 ARM Cortex M0 microprocessor ( Adafruit Feather M0 Adalogger ) and contains two nose-pokes , a pellet dispenser , a speaker for auditory stimuli , eight multi-color LEDs for visual stimuli , a screen for experimenter feedback , and a programmable analog output ( Figure 1A , B ) .","When mice interact with FED3 , the timing of each poke and pellet removal are logged to an internal microSD card for later analysis and summary data is displayed on the screen for immediate feedback to the researcher ( Figure 1B ) .","FED3 is small ( ~10 cm × 12 cm × 9 cm ) , battery powered , and completely self-contained , so it fits in most vivarium home-cages without modification or introducing wiring to the cage ( Figure 1C ) .","It is powered by a rechargeable battery that lasts ~1 week between charges ( exact battery life depends on the behavioral program being run and number of pellets dispensed ) .","FED3 also has magnetic mounts to facilitate wall-mounting on any plastic box to mimic a traditional operant setup ( see Video 1 ) .","We provide example programs for running rodents on multiple common behavioral paradigms , including free feeding , time-restricted free-feeding , fixed-ratio 1 ( FR1 ) , and progressive ratio ( PR ) operant tasks , and have written a FED3 Arduino library to ease development of custom programs .","FED3 also has an auxiliary I2C port for hardware customization and a programmable BNC output connection that allows synchronization with external equipment for aligning behavioral events with fiber-photometry recordings ( London et al . , 2018; Mazzone et al . , 2020 ) , electrophysiological recordings ( London et al . , 2018 ) , or other equipment such as video tracking systems ( Krynitsky et al . , 2020; Li et al . , 2019 ) .","This BNC output can report task events or control external hardware with delays of less than 200 µs , which compares favorably with computers that process information through Universal Serial Bus ( USB ) , which can introduce delays of several milliseconds .","Finally , we provide a graphical analysis package ( FED3VIZ ) written in Python that enables users to generate detailed plots from FED3 data ( Figure 2F ) .","FED3 is open-source and freely available online , including 3D design files , printed circuit board ( PCB ) files ( Figure 1—figure supplement 1A , B ) , build instructions ( Figure 1D ) , and code ( https://github . com/KravitzLabDevices/FED3 copy archived at swh:1:rev:ff8fc79d288a440d91566f4c6cd80011956f4be0 ) .","Ten FED3 devices were set to the ‘free-feeding’ program , which dispenses a pellet and monitors its presence in the pellet well ( Figure 2A ) .","In this paradigm , each time the pellet is removed , FED3 logs the date and time to the storage card and dispenses another pellet .","This allows for the reconstruction of detailed feeding records over multiple days .","Assuming calorie content of the pellets is known , total caloric intake can also be tracked over time .","Devices were placed with ten singly housed mice for six consecutive days with no additional food source .","We observed the expected circadian rhythm in food intake ( Figure 2B , C ) .","To test accuracy , we confirmed that the daily change in weight of the FED3 device correlated with the number of pellets removed multiplied by the weight of each pellet ( 20 mg pellets , Figure 2D ) .","The coefficient of determination ( R2 ) for the regression was 0 . 97 , indicating an error rate of ~3% between manual weighing and counting of pellets by FED .","Finally , we confirmed that the mice obtained all of their necessary daily calories from FED3 , evidenced by a stable body weight across these 6 days ( Figure 2E ) .","In other experiments , we have run FED3 for >1 month without observing weight loss .","FED3 can be used for multi-week experiments like this as it has a low rate of jamming .","This is facilitated by a unique angled pellet hopper and dispensing path that has fewer places where pellets can jam than traditional horizontal pellet dispensers ( Figure 2—figure supplement 1 ) , as well as ‘jam clearing’ movements in the code that vibrate and rotate the dispensing disk to dislodge stuck pellets when they are detected .","These are improvements over the first generation of this device ( Nguyen et al . , 2016 ) .","As a point on rodent safety , although FED3 is resistant to jamming it still must be checked for function at least once per day when being used as the only food source .","FED3 creates a large amount of data , particularly when run over multiple days .","To facilitate analysis of FED3 data , we created FED3VIZ , an open-source Python-based analysis program ( https://github . com/earnestt1234/FED3_Viz ) .","FED3VIZ offers plots for visualizing different aspects of FED3 data , including pellet retrieval , poke accuracy , pellet retrieval time , delay between consecutive pellet earnings ( inter pellet intervals ) , meal size , and progressive ratio breakpoint .","These metrics can be plotted for single files or group averages .","FED3VIZ’s averaging methods provide options for aggregating data recorded on different days , while preserving time-of day or phase of the light–dark cycle .","Furthermore , circadian activity patterns can also be visualized with the FED3VIZ ‘Chronogram’ and ‘Day/Night Plot’ functions ( see Figure 7 ) , which segment data based on a user-defined light cycle .","The processed data going into each plot can also be saved and used to create new visualizations or compute statistics in other programs , promoting reproducible , sharable analysis and visualization of FED3 data .","FED3 records the date and time that each pellet is removed , which can be used to quantify feeding patterns including the size and quantity of meals , eating rate within meals , and timing between meals .","Different parameters have been used by different groups to define meals , often based on numerical cut-offs ( Farley et al . , 2003; Kanoski et al . , 2013; Melhorn et al . , 2010 ) .","To complement these approaches , we aimed to develop an unbiased approach for understanding meal patterns , based on the distribution of time intervals between each pellet consumed .","For the 10 mice in the above experiment , we plotted the inter-pellet time interval histogram ( Figure 3A ) for both the dark and light cycles , based on an approach that was previously described in rats ( Cottone et al . , 2007 ) .","We observed a large peak at <1 min between pellets , showing that the majority of pellets were consumed within 1 min of another pellet .","We used this distribution to classify pellets eaten within the same minute as belonging to the same meal , and pellets with larger intervals between them belonging to different meals .","This approach revealed that animals eat fewer meals during the light cycle ( Figure 3B ) but eat similar number of pellets within each meal and obtain a similar fraction of their pellets within meals in each cycle .","Differences in meal patterning have been linked to obesity ( Farley et al . , 2003; Wald and Grill , 2019 ) , so this analytical approach may assist in understanding obesity and other disorders of feeding .","A unique feature of FED3 is that it is small and wire-free , allowing it to fit inside of traditional vivarium home-cages .","This facilitates quantification of circadian rhythms in both operant and feeding behavior ( Figure 4A ) .","To demonstrate operant capability , we quantified how nose-poking for pellets varied over the circadian cycle , by running mice on a FR1 task for six consecutive days .","Mice learned to nose-poke on the active port for a single pellet .","We tracked active ( rewarded ) and inactive ( unrewarded ) pokes .","As expected , both active and inactive pokes were higher during the dark cycle ( Figure 4B–D ) .","Surprisingly , however , poke efficiency ( % of total pokes on the active port ) was slightly higher during the light cycle ( Figure 4E ) , suggesting that operant responding is more efficient during the light cycle .","Multiple research groups currently use FED3 .","We therefore asked how mouse operant learning varies across different laboratories .","To do this , we obtained operant data from the first overnight FR1 session from seven laboratories , resulting in data from 122 mice ( mix of males and females , Figure 5A ) .","When comparing pellets earned in an overnight session across laboratories , we observed a significant effect of group , linked to significant differences in 4 of 21 post hoc comparisons ( Figure 5B ) .","When viewed as a single distribution ( Figure 5B inset ) , the distribution of pellets earned from all groups was consistent with a single Gaussian distribution ( p>0 . 05 , Shapiro-Wilk test for normality ) .","This highlights how FED3 enables high-throughput studies of operant behavior , and the power of large sample sizes to achieve adequate sampling of a distribution of behavior ( Figure 5B ) .","A unique capability of FED3 is that it has a sensor for detecting the presence of the pellet in the feeding well .","This enables time-stamping of when each pellet was removed for constructing feeding records , as well as measuring the time between the pellet dispense and removal , which we termed ‘retrieval time’ .","Changes in retrieval time may reflect learning , as this measure progressively decreased as animals gained experience with FED3 ( Figure 5E ) .","Accordingly , active active count and poke efficiency ( % of pokes on the active port ) increased over time , demonstrating that retrieval time decreased on the same time scale that mice learned which poke resulted in a pellet ( Figure 5C ) .","To compare learning rates in overnight training to traditional daily operant sessions , we ran a new group of eleven mice for sixteen daily 1 hr FR1 sessions with FED3 , returning them to the colony for the remainder of each day .","Learning rates did not differ between mice exposed to 16 daily 1 hr sessions or one 16 hr overnight session ( Figure 5F ) .","This suggests that the acquisition of this FR1 task may depend most strongly on the cumulative time mice interact with FED3 and that overnight training can greatly speed up task acquisition by giving them many hours of experience in one night .","Based on prior literature ( Steinhauer et al . , 1976 ) , we predicted that the speed of acquisition of an FR1 task would be further increased by prior magazine training .","To test this , we exposed 24 mice to magazine training using the free feeding FED3 paradigm for 1–3 days prior to FR1 training ( Figure 6A ) .","This paradigm dispenses a pellet into the feeding well of FED3 and replaces it whenever it is removed , providing an automatic method for magazine training animals .","In this way , magazine training allows mice to learn the location of the food source and associate food with the sound of the pellet dispenser operating and the pellet being dispensed , before commencing with FR1 training .","We compared the performance of the 24 magazine trained mice to 122 mice from Figure 5 ( which had not been magazine trained ) and found that magazine training resulted in significantly higher ( ~2 ) levels of pellet acquisition in the first night with the FR1 task ( Figure 6B , C ) .","Therefore , magazine training is recommended to speed up acquisition of nose-poking tasks .","FED3 can also be used for closed-economy feeding studies , in which all food is earned by operant nose-poking ( Figure 7 ) .","Closed-economy operant tasks have been used to quantify the economic demand for food ( Bauman , 1991; Chaney and Rowland , 2008 ) , as well as changes in economic demand due to manipulation of the dopamine system ( Beeler et al . , 2010; Mourra et al . , 2020 ) .","We tested seven mice on a repeating progressive ratio task ( adapted from Mourra et al . , 2020 ) , in which the nose-poking requirement began on FR1 and increased each time a pellet was earned .","When mice refrained from poking on either port for 30 min the ratio reset to FR1 ( Figure 7A , B ) .","Although pellets earned from this task were their only food source , mouse weights remained stable across days , demonstrating that mice earned their entire caloric need with the task ( Figure 7C ) .","Total pellets earned did not differ between light and dark cycles ( Figure 7D ) .","However , nose-poking rates were higher during the dark phase of their daily cycle ( Figure 7E ) , meaning mice performed more pokes to earn each pellet in this phase ( Figure 7F ) .","This suggests that motivation for food pellets varies over the circadian cycle such that mice are willing to work harder during the dark cycle vs . the light cycle .","When the data was visualized hourly across the circadian cycle , we further noted a ~3 hr window of enhanced nose-poking rates starting shortly after the onset of the dark cycle ( Figure 7F ) .","This time coincides with a circadian peak in extracellular dopamine levels in the striatum ( Ferris et al . , 2014 ) , and a peak in activity levels in mice ( Matikainen-Ankney et al . , 2020 ) .","Therefore , this task may be used to assay changes in accumbal dopamine function across the circadian cycle .","FED3 was also designed to synchronize with other experimental equipment through its programmable output port .","To demonstrate this capability , we programmed FED3 to act as a function generator and control an LED for an intra-cranial self-stimulation task .","The dorsal striatum contains two main classes of output neurons , differentiated by their expression of the dopamine D1 or D2 receptors ( Gerfen et al . , 1990 ) .","Dorsal striatal neurons that express the D1 receptor are highly reinforcing when optogenetically stimulated ( Kravitz et al . , 2012 ) .","To demonstrate how FED3 can be used to perform an optogenetic self-stimulation experiment , we used Cre-dependent viral expression to target channelrhodopsin-2 ( ChR2 ) to D1R-expressing neurons in the dorsal striatum ( Figure 8A ) .","FED3 was programmed to generate and trigger a 1 s train of 20 Hz pulses of 475 nm light upon each active nose-poke , and the session continued until they received 75 trains of stimulation ( Figure 8B ) .","Three mice were run on this task , resulting in significantly greater active vs inactive pokes ( Figure 8C , D ) , and demonstrating that FED3 can be used for optogenetic self-stimulation studies .","The programmable output is attached to an on-board digital-to-analog converter chip , meaning that it can be programmed to output pulses of any voltage from 0 to 3 V , allowing FED3 to represent multiple behavioral events with one output line .","The timing of the BNC output port is regulated by the SAMD21 microprocessor that has a documented pulsing precision of <1 µs ."],["Quantification of food intake is necessary to understand animal models of feeding disorders , as well as other medical conditions .","To quantify food intake in rodent home-cages , we previously published the FED version 1 , an open-source , stand-alone feeding device that can fit in a rack-mounted home-cage ( Nguyen et al . , 2016 ) .","FED records a time series of pellets removed , which can be used to reconstruct feeding records .","FED has been used by multiple research groups to understand feeding ( Brierley et al . , 2020; Burnett et al . , 2019; Chen et al . , 2020; Li et al . , 2019; London et al . , 2018 ) .","Since its original publication , we have redesigned the FED twice and here present the third version of FED , which revamps our original design and adds the ability to measure operant behavior , as well as a unique ‘angled’ pellet dispenser mechanism that is resistant to jamming , which enables long term studies .","We distributed the design for FED3 online , and it has been used to study how specific neural circuit manipulations alter food motivation ( Mazzone et al . , 2020; Sciolino et al . , 2019; Vachez et al . , 2021 ) , how weight-loss alters food motivation ( Matikainen-Ankney et al . , 2020 ) , and how food motivation is altered in a stress-susceptible mouse population ( Rodriguez et al . , 2020 ) .","FED3 is a stand-alone solution for home-cage operant training , enabling researchers to understand not just total food intake , but also motivation for and learning about food rewards .","FED3 has several unique benefits over comparable systems including: ( 1 ) FED3 is low cost .","The FED3 electronics cost ~$200 and the housing is 3D printed .","Even if 3D printing is done professionally this is >10× cheaper than most commercial solutions for measuring food intake or testing operant behavior; ( 2 ) FED3 is self-contained and fits within traditional vivarium caging , allowing for measures of true ‘home-cage’ feeding without modifying the cage .","Due to its small size , it can also be placed inside of other equipment where wires might be impractical , such as within an indirect calorimetry system; ( 3 ) FED3 has a programmable output that allows it to easily synchronize with other equipment .","Due to wiring , connecting FED3 to external hardware likely requires FED3 to be used outside of the home-cage , or to modify the home-cage system .","This has been done by multiple labs to synchronize the output of FED3 with fiber-photometry recordings ( London et al . , 2018; Mazzone et al . , 2020 ) , electrophysiological recordings ( London et al . , 2018 ) , optogenetic stimulation ( Vachez et al . , 2021 ) , and video tracking ( Krynitsky et al . , 2020; Li et al . , 2019 ) ; and ( 4 ) FED3 is open source and all design files and codes are freely available online .","This enables users to modify the code and hardware to achieve new functionality .","The purpose of this manuscript is to demonstrate the utility of FED3 for feeding research .","To this end , we demonstrate experiments that measured total food intake , operant responding , and optogenetic stimulation .","We further highlight how the high temporal resolution enables meal pattern analysis across multiple days .","Finally , we coordinated with six other research groups to compile a dataset of 122 mice across seven research sites , all running the same experimental FR1 program .","We observed similar patterns of acquisition across all sites , demonstrating that FED3 can be used for multi-site research studies on feeding .","Due to its low cost and open-source nature , we believe that multi-site studies with FED3 are more feasible than with other systems .","While FED3 has many strengths , it also has limitations .","One limitation is that FED3 uses internal microSD cards to store data .","While microSD cards are convenient , they are not ideal for large numbers of devices , where removing multiple cards can be cumbersome .","Wireless data logging is a potential solution to this problem , although there are challenges to implementing this in rodent cages .","A second limitation is that animals can ‘hoard’ pellets from FED3 , as animals can remove food without consuming it .","In our experience , this seemed to be a rare trait that specific mice engaged in ( <10% of mice hoard pellets ) .","Unfortunately , FED3 has no way to determine whether a mouse actually consumes each pellet after removal so we recommend checking for pellet hoarding and accounting for this in experimental conclusions .","A third limitation is that granular bedding can be kicked into the pellet well and interfere with pellet detection .","To avoid this , we recommend using ‘iso-pad’ bedding , or bedding pellets that are large enough to avoid this possibility .","A fourth limitation is that FED3 is not waterproof and would not be resistant to flooding , though the 3D printed housing is sufficient to protect electronics from typical wear associated with placement in a rodent cage .","A final limitation of FED3 is that it has no way of identifying individual mice in group housed environments , so feeding records must be collected in singly housed mice .","However , FED3 is open source , so it can be modified and improved with innovations from our group and the feeding community , and future iterations of the device may include methods for identifying multiple mice using radio-frequency identification ( RFID ) tags .","Being able to run studies on group housed mice would greatly increase throughput and allow for the study of interactions between social behavior and feeding .","We published the FED3 design as open-source and look forward to community contributions and modifications to enable new functionality and overcome these limitations ."],["The FED3 device is open-source , and design files and code are freely available online at: https://github . com/KravitzLabDevices/FED3 .","In addition , we have made all data and analysis code for the figures in this paper available at https://osf . io/hwxgv/ .","One hundred and fifty-nine C57Bl/6 mice were housed in a 12 hr light/dark cycle with ad libitum access to food and water except where described .","Mice were provided laboratory chow diet ( 5001 Rodent Diet; Lab Supply , Fort Worth , TX ) .","All procedures were approved by the Animal Care and Use Committee at Washington University in St Louis , the National Institutes of Health , Williams College , Virginia Tech , and Monash University .","Tutorial videos and other information on assembling FED3 are available at: https://github . com/KravitzLabDevices/FED3/wiki .","We demonstrate multiple operational modes for FED3 to highlight a range of functionality and applications .","The following programs are included with the standard FED3 code: Viral infections of male and female Drd1-Cre mice were conducted under 0 . 5–2 . 5% anesthesia on a stereotaxic apparatus .","Using a Nanoject injector , 500 nL of AAV2-DIO-hSyn-ChR2 was injected bilaterally ( 500 nL/hemisphere ) in the dorsomedial striatum .","Optical fibers were then implanted in the same region .","After letting ChR2 express for 4 weeks , animals were pre-trained on an FR1 schedule for pellets overnight in their home-cage .","Two days later , mice were placed in a box with a FED3 device connected to an LED driver .","The active poke for this paradigm was on the opposite side to the active poke of the pre-training session .","During the self-stimulation session , when the mouse poked on the active nose-poke , it received a 1 s train of 1 mW 475 nm light at 20 Hz .","Sessions were run for 60 min in a 550 cm2 arena .","CSV files generated by FED3 were processed and plotted with custom python scripts ( Python , version 3 . 6 . 7 , Python Software Foundation , Wilmington , DE ) .","All data and scripts are available on Open Science Framework ( https://osf . io/hwxgv/ ) .","Visualization was also completed using FED3VIZ GUI to generate plots .","FED3VIZ was written in Python’s standard library for developing GUIs ( tkinter ) .","FED3VIZ is a custom open-source graphical program for analyzing FED3 data .","FED3VIZ code , version history , installation instructions , and user manual are available on GitHub ( https://github . com/earnestt1234/FED3_Viz ) .","FED3VIZ offers plotting and data output for visualizing different aspects of FED3 data , including pellet retrieval , poke accuracy , pellet retrieval time , delay between consecutive pellet earnings ( inter-pellet intervals ) , meal size , and progressive ratio breakpoint .","Based on inter-pellet interval histograms ( Figure 3 ) , we defined meals as pellets eaten within 1 min of each other .","In addition , we defined a minimum size of 0 . 1 g ( five pellets ) to be counted as a meal .","Bartlett’s test for equal variances was performed; one- or two-way ANOVAs with Tukey post-tests were used to compare groups with equal variance ( p>0 . 05 ) where appropriate .","Linear mixed effects models were used to analyze groups with significantly difference variances ( p<0 . 05 ) .","p-value<0 . 05 was considered significant .","Statistical tests to compare means were run using statsmodels module in python ( Seabold and Perktold , 2010 ) .","Data sets are presented as mean ± SEM .","Numbers of animals per experiment is listed as n=number of animals .","Linear regression was used to determine correlative relationships .","T-tests or Mann–Whitney U tests were used to compare the means of two groups for parametric or nonparametric data distributions , respectively ."]],"string":"[\n [\n \"Feeding is critical for survival , and dysregulation of food intake underlies medical conditions such as obesity and anorexia nervosa .\",\n \"Quantifying food intake is necessary for understanding these disorders in animal models .\",\n \"However , it is challenging to accurately measure food intake in rodents due to the small volume of food that they eat .\",\n \"Researchers have devised multiple methods for quantifying food intake in rodents , each with advantages and drawbacks ( Ali and Kravitz , 2018 ) .\",\n \"Manual weighing of a food container is a simple and widely used method for quantifying food intake in a rodent home-cage .\",\n \"Yet this is time consuming to complete , is subject to error and variability , and does not allow for fine temporal analysis of consumption patterns ( Acosta-Rodríguez et al . , 2017; Reinert et al . , 2019 ) .\",\n \"Automated tools have been developed for measuring food intake in home-cages with high temporal resolution , although most require modified caging , powdered foods , or connected computers which limit throughput ( Ahloy-Dallaire et al . , 2019; Farley et al . , 2003; Moran , 2003; Yan et al . , 2011 ) .\",\n \"These include automated weighing ( Hulsey and Martin , 1991; Meguid et al . , 1990; Minematsu et al . , 1991 ) , pellet dispensers ( Aponte et al . , 2011; Gill et al . , 1989; Oh et al . , 2017 ) , or video detection-based systems ( Burnett et al . , 2016; Jhuang et al . , 2010; Salem et al . , 2015 ) .\",\n \"In addition to measuring total food intake , understanding neural circuits involved in feeding requires exploring why animals seek and consume food .\",\n \"Has their motivation for a specific nutrient changed ?\",\n \"Has their feeding gained a compulsive nature that is insensitive to satiety signals ?\",\n \"These questions can be answered with operant tasks , where rodents receive food contingent on their actions ( Curtis et al . , 2019; Mourra et al . , 2020; O'Connor et al . , 2015; Skinner , 1938; Thorndike , 1898; Wald et al . , 2020 ) .\",\n \"Typically , operant behavior is tested in dedicated chambers for a few hours each day .\",\n \"Commercial systems for testing operant behaviors typically comprise a dedicated arena equipped with levers , food or liquid dispensers , tones or spotlights , and video cameras .\",\n \"Programmable software interfaces allow experimenters to control this equipment to train animals on behavioral tasks such as fixed-ratio or progressive ratio responding .\",\n \"Some chambers are also equipped with video cameras and tracking software , so the location of the animal can be used as a trigger to control task events ( London et al . , 2018 ) .\",\n \"Training in dedicated operant chambers has limitations: tasks can take weeks for animals to learn , animals may be tested at different phases of their circadian cycle due to equipment availability , and food restriction can be necessary to get animals to seek food outside of their home-cage , which can confound feeding studies .\",\n \"To mitigate these issues , several researchers have begun to test operant behavior in rodent home-cages , resulting in both fewer interventions from the researcher and faster rates of learning ( Balzani et al . , 2018; Francis and Kanold , 2017; Lee et al . , 2020 ) .\",\n \"Here , we present a new solution for monitoring food intake and testing operant behavior in rodent home-cages: the Feeding Experimentation Device version 3 ( FED3 ) .\",\n \"Our goal was to develop a device for measuring food intake in rodent home-cages with high temporal resolution , while also measuring food motivation via operant behavior .\",\n \"FED3 is a stand-alone device that contains a pellet dispenser , two ‘nose-poke’ sensors for operant behavior , visual and auditory stimuli , and a screen for experimenter feedback .\",\n \"FED3 is compact and battery powered , fitting in most commercial vivarium home-cages without any connected computers or external wiring .\",\n \"FED3 also has a programmable output that can control other equipment , for example to trigger optogenetic stimulation after a nose-poke or pellet removal , or to synchronize feeding behavior with electrophysiological or fiber-photometry recordings , or with in vivo calcium imaging .\",\n \"Finally , FED3 is open source and was designed to be customized and re-programmed to perform novel tasks to help researchers understand food intake and food motivation .\",\n \"To this end , we have written a user-friendly Arduino library to facilitate custom behavioral programming of FED3 .\",\n \"Here , we describe the design and construction of FED3 and present several experiments that demonstrate its functionality .\",\n \"These include measuring circadian patterns of food intake over multiple days , performing meal pattern analysis , automated operant training , closed-economy motivational testing , and optogenetic self-stimulation .\",\n \"FED3 extends existing methods for quantifying food intake and operant behavior in rodents to help researchers achieve a deeper understanding of feeding and feeding disorders .\"\n ],\n [\n \"FED3 is controlled by a 48 mHz ATSAMD21G18 ARM Cortex M0 microprocessor ( Adafruit Feather M0 Adalogger ) and contains two nose-pokes , a pellet dispenser , a speaker for auditory stimuli , eight multi-color LEDs for visual stimuli , a screen for experimenter feedback , and a programmable analog output ( Figure 1A , B ) .\",\n \"When mice interact with FED3 , the timing of each poke and pellet removal are logged to an internal microSD card for later analysis and summary data is displayed on the screen for immediate feedback to the researcher ( Figure 1B ) .\",\n \"FED3 is small ( ~10 cm × 12 cm × 9 cm ) , battery powered , and completely self-contained , so it fits in most vivarium home-cages without modification or introducing wiring to the cage ( Figure 1C ) .\",\n \"It is powered by a rechargeable battery that lasts ~1 week between charges ( exact battery life depends on the behavioral program being run and number of pellets dispensed ) .\",\n \"FED3 also has magnetic mounts to facilitate wall-mounting on any plastic box to mimic a traditional operant setup ( see Video 1 ) .\",\n \"We provide example programs for running rodents on multiple common behavioral paradigms , including free feeding , time-restricted free-feeding , fixed-ratio 1 ( FR1 ) , and progressive ratio ( PR ) operant tasks , and have written a FED3 Arduino library to ease development of custom programs .\",\n \"FED3 also has an auxiliary I2C port for hardware customization and a programmable BNC output connection that allows synchronization with external equipment for aligning behavioral events with fiber-photometry recordings ( London et al . , 2018; Mazzone et al . , 2020 ) , electrophysiological recordings ( London et al . , 2018 ) , or other equipment such as video tracking systems ( Krynitsky et al . , 2020; Li et al . , 2019 ) .\",\n \"This BNC output can report task events or control external hardware with delays of less than 200 µs , which compares favorably with computers that process information through Universal Serial Bus ( USB ) , which can introduce delays of several milliseconds .\",\n \"Finally , we provide a graphical analysis package ( FED3VIZ ) written in Python that enables users to generate detailed plots from FED3 data ( Figure 2F ) .\",\n \"FED3 is open-source and freely available online , including 3D design files , printed circuit board ( PCB ) files ( Figure 1—figure supplement 1A , B ) , build instructions ( Figure 1D ) , and code ( https://github . com/KravitzLabDevices/FED3 copy archived at swh:1:rev:ff8fc79d288a440d91566f4c6cd80011956f4be0 ) .\",\n \"Ten FED3 devices were set to the ‘free-feeding’ program , which dispenses a pellet and monitors its presence in the pellet well ( Figure 2A ) .\",\n \"In this paradigm , each time the pellet is removed , FED3 logs the date and time to the storage card and dispenses another pellet .\",\n \"This allows for the reconstruction of detailed feeding records over multiple days .\",\n \"Assuming calorie content of the pellets is known , total caloric intake can also be tracked over time .\",\n \"Devices were placed with ten singly housed mice for six consecutive days with no additional food source .\",\n \"We observed the expected circadian rhythm in food intake ( Figure 2B , C ) .\",\n \"To test accuracy , we confirmed that the daily change in weight of the FED3 device correlated with the number of pellets removed multiplied by the weight of each pellet ( 20 mg pellets , Figure 2D ) .\",\n \"The coefficient of determination ( R2 ) for the regression was 0 . 97 , indicating an error rate of ~3% between manual weighing and counting of pellets by FED .\",\n \"Finally , we confirmed that the mice obtained all of their necessary daily calories from FED3 , evidenced by a stable body weight across these 6 days ( Figure 2E ) .\",\n \"In other experiments , we have run FED3 for >1 month without observing weight loss .\",\n \"FED3 can be used for multi-week experiments like this as it has a low rate of jamming .\",\n \"This is facilitated by a unique angled pellet hopper and dispensing path that has fewer places where pellets can jam than traditional horizontal pellet dispensers ( Figure 2—figure supplement 1 ) , as well as ‘jam clearing’ movements in the code that vibrate and rotate the dispensing disk to dislodge stuck pellets when they are detected .\",\n \"These are improvements over the first generation of this device ( Nguyen et al . , 2016 ) .\",\n \"As a point on rodent safety , although FED3 is resistant to jamming it still must be checked for function at least once per day when being used as the only food source .\",\n \"FED3 creates a large amount of data , particularly when run over multiple days .\",\n \"To facilitate analysis of FED3 data , we created FED3VIZ , an open-source Python-based analysis program ( https://github . com/earnestt1234/FED3_Viz ) .\",\n \"FED3VIZ offers plots for visualizing different aspects of FED3 data , including pellet retrieval , poke accuracy , pellet retrieval time , delay between consecutive pellet earnings ( inter pellet intervals ) , meal size , and progressive ratio breakpoint .\",\n \"These metrics can be plotted for single files or group averages .\",\n \"FED3VIZ’s averaging methods provide options for aggregating data recorded on different days , while preserving time-of day or phase of the light–dark cycle .\",\n \"Furthermore , circadian activity patterns can also be visualized with the FED3VIZ ‘Chronogram’ and ‘Day/Night Plot’ functions ( see Figure 7 ) , which segment data based on a user-defined light cycle .\",\n \"The processed data going into each plot can also be saved and used to create new visualizations or compute statistics in other programs , promoting reproducible , sharable analysis and visualization of FED3 data .\",\n \"FED3 records the date and time that each pellet is removed , which can be used to quantify feeding patterns including the size and quantity of meals , eating rate within meals , and timing between meals .\",\n \"Different parameters have been used by different groups to define meals , often based on numerical cut-offs ( Farley et al . , 2003; Kanoski et al . , 2013; Melhorn et al . , 2010 ) .\",\n \"To complement these approaches , we aimed to develop an unbiased approach for understanding meal patterns , based on the distribution of time intervals between each pellet consumed .\",\n \"For the 10 mice in the above experiment , we plotted the inter-pellet time interval histogram ( Figure 3A ) for both the dark and light cycles , based on an approach that was previously described in rats ( Cottone et al . , 2007 ) .\",\n \"We observed a large peak at <1 min between pellets , showing that the majority of pellets were consumed within 1 min of another pellet .\",\n \"We used this distribution to classify pellets eaten within the same minute as belonging to the same meal , and pellets with larger intervals between them belonging to different meals .\",\n \"This approach revealed that animals eat fewer meals during the light cycle ( Figure 3B ) but eat similar number of pellets within each meal and obtain a similar fraction of their pellets within meals in each cycle .\",\n \"Differences in meal patterning have been linked to obesity ( Farley et al . , 2003; Wald and Grill , 2019 ) , so this analytical approach may assist in understanding obesity and other disorders of feeding .\",\n \"A unique feature of FED3 is that it is small and wire-free , allowing it to fit inside of traditional vivarium home-cages .\",\n \"This facilitates quantification of circadian rhythms in both operant and feeding behavior ( Figure 4A ) .\",\n \"To demonstrate operant capability , we quantified how nose-poking for pellets varied over the circadian cycle , by running mice on a FR1 task for six consecutive days .\",\n \"Mice learned to nose-poke on the active port for a single pellet .\",\n \"We tracked active ( rewarded ) and inactive ( unrewarded ) pokes .\",\n \"As expected , both active and inactive pokes were higher during the dark cycle ( Figure 4B–D ) .\",\n \"Surprisingly , however , poke efficiency ( % of total pokes on the active port ) was slightly higher during the light cycle ( Figure 4E ) , suggesting that operant responding is more efficient during the light cycle .\",\n \"Multiple research groups currently use FED3 .\",\n \"We therefore asked how mouse operant learning varies across different laboratories .\",\n \"To do this , we obtained operant data from the first overnight FR1 session from seven laboratories , resulting in data from 122 mice ( mix of males and females , Figure 5A ) .\",\n \"When comparing pellets earned in an overnight session across laboratories , we observed a significant effect of group , linked to significant differences in 4 of 21 post hoc comparisons ( Figure 5B ) .\",\n \"When viewed as a single distribution ( Figure 5B inset ) , the distribution of pellets earned from all groups was consistent with a single Gaussian distribution ( p>0 . 05 , Shapiro-Wilk test for normality ) .\",\n \"This highlights how FED3 enables high-throughput studies of operant behavior , and the power of large sample sizes to achieve adequate sampling of a distribution of behavior ( Figure 5B ) .\",\n \"A unique capability of FED3 is that it has a sensor for detecting the presence of the pellet in the feeding well .\",\n \"This enables time-stamping of when each pellet was removed for constructing feeding records , as well as measuring the time between the pellet dispense and removal , which we termed ‘retrieval time’ .\",\n \"Changes in retrieval time may reflect learning , as this measure progressively decreased as animals gained experience with FED3 ( Figure 5E ) .\",\n \"Accordingly , active active count and poke efficiency ( % of pokes on the active port ) increased over time , demonstrating that retrieval time decreased on the same time scale that mice learned which poke resulted in a pellet ( Figure 5C ) .\",\n \"To compare learning rates in overnight training to traditional daily operant sessions , we ran a new group of eleven mice for sixteen daily 1 hr FR1 sessions with FED3 , returning them to the colony for the remainder of each day .\",\n \"Learning rates did not differ between mice exposed to 16 daily 1 hr sessions or one 16 hr overnight session ( Figure 5F ) .\",\n \"This suggests that the acquisition of this FR1 task may depend most strongly on the cumulative time mice interact with FED3 and that overnight training can greatly speed up task acquisition by giving them many hours of experience in one night .\",\n \"Based on prior literature ( Steinhauer et al . , 1976 ) , we predicted that the speed of acquisition of an FR1 task would be further increased by prior magazine training .\",\n \"To test this , we exposed 24 mice to magazine training using the free feeding FED3 paradigm for 1–3 days prior to FR1 training ( Figure 6A ) .\",\n \"This paradigm dispenses a pellet into the feeding well of FED3 and replaces it whenever it is removed , providing an automatic method for magazine training animals .\",\n \"In this way , magazine training allows mice to learn the location of the food source and associate food with the sound of the pellet dispenser operating and the pellet being dispensed , before commencing with FR1 training .\",\n \"We compared the performance of the 24 magazine trained mice to 122 mice from Figure 5 ( which had not been magazine trained ) and found that magazine training resulted in significantly higher ( ~2 ) levels of pellet acquisition in the first night with the FR1 task ( Figure 6B , C ) .\",\n \"Therefore , magazine training is recommended to speed up acquisition of nose-poking tasks .\",\n \"FED3 can also be used for closed-economy feeding studies , in which all food is earned by operant nose-poking ( Figure 7 ) .\",\n \"Closed-economy operant tasks have been used to quantify the economic demand for food ( Bauman , 1991; Chaney and Rowland , 2008 ) , as well as changes in economic demand due to manipulation of the dopamine system ( Beeler et al . , 2010; Mourra et al . , 2020 ) .\",\n \"We tested seven mice on a repeating progressive ratio task ( adapted from Mourra et al . , 2020 ) , in which the nose-poking requirement began on FR1 and increased each time a pellet was earned .\",\n \"When mice refrained from poking on either port for 30 min the ratio reset to FR1 ( Figure 7A , B ) .\",\n \"Although pellets earned from this task were their only food source , mouse weights remained stable across days , demonstrating that mice earned their entire caloric need with the task ( Figure 7C ) .\",\n \"Total pellets earned did not differ between light and dark cycles ( Figure 7D ) .\",\n \"However , nose-poking rates were higher during the dark phase of their daily cycle ( Figure 7E ) , meaning mice performed more pokes to earn each pellet in this phase ( Figure 7F ) .\",\n \"This suggests that motivation for food pellets varies over the circadian cycle such that mice are willing to work harder during the dark cycle vs . the light cycle .\",\n \"When the data was visualized hourly across the circadian cycle , we further noted a ~3 hr window of enhanced nose-poking rates starting shortly after the onset of the dark cycle ( Figure 7F ) .\",\n \"This time coincides with a circadian peak in extracellular dopamine levels in the striatum ( Ferris et al . , 2014 ) , and a peak in activity levels in mice ( Matikainen-Ankney et al . , 2020 ) .\",\n \"Therefore , this task may be used to assay changes in accumbal dopamine function across the circadian cycle .\",\n \"FED3 was also designed to synchronize with other experimental equipment through its programmable output port .\",\n \"To demonstrate this capability , we programmed FED3 to act as a function generator and control an LED for an intra-cranial self-stimulation task .\",\n \"The dorsal striatum contains two main classes of output neurons , differentiated by their expression of the dopamine D1 or D2 receptors ( Gerfen et al . , 1990 ) .\",\n \"Dorsal striatal neurons that express the D1 receptor are highly reinforcing when optogenetically stimulated ( Kravitz et al . , 2012 ) .\",\n \"To demonstrate how FED3 can be used to perform an optogenetic self-stimulation experiment , we used Cre-dependent viral expression to target channelrhodopsin-2 ( ChR2 ) to D1R-expressing neurons in the dorsal striatum ( Figure 8A ) .\",\n \"FED3 was programmed to generate and trigger a 1 s train of 20 Hz pulses of 475 nm light upon each active nose-poke , and the session continued until they received 75 trains of stimulation ( Figure 8B ) .\",\n \"Three mice were run on this task , resulting in significantly greater active vs inactive pokes ( Figure 8C , D ) , and demonstrating that FED3 can be used for optogenetic self-stimulation studies .\",\n \"The programmable output is attached to an on-board digital-to-analog converter chip , meaning that it can be programmed to output pulses of any voltage from 0 to 3 V , allowing FED3 to represent multiple behavioral events with one output line .\",\n \"The timing of the BNC output port is regulated by the SAMD21 microprocessor that has a documented pulsing precision of <1 µs .\"\n ],\n [\n \"Quantification of food intake is necessary to understand animal models of feeding disorders , as well as other medical conditions .\",\n \"To quantify food intake in rodent home-cages , we previously published the FED version 1 , an open-source , stand-alone feeding device that can fit in a rack-mounted home-cage ( Nguyen et al . , 2016 ) .\",\n \"FED records a time series of pellets removed , which can be used to reconstruct feeding records .\",\n \"FED has been used by multiple research groups to understand feeding ( Brierley et al . , 2020; Burnett et al . , 2019; Chen et al . , 2020; Li et al . , 2019; London et al . , 2018 ) .\",\n \"Since its original publication , we have redesigned the FED twice and here present the third version of FED , which revamps our original design and adds the ability to measure operant behavior , as well as a unique ‘angled’ pellet dispenser mechanism that is resistant to jamming , which enables long term studies .\",\n \"We distributed the design for FED3 online , and it has been used to study how specific neural circuit manipulations alter food motivation ( Mazzone et al . , 2020; Sciolino et al . , 2019; Vachez et al . , 2021 ) , how weight-loss alters food motivation ( Matikainen-Ankney et al . , 2020 ) , and how food motivation is altered in a stress-susceptible mouse population ( Rodriguez et al . , 2020 ) .\",\n \"FED3 is a stand-alone solution for home-cage operant training , enabling researchers to understand not just total food intake , but also motivation for and learning about food rewards .\",\n \"FED3 has several unique benefits over comparable systems including: ( 1 ) FED3 is low cost .\",\n \"The FED3 electronics cost ~$200 and the housing is 3D printed .\",\n \"Even if 3D printing is done professionally this is >10× cheaper than most commercial solutions for measuring food intake or testing operant behavior; ( 2 ) FED3 is self-contained and fits within traditional vivarium caging , allowing for measures of true ‘home-cage’ feeding without modifying the cage .\",\n \"Due to its small size , it can also be placed inside of other equipment where wires might be impractical , such as within an indirect calorimetry system; ( 3 ) FED3 has a programmable output that allows it to easily synchronize with other equipment .\",\n \"Due to wiring , connecting FED3 to external hardware likely requires FED3 to be used outside of the home-cage , or to modify the home-cage system .\",\n \"This has been done by multiple labs to synchronize the output of FED3 with fiber-photometry recordings ( London et al . , 2018; Mazzone et al . , 2020 ) , electrophysiological recordings ( London et al . , 2018 ) , optogenetic stimulation ( Vachez et al . , 2021 ) , and video tracking ( Krynitsky et al . , 2020; Li et al . , 2019 ) ; and ( 4 ) FED3 is open source and all design files and codes are freely available online .\",\n \"This enables users to modify the code and hardware to achieve new functionality .\",\n \"The purpose of this manuscript is to demonstrate the utility of FED3 for feeding research .\",\n \"To this end , we demonstrate experiments that measured total food intake , operant responding , and optogenetic stimulation .\",\n \"We further highlight how the high temporal resolution enables meal pattern analysis across multiple days .\",\n \"Finally , we coordinated with six other research groups to compile a dataset of 122 mice across seven research sites , all running the same experimental FR1 program .\",\n \"We observed similar patterns of acquisition across all sites , demonstrating that FED3 can be used for multi-site research studies on feeding .\",\n \"Due to its low cost and open-source nature , we believe that multi-site studies with FED3 are more feasible than with other systems .\",\n \"While FED3 has many strengths , it also has limitations .\",\n \"One limitation is that FED3 uses internal microSD cards to store data .\",\n \"While microSD cards are convenient , they are not ideal for large numbers of devices , where removing multiple cards can be cumbersome .\",\n \"Wireless data logging is a potential solution to this problem , although there are challenges to implementing this in rodent cages .\",\n \"A second limitation is that animals can ‘hoard’ pellets from FED3 , as animals can remove food without consuming it .\",\n \"In our experience , this seemed to be a rare trait that specific mice engaged in ( <10% of mice hoard pellets ) .\",\n \"Unfortunately , FED3 has no way to determine whether a mouse actually consumes each pellet after removal so we recommend checking for pellet hoarding and accounting for this in experimental conclusions .\",\n \"A third limitation is that granular bedding can be kicked into the pellet well and interfere with pellet detection .\",\n \"To avoid this , we recommend using ‘iso-pad’ bedding , or bedding pellets that are large enough to avoid this possibility .\",\n \"A fourth limitation is that FED3 is not waterproof and would not be resistant to flooding , though the 3D printed housing is sufficient to protect electronics from typical wear associated with placement in a rodent cage .\",\n \"A final limitation of FED3 is that it has no way of identifying individual mice in group housed environments , so feeding records must be collected in singly housed mice .\",\n \"However , FED3 is open source , so it can be modified and improved with innovations from our group and the feeding community , and future iterations of the device may include methods for identifying multiple mice using radio-frequency identification ( RFID ) tags .\",\n \"Being able to run studies on group housed mice would greatly increase throughput and allow for the study of interactions between social behavior and feeding .\",\n \"We published the FED3 design as open-source and look forward to community contributions and modifications to enable new functionality and overcome these limitations .\"\n ],\n [\n \"The FED3 device is open-source , and design files and code are freely available online at: https://github . com/KravitzLabDevices/FED3 .\",\n \"In addition , we have made all data and analysis code for the figures in this paper available at https://osf . io/hwxgv/ .\",\n \"One hundred and fifty-nine C57Bl/6 mice were housed in a 12 hr light/dark cycle with ad libitum access to food and water except where described .\",\n \"Mice were provided laboratory chow diet ( 5001 Rodent Diet; Lab Supply , Fort Worth , TX ) .\",\n \"All procedures were approved by the Animal Care and Use Committee at Washington University in St Louis , the National Institutes of Health , Williams College , Virginia Tech , and Monash University .\",\n \"Tutorial videos and other information on assembling FED3 are available at: https://github . com/KravitzLabDevices/FED3/wiki .\",\n \"We demonstrate multiple operational modes for FED3 to highlight a range of functionality and applications .\",\n \"The following programs are included with the standard FED3 code: Viral infections of male and female Drd1-Cre mice were conducted under 0 . 5–2 . 5% anesthesia on a stereotaxic apparatus .\",\n \"Using a Nanoject injector , 500 nL of AAV2-DIO-hSyn-ChR2 was injected bilaterally ( 500 nL/hemisphere ) in the dorsomedial striatum .\",\n \"Optical fibers were then implanted in the same region .\",\n \"After letting ChR2 express for 4 weeks , animals were pre-trained on an FR1 schedule for pellets overnight in their home-cage .\",\n \"Two days later , mice were placed in a box with a FED3 device connected to an LED driver .\",\n \"The active poke for this paradigm was on the opposite side to the active poke of the pre-training session .\",\n \"During the self-stimulation session , when the mouse poked on the active nose-poke , it received a 1 s train of 1 mW 475 nm light at 20 Hz .\",\n \"Sessions were run for 60 min in a 550 cm2 arena .\",\n \"CSV files generated by FED3 were processed and plotted with custom python scripts ( Python , version 3 . 6 . 7 , Python Software Foundation , Wilmington , DE ) .\",\n \"All data and scripts are available on Open Science Framework ( https://osf . io/hwxgv/ ) .\",\n \"Visualization was also completed using FED3VIZ GUI to generate plots .\",\n \"FED3VIZ was written in Python’s standard library for developing GUIs ( tkinter ) .\",\n \"FED3VIZ is a custom open-source graphical program for analyzing FED3 data .\",\n \"FED3VIZ code , version history , installation instructions , and user manual are available on GitHub ( https://github . com/earnestt1234/FED3_Viz ) .\",\n \"FED3VIZ offers plotting and data output for visualizing different aspects of FED3 data , including pellet retrieval , poke accuracy , pellet retrieval time , delay between consecutive pellet earnings ( inter-pellet intervals ) , meal size , and progressive ratio breakpoint .\",\n \"Based on inter-pellet interval histograms ( Figure 3 ) , we defined meals as pellets eaten within 1 min of each other .\",\n \"In addition , we defined a minimum size of 0 . 1 g ( five pellets ) to be counted as a meal .\",\n \"Bartlett’s test for equal variances was performed; one- or two-way ANOVAs with Tukey post-tests were used to compare groups with equal variance ( p>0 . 05 ) where appropriate .\",\n \"Linear mixed effects models were used to analyze groups with significantly difference variances ( p<0 . 05 ) .\",\n \"p-value<0 . 05 was considered significant .\",\n \"Statistical tests to compare means were run using statsmodels module in python ( Seabold and Perktold , 2010 ) .\",\n \"Data sets are presented as mean ± SEM .\",\n \"Numbers of animals per experiment is listed as n=number of animals .\",\n \"Linear regression was used to determine correlative relationships .\",\n \"T-tests or Mann–Whitney U tests were used to compare the means of two groups for parametric or nonparametric data distributions , respectively .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Feeding is critical for survival , and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa .","It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders .","Operant behavioral testing can be used to measure the motivational component to feeding , but most food intake monitoring systems do not measure operant behavior .","Here , we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 ( FED3 ) .","FED3 measures food intake and operant behavior in rodent home-cages , enabling longitudinal studies of feeding behavior with minimal experimenter intervention .","It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings .","Finally , FED3 design files are open-source and freely available , allowing researchers to modify FED3 to suit their needs ."],"string":"[\n \"Feeding is critical for survival , and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa .\",\n \"It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders .\",\n \"Operant behavioral testing can be used to measure the motivational component to feeding , but most food intake monitoring systems do not measure operant behavior .\",\n \"Here , we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 ( FED3 ) .\",\n \"FED3 measures food intake and operant behavior in rodent home-cages , enabling longitudinal studies of feeding behavior with minimal experimenter intervention .\",\n \"It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings .\",\n \"Finally , FED3 design files are open-source and freely available , allowing researchers to modify FED3 to suit their needs .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Obesity and anorexia nervosa are two health conditions related to food intake .","Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is , why animals seek and consume food .","Measuring an animal’s food intake is usually done by weighing food containers .","However , this can be inaccurate due to the small amount of food that rodents eat .","As for studying feeding motivation , this can involve calculating the number of times an animal presses a lever to receive a food pellet .","These tests are typically conducted in hour-long sessions in temporary testing cages , called operant boxes .","Yet , these tests only measure a brief period of a rodent's life .","In addition , it takes rodents time to adjust to these foreign environments , which can introduce stress and may alter their feeding behaviour .","To address this , Matikainen-Ankney , Earnest , Ali et al . developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention .","This ‘Feeding Experimentation Device’ ( FED3 ) features a pellet dispenser and two ‘nose-poke’ sensors to measure total food intake , as well as motivation for and learning about food rewards .","The battery-powered , wire-free device fits in standard home cages , enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals .","This means researchers can relate data to circadian rhythms and meal patterns , as Matikainen-Ankney did here .","Moreover , the device software is open-source so researchers can customise it to suit their experimental needs .","It can also be programmed to synchronise with other instruments used in animal experiments , or across labs running the same behavioural tasks for multi-site studies .","Used in this way , it could help improve reproducibility and reliability of results from such studies .","In summary , Matikainen-Ankney et al . have presented a new practical solution for studying food-related behaviours in mice and rats .","Not only could the device be useful to researchers , it may also be suitable to use in educational settings such as teaching labs and classrooms ."],"string":"[\n \"Obesity and anorexia nervosa are two health conditions related to food intake .\",\n \"Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is , why animals seek and consume food .\",\n \"Measuring an animal’s food intake is usually done by weighing food containers .\",\n \"However , this can be inaccurate due to the small amount of food that rodents eat .\",\n \"As for studying feeding motivation , this can involve calculating the number of times an animal presses a lever to receive a food pellet .\",\n \"These tests are typically conducted in hour-long sessions in temporary testing cages , called operant boxes .\",\n \"Yet , these tests only measure a brief period of a rodent's life .\",\n \"In addition , it takes rodents time to adjust to these foreign environments , which can introduce stress and may alter their feeding behaviour .\",\n \"To address this , Matikainen-Ankney , Earnest , Ali et al . developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention .\",\n \"This ‘Feeding Experimentation Device’ ( FED3 ) features a pellet dispenser and two ‘nose-poke’ sensors to measure total food intake , as well as motivation for and learning about food rewards .\",\n \"The battery-powered , wire-free device fits in standard home cages , enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals .\",\n \"This means researchers can relate data to circadian rhythms and meal patterns , as Matikainen-Ankney did here .\",\n \"Moreover , the device software is open-source so researchers can customise it to suit their experimental needs .\",\n \"It can also be programmed to synchronise with other instruments used in animal experiments , or across labs running the same behavioural tasks for multi-site studies .\",\n \"Used in this way , it could help improve reproducibility and reliability of results from such studies .\",\n \"In summary , Matikainen-Ankney et al . have presented a new practical solution for studying food-related behaviours in mice and rats .\",\n \"Not only could the device be useful to researchers , it may also be suitable to use in educational settings such as teaching labs and classrooms .\"\n]","isConversational":false},"year":{"kind":"string","value":"2021"}}},{"rowIdx":289,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["developmental biology"],"string":"[\n \"developmental biology\"\n]","isConversational":false},"title":{"kind":"string","value":"A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus"},"id":{"kind":"string","value":"elife-03229-v2"},"sections":{"kind":"list like","value":[["An understanding of ecological interactions and evolutionary history can be beneficial to the understanding of the cellular and developmental processes of an organism .","For example , the genes responsible for fruit and bacterial odor preferences in Drosophila melanogaster and Caenorhabditis elegans are most informative when considering the natural ecologies in rotting fruits and vegetation ( Bargmann et al . , 1993; Bargmann , 2006; Hallem and Carlson , 2006; Kiontke and Sudhaus , 2006 ) .","In Pristionchus pacificus , an emerging model organism for the study of development and behavior ( Hong and Sommer , 2006b ) , a systematic effort to identify the natural ecology of Pristionchus nematodes revealed that P . pacificus host preferences include the oriental beetle ( Exomala orientalis ) found in Japan and northeastern United States ( Herrmann et al . , 2007 ) .","Unlike several fructivorous Caenorhabditis species , Pristionchus species are considered to be necromenic nematodes that have species-specific beetle host preferences and feed on the microorganisms that emerge from the beetle carcass ( Herrmann et al . , 2006a , 2006b; Brown et al . , 2011 ) .","Pristionchus species are members of the Diplogasteridae family of nematodes whose common ancestor with the Rhabditidae family ( e . g . , C . elegans ) diverged ∼300 million years ago ( Dieterich et al . , 2008 ) .","Given that beetles ( Coleoptera ) represent a group with the most described species , the possibility that most beetle species can harbor specific entomophilic nematode associations underscores the vast number of nematode species remaining to be described .","Although biologists have been aware of such species-specific insect–nematode associations for some time , the genetic basis for such interactions remains largely unknown .","Previous studies suggest that the beetle host specificity of Pristionchus species is in part due to nematode attraction to various plant volatile compounds released during beetle feeding , as well as to beetle aggregation and sex pheromones during mating ( Hong and Sommer , 2006a; Hong et al . , 2008b , 2008a ) .","For example , Pristionchus maupasi associates primarily with the European May beetle Melolontha spp .","and is attracted to the blend of the green leaf alcohol from plants and the phenol pheromone from beetles ( Hong et al . , 2008b ) .","Thus , insect and plant odors can synergize to provide nematodes with information on host presence , sex , and maturity .","By contrast , C . elegans attractants such as 2 , 3-butanedione ( diacetyl ) , 2 , 3-pentanedione , and isoamyl alcohol are significantly less attractive to P . pacificus ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .","This piggybacking of intra-species communication for inter-species interactions between Pristionchus and beetles can also evolve rapidly at the population level , as exhibited by the natural variation of P . pacificus odor preference for insect pheromones ( Hong et al . , 2008a; McGaughran et al . , 2013b ) .","In particular , P . pacificus Washington strain ( PS1843 ) is attracted to the lepidopteran sex pheromone E-11-tetradecenyl acetate ( ETDA ) and the oriental beetle sex pheromone z-7-tetradece-2-one ( ZTDO ) ( Herrmann et al . , 2007 ) , but the wild-type reference strain from California ( PS312 ) requires exogenous cGMP treatment to become attracted to these insect pheromones ( Hong et al . , 2008a ) .","These natural variations in insect pheromone attraction , combined with regional-specific P . pacificus host preferences , may offer support for the geographic mosaic theory of co-evolution ( Thompson , 2005 ) .","Although the distinct chemosensory profiles of P . pacificus populations suggest insect pheromone attraction to be an important and rapidly evolving trait , other studies on insect–nematode interactions show that there are other potential roles host chemicals can play in coordinating host–parasite development and nematode dispersal behavior .","For example , the pinewood nematode Bursaphelenchus xylophilus is intimately associated with its Monochamus beetle vector , and fatty acid ethyl esters exuded during beetle eclosion promote the formation of the dispersal fourth larval stage B . xylophilus ( Zhao et al . , 2013 ) .","Similarly , the species-specific phoretic association between the burrower bug Parastrachia japonensis and the nematode Caenorhabditis japonica may be mediated by the beetle cuticular compounds that not only attract C . japonica but also assist infestation by inhibiting C . japonica dispersal ( Okumura et al . , 2013 ) .","P . pacificus populations have been found on various beetle hosts worldwide , but the association with the oriental beetle was the first to be identified and the only one whose sex pheromone is a strong attractant ( Herrmann et al . , 2007; McGaughran et al . , 2013b ) .","Therefore , the oriental beetle sex pheromone represents the single most ecologically relevant semiochemical by which we could repeatedly interrogate the P . pacificus genes involved for host sensing and other unexplored developmental roles .","In this study , we show that although the beetle pheromone , ZTDO , is attractive to P . pacificus adults , the beetle host pheromone may moderate nematode infection by arresting embryonic development , inducing larval paralysis , and inhibiting dauer exit .","We leveraged the ability to use exogenous cGMP to potentiate the California reference strain for ZTDO attraction to find additional genes important for ZTDO sensitivity by performing an unbiased forward genetic screen for mutants with altered responses to ZTDO .","We found that a putative lipid-binding protein Ppa-OBI-1 mediates both beetle pheromone attraction as well as a previously unknown role of the beetle pheromone to regulate P . pacificus development .","Our results suggest that the host pheromone may play a complex role in reducing infection or coordinating development for the dispersal cycle of the necromenic lifestyle ."],["P . pacificus and C . elegans have diametrically opposed odor preference profiles .","Pristionchus species are generally attracted to plant volatiles , insect pheromones , and cuticular hydrocarbons , but each Pristionchus species can have its own chemosensory profile reflective of its preferred beetle hosts ( Hong and Sommer , 2006a; McGaughran et al . , 2013a ) .","P . pacificus populations found on the oriental beetle E . orientalis in Japan and northeastern United States are attracted to the sex pheromone of the beetle , z-7-tetradecen-2-one ( ZTDO ) ( Zhang et al . , 1994; Herrmann et al . , 2007 ) .","In contrast , the compost-dwelling C . elegans is less attracted to or even repelled by plant and insect odors , including ZTDO , but is strongly attracted to small compounds released by microbes ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .","To identify genes required to mediate ZTDO attraction in a pheromone-insensitive reference strain , California PS312 , we performed a behavior-based genetic screen for mutants that are insensitive toward ZTDO following a brief 1-hr treatment with the cell-permeable 8-br-cGMP .","We isolated two Oriental Beetle pheromone Insensitive mutants , Ppa-obi-1 ( tu404 ) and Ppa-obi-3 ( tu405 ) .","The chemosensory phenotype of Ppa-obi-1 and Ppa-obi-3 is strongly specific for ZTDO and does not affect the chemoattraction towards other insect pheromones ETDA and myristate nor plant volatiles β-caryophyllene and nicotine ( Figure 1 ) .","Odor avoidance in obi mutants towards 1-octanol , a repellant common between P . pacificus and C . elegans , also resembles wild type .","These results indicate that both obi mutants are specifically defective in sensing a beetle pheromone rather than more generally defective in chemosensation .","Ppa-obi-1 ( tu404 ) and Ppa-obi-3 ( tu405 ) are non-allelic recessive mutants that complement each other in their physical and behavioral phenotypes .","We proceeded to characterize Ppa-obi-1 ( tu404 ) based on its altered response to ZTDO attraction and its unexpected function in protecting larvae . 10 . 7554/eLife . 03229 . 003Figure 1 . Chemosensory behavior of oriental beetle pheromone insensitive mutants . Ppa-obi-1 and Ppa-obi-3 mutant animals are specifically defective for sensing the host oriental beetle sex pheromone , Z-7-tetradece-2-one .","The ZTDO structure is depicted below the beetle .","In contrast , obi mutants' response is wild type towards another cGMP-dependent attractant , E-11-tetradecenyl acetate ( ETDA , a lepidopteran pheromone ) , as well as myristate ( methyl tetradecanoate , an insect allomone ) , plant volatiles beta-caryophyllene and nicotinic acid , and the repellent 1-octanol .","N ≥ 15 replicates in ≥3 trials .","Significant differences to wild type are indicated by p values ( Dunnett's test ) and error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 003 Necromeny occupies a midpoint in the continuum of nematode–insect interactions that span from mutualism to pathogenicity .","To address possible developmental effects of long-term exposure to ZTDO , we attempted to grow worms in the presence of the ZTDO by placing eggs onto OP50 bacteria in plates containing 10 µl of 0 . 5% ZTDO suspended under the plate lids , which is approximately a third of the amount used in chemotaxis assays .","Surprisingly , very few of the synchronized eggs from wild type and Ppa-obi-1 ( tu404 ) hatched after 36 hr , compared to ∼94% of the untreated eggs ( Figure 2A ) .","ZTDO therefore may function not just as a volatile attractant but also as an antagonistic host-derived developmental regulator . 10 . 7554/eLife . 03229 . 004Figure 2 . The oriental beetle pheromone ZTDO inhibits P . pacificus development and induces paralysis .","( A ) Ppa-obi-1 mutant is hypersensitive to 0 . 10% ZTDO compared to wild type PS312 ( California ) .","However , PS312 is more sensitive to 0 . 5% ZTDO than another strain , PS1843 ( Washington ) .","PS312 ( n = 372 , 82 , 90 , 320 ) ; PS1843 ( n = 309 , 88 , 92 , 237 ) ; Ppa-obi-1 ( n = 332 , 69 , 71 , 389 ) ; C . elegans wild-type N2 ( n = 316 , 63 , 84 , 174 ) .","( B ) Both wild-type PS312 and Ppa-obi-1 mutant J2 larvae are more sensitive to ZTDO than PS1843 .","PS312 ( n = 154 , 115 ) ; PS1843 ( n = 110 , 102 ) ; Ppa-obi-1 ( n = 104 , 100 ) ; C . elegans wild-type N2 ( n = 23 , 73 ) .","( C ) Ppa-obi-1 J4 larvae is uniquely sensitive to ZTDO compared to PS312 ( p < 0 . 0001 ) .","PS312 ( n = 209 , 290 ) ; PS1843 ( n = 102 , 99 ) ; obi-1 ( n = 244 , 237 ) ; C . elegans wild-type N2 ( n = 69 , 109 ) .","( D ) 90 min exposure of a Ppa-obi-1 ( tu404 ) J4 larvae to ethanol control compared to ( E ) 0 . 50% ZTDO resulted in a rigid fully paralyzed posture .","( F ) Adults are not paralyzed by ZTDO .","PS312 ( n = 108 , 113 ) ; PS1843 ( n = 114 , 107 ) ; Ppa-obi-1 ( n = 109 , 109 ) ; C . elegans wild-type N2 ( n = 62 , 72 ) .","( G ) 0 . 01% ZTDO prevents resumption of post-dauer development in approximately 40% of wild-type animals and nearly all Ppa-obi-1 ( tu404 ) mutants .","This ZTDO inhibition of dauer exit is dependent on the cGMP-dependent PKG , Ppa-egl-4 .","PS312 ( n = 384 , 654 , 267 ) ; PS1843 ( n = 195 , 139 , 101 ) ; Ppa-obi-1 ( n = 339 , 343 , 146 ) ; Ppa-egl-4 ( tu374 ) ( n = 123 , 181 , 77 ) ; Ppa-obi-1;Ppa-egl-4 ( n = 132 , 144 , 63 ) ; N2 ( n = 131 , 109 , 115 ) .","( H ) Exposure to 0 . 50% ETDA moth pheromone from Spodoptera littoralis does not paralyze Ppa-obi-1 mutant and wild-type J4 larvae .","PS312 ( n = 40 , 103 ) ; PS1843 ( n = 40 , 64 ) ; Ppa-obi-1 ( n = 40 , 193 ) .","p values indicate Dunnett's multiple comparisons test to wild-type PS312 .","***p < 0 . 001; **p < 0 . 01 indicate difference between ethanol control and ZTDO exposed groups ( pairwise unpaired t test ) .","Error bars are SEM and n is the number of nematodes tested . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 004 Detailed examination revealed that Ppa-obi-1 ( tu404 ) eggs had significantly lower hatching rate in 0 . 10% ZTDO compared to wild-type PS312 .","Interestingly , the Washington strain that has natural cGMP-independent attraction to ZTDO was more resistant to 0 . 25–0 . 50% ZTDO than the ZTDO-insensitive California strain .","Although ZTDO exposure had no noticeable effect on egg laying output by adult hermaphrodites , eggs continuously exposed to ZTDO for 2 days at an early stage remain arrested at the pre-comma globular embryonic stage , before gastrulation .","The embryocidal effect of ZTDO was concentration-dependent and species-specific , since wild-type C . elegans eggs were completely unaffected ( Figure 2A ) .","Ppa-obi-1 ( tu404 ) hypersensitivity to the developmental and paralyzing effects of ZTDO also continued into the subsequent J2 and J4 larval stages ( Figure 2B , C ) .","Both wild-type PS312 and Ppa-obi-1 ( tu404 ) J2 larvae exhibited ZTDO-induced paralysis , while the Washington strain was significantly more resistant to paralysis .","However , by the J4 larval stage , only Ppa-obi-1 ( tu404 ) was sensitive to ZTDO compared to both wild-type P . pacificus strains ( Figure 2D–E ) .","Ppa-obi-1 ( tu404 ) J4 larvae exposed to ZTDO displayed initial agitation , followed by full body paralysis between 90 and 120 min .","In contrast , wild-type C . elegans J4 larvae displayed only minute sensitivity to ZTDO .","To determine if the Ppa-obi-1 ( tu404 ) mutation renders it susceptible to paralysis from other insect pheromones , we also tested the moth pheromone ETDA and found that the J4 larvae of Ppa-obi-1 ( tu404 ) and the wild-type strains were not affected ( Figure 2H ) .","All strains , including Ppa-obi-1 ( tu404 ) , were insensitive to ZTDO-induced paralysis when they reach adulthood ( Figure 2F ) , the dominant stage used in our chemotaxis assays .","Taken together , our findings show that ZTDO is capable of arresting wild-type P . pacificus embryogenesis and severely paralyzing J2 larvae , indicating that ZTDO is a potential host-specific developmental regulator .","We speculate that wild-type Ppa-OBI-1 may serve to protect P . pacificus embryos and reproductive larvae against the volatile anesthetic-like effects of ZTDO from the adult beetle host .","The dauer larva ( DL ) is an alternative , non-reproductive , long-lasting stage in nematodes capable of withstanding starvation , as well as the infective stage in parasitic nematodes ( Lee , 2002 ) .","To explore the possibility that ZTDO also acts to restrain the dispersive stage of P . pacificus development after hatching , we asked if ZTDO can also regulate DL exit in the presence of food using culturing plates containing 0 . 001–0 . 01% ZTDO and bacteria .","Nearly all untreated wild-type P . pacificus DL developed into J4 larvae after 2 days on food , whereas ∼40% of the wild-type worms remained as active DL on 0 . 01% ZTDO with OP50 food that would have otherwise promoted reproductive development ( Figure 2G ) .","However for Ppa-obi-1 DL , this dauer exit defect is exacerbated by ZTDO due to paralysis .","Approximately 80% of the developmentally blocked Ppa-obi-1 ( tu404 ) DL show immobility on 0 . 001% ZTDO , and nearly 95% of the Ppa-obi-1 ( tu404 ) DL became paralyzed on 0 . 01% ZTDO .","Although DL is the primary infective stage in many entomophilic nematodes found on live beetles ( Weller et al . , 2010 ) , we were unable to assess DL's chemotaxis response to ZTDO because of its immobilizing effect on DL .","The negative regulation of DL exit by ZTDO is specific to P . pacificus and barely affected C . elegans even at 0 . 01% ZTDO .","These results show that 0 . 01% ZTDO can block active wild-type P . pacificus DL from progressing to the reproductive stage without causing paralysis , but 0 . 001–0 . 01% ZTDO significantly reduced dauer exit in Ppa-obi-1 DL by primarily inducing paralysis and death .","Since Ppa-obi-1 ( tu404 ) has a defect in DL exit that is further enhanced by exposure to ZTDO , we asked if DL arrest in Ppa-obi-1 ( tu404 ) by ZTDO is similarly dependent on Ppa-EGL-4 function .","Egl-4 mutants are defective in a cGMP-dependent protein kinase in C . elegans , which affect dauer formation as well as chemosensation ( Daniels et al . , 2000; L'Etoile et al . , 2002 ) .","We found that while the Ppa-egl-4 ( tu374 ) loss-of-function mutation did not significantly affect the ability to exit the dauer stage , the Ppa-egl-4 ( tu374 ) mutation in the Ppa-obi-1 ( tu404 ) background did partially suppress the inhibitory effect of ZTDO , such that Ppa-egl-4;Ppa-obi-1 post-dauer development on 0 . 001% ZTDO is higher than Ppa-obi-1 ( tu404 ) ( Figure 2G ) .","The Ppa-egl-4 ( tu374 ) mutation did not , however , reduce the ZTDO inhibition of DL exit in the presence of a higher amount of 0 . 01% ZTDO .","This outcome suggests that the susceptibility of Ppa-obi-1 ( tu404 ) DL to ZTDO is partially dependent on Ppa-egl-4 .","While the loss of Ppa-obi-1 alone can cause reduced rate of DL exit on food independent of ZTDO , the aggravated DL arrest by ZTDO in Ppa-obi-1 ( tu404 ) is partially mediated by Ppa-egl-4 .","Although wild-type P . pacificus adult animals are attracted to ZTDO , the host beetle sex pheromone regulates P . pacificus development by inhibiting embryogenesis , restricting DL exit , and inducing paralysis in larval stages .","This ZTDO sensitivity is the most acute at the embryonic stage and is likely to be buffered by wild-type Ppa-OBI-1 in later developmental stages as the strongest hypersensitivity to ZTDO was observed in the J4 stage of the Ppa-obi-1 ( tu404 ) mutant .","By contrast , the PS1843 Washington strain that is naturally attracted to ZTDO is more resistant to ZTDO in non-dauer stages compared to the PS312 California wild type .","Further studies in other natural populations are needed to determine the correlation between ZTDO attraction and resistance to ZTDO-induced paralysis in P . pacificus .","In addition to the specific chemosensory defect in Ppa-obi-1 ( tu404 ) mutants , we also noticed a linked phenotype in its body morphology .","Detailed measurements indicate that Ppa-obi-1 ( tu404 ) animals are significantly longer and thinner than wild type ( Figure 3 ) , resulting in a slim body phenotype with adult Ppa-obi-1 ( tu404 ) hermaphrodites ∼20% longer than wild-type animals .","The slim body phenotype may also contribute to Ppa-obi-1 ( tu404 ) 's moderate egg-laying defect ( egl; Table 1 ) and reduction in hatching rate similar to Ppa-egl-4 ( tu374 ) mutants , possibly due to defects in vulva formation and cuticle development .","Using molecular markers and the slim phenotype , we were able to delineate the Ppa-obi-1 lesion to an ∼138 kb region on Supercontig 1 of chromosome I between markers SSLP21 and SSLP17 ( Figure 4A ) .","We subsequently identified all polymorphic sites between PS312 and Ppa-obi-1 ( tu404 ) in the fine mapping interval by Illumina whole genome sequencing and focused on disruptions of predicted open reading frames .","We found a single base pair deletion in a predicted coding region orthologous to C06G1 . 1 in C . elegans and confirmed this tu404 deletion by PCR amplification and Sanger sequencing of both wild type and Ppa-obi-1 ( tu404 ) genomic and cDNA .","The tu404 allele contains a single base pair deletion in exon 9 .","The resulting frame shift and nonsense mutation are predicted to stop translation half way into the reading frame to result in a hypomorphic allele .","In the two Ppa-obi-1 splice forms , the longer transcript contains a VKDDDPR peptide that may be the result of an alternative splice donor site .","Both splice variants were isolated from the California and Washington strains in equal abundance by sub-cloning RT-PCR products ( data not shown ) , and no differences in nucleotide sequence were found in the coding region of these two strains .","Therefore , splice form variation in Ppa-OBI-1 is unlikely to contribute to the difference in insect pheromone attraction between these two strains .","The region encoding for VKDDDPR is also absent in all the Ppa-obi-1 orthologs we have examined in available databases ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 03229 . 005Figure 3 . Ppa-obi-1 mutants are long and slim .","( A and B )","Wild-type J4 larva and young adult .","( C and D )","Ppa-obi-1 J4 larva and young adult .","( E and F )","The body lengths and widths of wild-type ( dotted red line ) versus Ppa-obi-1 mutants ( solid black line ) recorded over 4 days post J4 stage ( μm ) ; n = 15–30 animals for each time point .","Error bars are SEM .","Scale bars denote 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00510 . 7554/eLife . 03229 . 006Table 1 . Ppa-obi-1 affects hatching rate and brood sizeDOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 006Hatching rate ( % ) Brood size ( 3 days ) nWild-type ( PS312 ) 95 ± 3156 ± 1618Ppa-obi-1 ( tu404 ) 85 ± 3103 ± 171510 . 7554/eLife . 03229 . 007Figure 4 . Ppa-obi-1 gene structure and protein comparisons .","( A ) The order of mapping markers is depicted on top ( not to scale ) .","The Ppa-obi-1 mRNA is SL1 trans-spliced and contains 19 exons spanning ∼19 kb .","The 50 bp 5′ UTR and 341 bp 3′ UTR are indicated in light blue .","The tu404 allele contains a single base pair deletion in exon 9 that results in a frameshift , converting a codon to a stop codon ( inverted triangles ) .","The red dot represents the splice variant .","( B ) A diagram of the secretion and two BPI lipid-binding domains ( PFAM and SMART ) .","( C–E )","Transgenic rescue of the Ppa-obi-1 ( tu404 ) .","( C ) A 4 . 3 kb promoter driving Ppa-obi-1 cDNA completely rescued cGMP-dependent ZTDO attraction in Ppa-obi-1 ( tu404 ) mutants .","PS312 ( N = 21 ) ; obi-1 ( N = 15 ) ; obi-1 rescue ( N = 12 ) .","The Ppa-obi-1 transgenic rescue ( D ) restored the body length defect in Ppa-obi-1 ( tu404 ) mutants , PS312 ( n = 15 ) ; obi-1 ( n = 15 ) ; obi-1 rescue ( n = 19 ) as well as ( E ) significantly reduced ZTDO-induced paralysis in Ppa-obi-1 ( tu404 ) J4 larvae .","PS312 ( N = 11 ) ; obi-1 rescue rfp+ ( N = 12 ) ; obi-1 rescue rfp− ( N = 5 ) .","**p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 ( Tukey's multiple comparisons test ) .","Error bars represent SEM .","N is the number of assays performed and n is the number of individual worms scored for each time point . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00710 . 7554/eLife . 03229 . 008Figure 4—figure supplement 1 . An alignment of putative Ppa-OBI-1 protein orthologs . Caenorhabditis elegans ( C06G1 . 1 ) , C . briggsae ( CBP03891 ) , C . remanei ( RP35181 ) , C . japonica ( JA26805 ) , Brugia malayi ( BM32564 ) , and a human Bactericidal Permeability Increasing protein ( NP_001716 ) .","The predicted protein secretion signal peptides for Ppa-OBI-1 ( PP33427 ) and C06G1 . 1 ( CE27054 ) are 17–18 amino acids long at the N terminus ( red bar ) .","The longest predicted protein splice forms are used in the alignment , with orthologs in Caenorhabditis species containing ‘GS’ ( red boxed ) and Ppa-OBI-1 in P . pacificus containing ‘VKDDDPR’ peptides ( red boxed with a red dot ) .","The single base pair deletion in Ppa-obi-1 ( tu404 ) converts the leucine codon to a stop codon ( inverted red triangle ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00810 . 7554/eLife . 03229 . 009Figure 4—figure supplement 2 . A maximum likelihood relationship of lipid-binding proteins in P . pacificus and C . elegans . The ‘PP’ and ‘CE’ prefixes denote P . pacificus and C . elegans proteins , respectively ( www . wormbase . org ) .","10 orthologous protein pairs are circled .","Ppa-OBI-1 ( PP33427 ) and the 12 other paralogs are also represented in the P . pacificus proteome ( Pristionchus . org ) .","A human BPI protein ( GenBank NP_001716 . 2 ) was used as outgroup .","Bootstrap values are indicated along branches . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00910 . 7554/eLife . 03229 . 010Figure 4—figure supplement 3 . Chemosensory behavior of C . elegans mutants C06G1 . 1 and nrf-5 . A mutation in C06G1 . 1 ( vc20357 ) , the ortholog of Ppa-obi-1 , does not alter chemotaxis behavior towards 10% ZTDO , 1% 2 , 3-pentanedione , 1% 2 , 3-butanedione ( diacetyl ) , and 1% isoamyl alcohol .","However , nrf-5 ( sa513 ) mutants have reduced attraction to 1% 2 , 3-butanedione and 1% isoamyl alcohol .","nrf-5 ( sa513 ) was not tested for response to 2 , 3-pentanedione .","Significant differences to wild-type are indicated by p values ( two sampled t test ) .","N ≥ 10 replicates .","Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01010 . 7554/eLife . 03229 . 011Figure 4—figure supplement 4 . Reduction of C06G1 . 1 function produce a slimmer body phenotype .","( A ) RNAi using two dsRNA fragments of C06G1 . 1 fed to C . elegans rrf-3 ( pk1426 ) resulted in a longer and thinner body proportion .","( B ) Genetic mutation in C06G1 . 1 ( VC20357 ) resulted in significantly slimmer body proportion ( increase length/width ) similar to RNAi against C06G1 . 1 and the Ppa-obi-1 ( tu404 ) mutant .","n ≥ 13 animals .","Note that y-axes do not begin at zero .","Significant differences to wild-type are indicated by p values ( two sampled t-test ) .","Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 011 To confirm that Ppa-obi-1 is indeed responsible for the lack of ZTDO attraction in adults and for hypersensitivity to ZTDO-induced paralysis in the J4 larvae , we made transgenic lines containing a 4 . 3 kb Ppa-obi-1 promoter driving the Ppa-obi-1 full-length cDNA ( Schlager et al . , 2009 ) .","The introduction of the wild-type Ppa-obi-1 transgene restored the cGMP-dependent ZTDO attraction in Ppa-obi-1 ( tu404 ) adults compared to Ppa-obi-1 ( tu404 ) adults and sibling animals that did not segregate for the rescue construct ( Figure 4C ) .","Furthermore , the Ppa-obi-1 rescue transgene significantly shortened the body length of Ppa-obi-1 ( tu404 ) adults ( Figure 4D ) , confirming that both the ZTDO chemosensory and the long body length phenotype used for subsequent positional mapping were due to the same mutation in Ppa-obi-1 ( tu404 ) .","Lastly , we tested the ability of the wild-type Ppa-obi-1 transgene to reduce the ZTDO hypersensitivity and found that it significantly reduced the percentage of ZTDO-induced paralysis in Ppa-obi-1 ( tu404 ) J4 larvae ( Figure 4E ) .","Ppa-obi-1 therefore mediates ZTDO chemosensation , body dimensions , and ZTDO susceptibility .","Sequence analyses of coding regions show that Ppa-obi-1 and C06G1 . 1 encode for proteins that represent a class of highly conserved but functionally diverse group of secreted lipid-transfer/lipid-binding proteins present in almost all metazoans except Drosophila ( Beamer et al . , 1998 ) , including lipopolysaccharide-binding proteins ( LBP ) ( Tobias et al . , 1997 ) , bactericidal permeability-increasing proteins ( BPI ) ( Weiss et al . , 1978 ) , phospholipid transfer proteins ( PLTP ) ( Gautier and Lagrost , 2011 ) , and cholesteryl ester transfer proteins in mammals ( CETP ) ( Kawano et al . , 2000 ) .","The LBP/BPI/CETP/PTLP family in humans is known to play a role in immunological responses to bacterial pathogens and mediate the transfer of cholesteryl ester and triglycerides in the blood plasma ( Bruce et al . , 1998; Kawano et al . , 2000; Schultz and Weiss , 2007 ) .","Ppa-OBI-1 and C06G1 . 1 each contain two predicted lipid-binding domains as well as a predicted secretion peptide sequence in the N-terminal regions ( PFAM and SMART; SignalP 4 . 0 Server ) ( Figure 4—figure supplement 1 ) .","To identify all members of the LBP family in the sequenced genome and transcriptome of P . pacificus , we performed reciprocal BLASTP searches ( WormBase WS221 ) , which indicated 12 members predicted to be encoded in the P . pacificus genome .","Maximum likelihood phylogenetic analysis of the OBI-1/C06G1 . 1 LBP protein family indicates 10 closely related orthologous pairs in the two species .","Interestingly , the lengths of the branches suggest that all the orthologs between P . pacificus and C . elegans share similar divergence times , with the exception of the OBI-1/C06G1 . 1 pair that seemed to have undergone faster divergence relative to other paralogs ( Figure 4—figure supplement 2 ) .","Consistent with their roles in immune response in mammals , a member of the LBP family in C . elegans , F44A2 . 3 , was found to be up-regulated in response to several species of pathogenic bacteria ( Wong et al . , 2007 ) .","Another C . elegans LBP member , NRF-5 , is partially necessary for nose contraction in response to the anti-depressant fluoxetine ( Prozac ) ( Choy et al . , 2006 ) , and for mediating phosphatidylserine transfer from apoptotic cells to engulfing cells during cell corpse removal ( Zhang et al . , 2012 ) .","We found that nrf-5 ( sa513 ) mutants are also defective in attraction to 2 , 3-butanedione ( diacetyl ) , and isoamyl alcohol , although the C06G1 . 1 ( vc20357 ) allele with a missense mutation did not show chemosensory defects ( Figure 4—figure supplement 3 ) .","Thus LBP members may have a conserved previously uncharacterized role in chemosensation in diverse nematodes .","To determine if C06G1 . 1 can also regulate body morphology in C . elegans , we performed C06G1 . 1 RNAi by feeding .","C06G1 . 1 RNAi was previously observed to generate a long body phenotype in a large-scale RNAi screen in C . elegans ( Simmer et al . , 2003 ) .","We used this fragment ( Fragment 1 ) , along with another C06G1 . 1 fragment more proximal to the 3′ end ( Fragment 2 ) , to quantify the effect of C06G1 . 1 RNAi in the enhanced RNAi rrf-3 ( pk1426 ) strain .","Although C06G1 . 1 RNAi did not significantly change the absolute body length of adult worms , we found RNAi against C06G1 . 1 using both fragments resulted in a significantly slimmer body proportion ( length/width ratio ) similar to the Ppa-obi-1 ( tu404 ) mutant ( Figure 4—figure supplement 4 ) .","A missense mutation in C06G1 . 1 ( VC20357 ) also resulted in a similar slim body phenotype .","Thus both Ppa-OBI-1 and its C . elegans ortholog also share a conserved function in regulating body proportions .","To characterize which tissue types Ppa-OBI-1 functions in , we sought to determine the Ppa-obi-1 expression pattern using a Ppa-obi-1p::gfp transcriptional reporter gene containing a 4 . 3 kb promoter fragment upstream of the Ppa-obi-1 ATG initiation codon .","Our analysis was based on a stably integrated transgenic line csuIs01 and parsimonious inferences to homologous C . elegans cell positions and known cellular functions .","Detailed analyses of all developmental stages show that the earliest Ppa-obi-1 expression occurs in at least one cell in the pre-comma embryonic stage but soon disappears until weak expression reappears in the hyp7 and seam cells in J4 larvae ( Figure 5 ) .","The most robust and diverse Ppa-obi-1 expression was found in mid-J4 hermaphrodites , which is consistent with real-time quantitative PCR results indicating that transcript levels in J2 and J3 larvae are less than in J4 larvae ( data not shown ) .","In the mid-J4 stage , Ppa-obi-1 is strongly expressed in the major hypodermal syncytium and the lateral hypodermal cells that cover most of the body ( hyp7 and seam cells ) as well as the ventral tail ( hyp 8 and 9 ) ( Figure 5 ) .","In contrast to young adult C . elegans , which have 16 seam cells on each lateral surface ( H0–H3 , V1–V6 , T descendants; The WormAtlas ) , we counted only 15 seam cells per side in P . pacificus J4 larvae ( n = 20 ) .","Ppa-obi-1 is also strongly expressed in the P ( 5–7 ) .","p cell descendants that form the vulva lumen ( vulC , D , E ) during the mid-J4 stage until just prior to vulval eversion as well as in the vulva muscles ( likely vm1 , 2 ) ( Figure 5B inset; Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 03229 . 012Figure 5 . Ppa-obi-1 and C06G1 . 1 expression in their respective nematode species .","( A–D )","An integrated Ppa-obi-1 promoter::gfp transgene ( csuIs01 ) in P . pacificus .","( A ) Lateral view of a single animal showing prominent expression in an amphid sheath cell , seam cells , and the syncytial hypodermal cell ( hyp7 ) in a J4 hermaphrodite .","( B ) Medial view of a transgenic animal with expression in the putative dorsal and ventral inner labial ( IL ) or outer labial ( OL ) neuronal support cells ( socket or sheath ) , the duct and excretory cells on the ventral side , the amphid sheath cell on the dorsal side ( arrow head ) , and the vulval muscles .","The inset shows a ventral view of the young adult vulva in another animal .","Autofluorescence is visible in the intestine .","( C ) A DiI-labeled neuron ( red arrow ) anterior to a Ppa-obi-1p::gfp expressing amphid sheath cell ( white arrow ) is dorsal to an excretory cell ( arrow head ) .","A putative IL or OL socket or sheath cell ( arrow ) is also visible .","( D ) DIC and fluorescent overlay image of expression in early P . pacificus embryo .","( E–I )","C06G1 . 1 promoter::gfp transgenes ( csuEx02 , csuEx03 ) in C . elegans .","( E ) Lateral view of a seam cell expression in a L3 hermaphrodite .","( F ) Medial view of putative inner or outer labial neuron support cells ( arrow ) and excretory cells ( arrowhead ) .","No amphid sheath shows gfp expression .","( G ) Ventral view of head region shows prominent expression in excretory cells and canal ( arrow ) .","( H ) Ventral view of young adult vulva .","( I ) Embryonic expression in C . elegans embryos appears later , stronger , and in more cells than in Ppa-obi-1 .","Anterior is left and the scale bars represent 50 μm .","Each panel is representative of ≥50 animals . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01210 . 7554/eLife . 03229 . 013Figure 5—figure supplement 1 . Ppa-obi-1 is expressed in putative amphid socket cells , amphid sheath cells , and vulval cells . Dorsal and ventral views of Ppa-obi-1p::gfp expression in roller prl-1 ( tu92 ) background with merged DIC and fluorescence ( A , C , E ) and corresponding fluorescence images ( B , D , F ) .","( A and B )","Dorso-lateral view of head region shows possible Ppa-obi-1 expression in a socket cell ( top ) , an IL sheath cell ( bottom ) , two seam cells ( asterisks ) , and two amphid sheath cells ( arrows ) .","( C and D )","Ventral view of a mid-J4 larvae vulva shows Ppa-obi-1p::gfp expression in P ( 5–7 ) .","p vulval epidermal cell descendants , a putative vulval muscle ( triangle ) , and a hyp7 cell ( asterisk ) .","( E and F )","Ventral view of a young adult vulva ( top left ) shows Ppa-obi-1p::gfp expression in P ( 5–7 ) .","p vulval epidermal cell descendents and four vulval muscles ( triangles ) and a lateral view of a mid-J4 vulva ( bottom right ) in another animal .","Anterior is left and the scale bars represent 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01310 . 7554/eLife . 03229 . 014Figure 5—figure supplement 2 . Targeted misexpression of Ppa-obi-1 in C . elegans . L2 larvae show amphid sheath cell expression using ( A ) Cel-T02B11 . 3 promoter::Ppa-obi-1:gfp ( csuEx29 ) and ( B ) Cel-F16F9 . 3 promoter::Ppa-obi-1:gfp ( csuEx30 ) .","The co-injection marker myo-2p::DsRED is visible in the anterior pharyngeal bulb due to bleeding over into the GFP spectrum .","Arrows indicate amphid sheath cell bodies .","( C ) Chemotaxis response to the ZTDO beetle pheromone remains unchanged towards 2% or 10% ZTDO .","( D ) ZTDO-induced paralysis in L4 larvae shows that transgenic Cel-F16F9 . 3p::Ppa-obi-1::gfp are better protected than wild-type animals in a 16 hr assay .","N2 ( N = 8 ) , T02B11 . 3p::Ppa-obi-1 ( N = 6 ) , F16F9 . 3p::Ppa-obi-1 ( N = 6 ) .","Anterior is left and the scale bar represents 25 μm .","p value indicates Dunnett's multiple comparisons test to wild-type N2 .","Error bars are SEM and N is the number of assays performed . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 014 Most notably , Ppa-obi-1 expression was observed in the two bilaterally symmetrical specialized epithelial cells that envelope the chemosensory neurons , known as the amphid sheath cells ( Figure 5A–B , arrows ) .","The amphid sheath cells are support cells for the sensory amphid neurons and completely enclose the ciliated endings of the AWA , AWB , and AWC chemosensory neurons in C . elegans .","Ppa-obi-1p::gfp expression in the putative amphid sheath cells lie dorsal–lateral and posterior to the DiI-labeled amphid neurons ASK , ADL , and ASI ( Figure 5C ) .","DiI staining of amphid neurons in Ppa-obi-1 mutants is superficially wild type , suggesting that Ppa-OBI-1 is unlikely to be involved in the development of the amphid neurons ( data not shown ) .","We also noticed expression in four unknown cell types anterior to the metacarpus ( anterior pharyngeal bulb ) , which could be putative neuronal support cells for the inner labial or outer labial quadrant sensilla ( IL or OLQ ) ( Figure 5—figure supplement 1 ) .","Ppa-obi-1 is also expressed in putative duct and excretory cells on the ventral anterior side that form part of the excretory system ( Figure 5B , arrowhead ) .","Based on conserved DiI labeling of P . pacificus amphid neurons and cell positions , the Ppa-obi-1 expression profile and secretion peptide sequence suggest that Ppa-OBI-1 is likely an extracellular protein involved in sensory neuron support , epithelial cell development , and osmoregulation .","Although Ppa-OBI-1 and its ortholog C06G1 . 1 in C . elegans share 61% amino acid identity , P . pacificus and C . elegans have opposite responses to the pheromone ZTDO .","While small changes in sequence can lead to large changes in protein function , the absence of expression of the C . elegans obi-1 ortholog in amphid sheath cells suggests that changes in gene expression pattern may also play a role in determining ZTDO sensitivity .","We therefore sought to determine if the Ppa-obi-1 ortholog C06G1 . 1 have shared tissue expression with Ppa-obi-1 .","We examined endogenous C06G1 . 1p::gfp expression in C . elegans in five independent transgenic lines with extrachromosomal arrays and found strong mid-L4 larvae expression in the seam cells , two tail hypodermal cells , four cells in the excretory duct system ( duct , excretory , gland cells ) , vulval muscles , and possible IL or OLQ sheath or socket neuronal support cells similar to the expression pattern observed for Ppa-obi-1 ( Figure 5E–H ) .","In contrast to the earliest Ppa-obi-1 expression in pre-comma stage , C06G1 . 1 expression is detectable during late embryogenesis in putative seam cells ( Figure 5I ) .","This seam cell expression is earlier than Ppa-obi-1p::gfp in the L1 stage , and is maintained until the young adult stage .","A reason for this earlier seam cell expression could be that the equivalent P . pacificus J1 larvae remain inside the egg while C . elegans eggs hatch as L1 larvae ( Fürst von Lieven , 2005 ) .","Like Ppa-obi-1 , C06G1 . 1 expression is largely absent in all tissue types when the transgenic animals become 1-day old adult hermaphrodites , coinciding with the fusion of seam cells into a syncytium .","This observation indicates that C06G1 . 1 expression in the excretory and epithelial system may be coordinated and involved in seam cell development .","Our expression analysis of obi-1 orthologs in the their respective nematode species revealed that the most striking difference is the lack of expression in the amphid sheath cells in C . elegans , suggesting that unlike Ppa-obi-1 , C06G1 . 1 is not likely to function directly with chemosensory neurons in C . elegans .","In addition to differences in gene expression pattern , the possibility that there is a ZTDO receptor that interacts with Ppa-OBI-1 in P . pacificus but not in C . elegans may also help to explain how Ppa-obi-1 orthologues confer different ZTDO sensitivities in the two species during certain developmental stages .","To explore yet another possibility that changes in both protein function and expression may lead to differences in ZTDO sensitivity , we expressed the Ppa-obi-1:cDNA under the C . elegans amphid sheath cell-specific promoters T02B11 . 3 and F16F9 . 3 in C . elegans ( Bacaj et al . , 2008; Kramer et al . , 2010; Oikonomou et al . , 2011; Procko et al . , 2011 ) .","Both T02B11 . 3p::Ppa-obi-1:gfp ( csuEx29 ) and F16F9 . 3p::Ppa-obi-1:gfp ( csuEx30 ) transgenic animals showed strong GFP expression in the amphid sheath cells ( Figure 5—figure supplement 2 ) as well as phasmid sheath cells in the tail region ( not shown ) .","While the targeted misexpression of Ppa-obi-1 in C . elegans did not alter the strong avoidance behavior towards either 2% or 10% ZTDO , Ppa-obi-1 expression in C . elegans did reduce the paralytic effects of ZTDO ( Figure 5—figure supplement 2 ) .","Because wild-type C . elegans N2 is not susceptible to ZTDO-induced paralysis at 0 . 5% ZTDO that P . pacificus is , we increased the stringency of the assay by using 10% ZTDO , incubating for 16 hr , and exposing the animals in the absence of OP50 food .","Under this enhanced ZTDO paralysis assay , we found that F16F9 . 3p::Ppa-obi-1:gfp ( csuEx30 ) transgenic L4 larvae but not T02B11 . 3p::Ppa-obi-1:gfp ( csuEx29 ) exhibited a small but statistically significant increase in protection from ZTDO-induced paralysis compared to wild-type N2 ( Figure 5—figure supplement 2C , D ) .","Because we could not detect apparent differences in GFP expression patterns between the two amphid promoters , the ability of F16F9 . 3p::Ppa-obi-1:gfp to reduce ZTDO paralysis may be due to subtle transient expression differences .","These results suggest that while the expression of the Ppa-OBI-1 protein in the C . elegans amphid sheath cells is not sufficient to reduce avoidance to ZTDO , it can contribute to the obi-1-dependent protection from ZTDO-induced paralysis ."],["Sensitivity to ZTDO-induced paralysis/arrest is Ppa-obi-1-dependent predominantly during the J4 larval stage and , to a lesser degree , in the embryos and dauers .","Given the diversity of tissues expressing Ppa-obi-1 in the J4 larvae , it is not apparent which tissues require Ppa-obi-1 expression for proper chemosensation and if the same tissues are involved with protection from ZTDO-induced paralysis in wild-type animals ( Figure 6 ) .","Our gene expression studies show that despite coding highly similar proteins , P . pacificus Ppa-obi-1 is uniquely expressed in the amphid sheath cells compared to its C . elegans ortholog C06G1 . 1 .","Based on this species-specific expression pattern , we are tempted to speculate that these chemosensory glial cells , the IL and amphid sheath cells , play a dominant role in mediating ZTDO sensitivity .","However , additional experiments will be needed to test the possibility that epidermal cells such as the vulva , hypodermis , and seam cells , or cells in the excretory system may also mediate ZTDO sensing . 10 . 7554/eLife . 03229 . 015Figure 6 . Summary and model of the oriental beetle pheromone ZTDO on P . pacificus development and lifecycle .","( Top )","Peak Ppa-obi-1 expression in the wild-type J4 larvae coincides with the least sensitive developmental stage to the paralytic effects of the beetle pheromone ZTDO ( at 0 . 5% ) .","Ppa-obi-1 mutants are most sensitive to ZTDO-induced paralysis in the J4 stage .","Our working model on Ppa-OBI-1 functions in the J4 larvae hypothesizes that Ppa-OBI-1 proteins in the extracellular space , such as the lumen of the amphid sheath cell , may be required to remove ZTDO bound to odor receptors on neurons .","( Bottom )","This working model shows both P . pacificus adults and dauer larvae from non-beetle populations associate with the host oriental beetle through attraction to the beetle's sex pheromone ZTDO , whereby the progeny born on the beetle are arrested as eggs or dauer larvae .","Death of the host allows microorganism to grow , which then encourages dauer larvae to exit and resume reproductive development .","Several iterations of asynchronous nematode populations likely complete each dispersal cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 015 Given the growing evidence that the amphid sheath cells in C . elegans are critical for proper neuronal development and function of the 12 pairs of chemosensory amphid neurons ( Shaham , 2006 ) , we propose the following models on how the Ppa-OBI-1 protein can act in the sheath cells to directly or indirectly modulate ZTDO chemosensation .","In C . elegans , many proteins have enriched expression in the amphid sheath , such as DAF-6 and the secreted venom allergen-like VAP-1 , and these secretions are corroborated by dense populations of vesicles in amphid sheath cells under electron microscopy ( Bacaj et al . , 2008; Doroquez et al . , 2014 ) .","However , it is not yet known which amphid proteins , if any , are required in processing odors using behavioral assays .","Interestingly , DAF-6 is a Patched-related protein with expression pattern that overlaps with Ppa-obi-1 expression ( amphid lumen , excretory canal , vulva lumen ) and is required for chemotaxis and dauer formation ( Perens and Shaham , 2005 ) .","Similarly , Ppa-obi-1 expression in the putative sheath or socket cells of the inner labial neurons may also be involved in ZTDO sensing , as recent ultrastructural electron micrograph reconstructions indicate that BAG ( gas sensing ) and FLP ( nociceptive ) neurons in C . elegans associate closely with the socket cells of the inner labial sensillae ( Doroquez et al . , 2014 ) .","One possible mode of action for ZTDO may be its ability to change cell membrane permeability , thereby indirectly affecting the functions of the glia-associated neurons .","Hence , Ppa-OBI-1 may function to counter the cell membrane permeability changes brought about by ZTDO by interacting with cell membrane lipids .","Alternatively , Ppa-OBI-1 could mediate pheromone binding directly .","In lepidopterans such as the wild silk moth Antheraea polyphemus and the cotton leafworm Spodoptera littoralis , pheromone-degrading and odorant-degrading enzymes ( PDEs and ODEs ) are esterases specifically expressed in the male antennal sensillar lymph analogous to the amphid lumen ( Ishida and Leal , 2005; Durand et al . , 2011 ) .","The termination of the signal by removing odors that have already triggered a response is an important process for preventing odor adaptation and increasing odor sensitivity .","Thus , it is possible that Ppa-OBI-1 acts as a solubilizing factor for the lipid pheromones in the excretion-filled amphid sheath lumen ( Figure 6 ) .","The weak ability of amphid sheath-specific expression of Ppa-OBI-1 to confer ZTDO protection in C . elegans suggests that both expression pattern and protein function could be important factors in ZTDO sensing in P . pacificus .","Future work will be necessary to determine if Ppa-obi-1 expression restricted to the amphid sheath is sufficient to rescue the ZTDO attraction defect in P . pacificus and if the amphid sheath cells are required to mediate ZTDO sensing .","Given that Ppa-obi-1-independent susceptibility to ZTDO occurs during the J2 larval stage when Ppa-obi-1 is expressed in the seam and hypodermal cells rather than in the amphid sheath that is only active in the J4 larvae , ZTDO-induced paralysis likely acts through the extracellular matrix of the nematode cuticle .","P . pacificus adults , whose cuticle may be compositionally different from the larval cuticle , are not susceptible to ZTDO .","However in P . pacificus embryos , the susceptibility of ZTDO may be due to ZTDO limiting gas or ion exchange with the environment rather than direct interaction between ZTDO and cellular components , given that nematode egg shells are considered to have very limited permeability accessible only to small molecular weight solutes and gas molecules ( Chitwood and Chitwood , 1974 ) .","Since ZTDO exposure does not lead to embryonic arrest and paralysis in C . elegans , ZTDO must also interact with other non-conserved P . pacificus-specific molecules that have yet to be identified .","There are very few known nematocides from natural compounds .","One rare example comes from the oyster mushroom Pleurotus ostreatus , which secretes droplets that can immobilize rhabditid nematodes in an hour , though its effects on embryos have not been investigated ( Barron and Thorn , 1987; Stadler et al . , 1994 ) .","Future work on the different susceptibility to ZTDO may help to answer if there is a wider pattern of negative correlation between ZTDO attraction and ZTDO-induced paralysis in other natural isolates , as observed in the California and Washington strains .","Such antagonistic traits may reveal possible co-evolution between host finding and host tolerance in necromenic associations .","We further speculate that in the natural ecology of P . pacificus , ZTDO may enable the beetle host to limit the nematode number and to thus synchronize the developmental stage of infective P . pacificus by arresting embryogenesis and lethally paralyzing J2 larvae .","The unexpected role of a P . pacificus attractant to act as a developmental regulator against the younger larval stages , particularly the dauer larvae ( DL ) , suggests that the ZTDO-resistant adult stage is a potential dispersal stage .","This attenuated antagonistic interaction also revealed that nematode larvae and adults may be susceptible to distinct selective pressures .","P . pacificus DL does not disperse readily , likely due to the secretion of the wax ester nematoil that enables P . pacificus to form dauer towers when potential hosts are nearby ( Penkov et al . , 2014 ) .","Therefore under natural conditions , lower concentrations of ZTDO than the concentrations we have tested may serve as a close-range host dwelling strategy by inhibiting DL dispersal to form dauer aggregates .","The developmental role of ZTDO may be part of a mechanism to limit nematode dispersal until the beetles mature .","In these respects , the promotion of a non-DL dispersal stage and inhibition of DL dispersal by ZTDO resemble other known host-derived compounds found in the entomophilic nematode B . xylophilus and the phoretic nematode C . japonica ( Okumura et al . , 2013; Zhao et al . , 2013 ) .","Attraction and paralysis by ZTDO may have co-evolved in the necromenic association to allow successive populations of founder P . pacificus DL and adults to infect beetles , culminating in arrested DL remaining on sexually mature beetles during the dispersal cycle .","The pheromone arrested DL state may also help to account the observation that Pristionchus nematodes emerge several days later than rhabditids from the same species of host beetles .","Our findings that a lipid-binding protein has a role in coordinating chemosensation , body proportions , and protection from a host pheromone encourages fresh inquiries on additional genes involved in ZTDO sensory signaling and its mechanism of action ."],["All nematodes were raised on NGM media containing OP50 E . coli at ∼23°C as previously described ( Hong and Sommer , 2006a; Hong et al . , 2008a ) .","The following strains were used: P . pacificus: California ( PS312 ) wild type , Washington ( PS1843 ) , Ppa-obi-1 ( tu404 ) I; Ppa-egl-4 ( tu374 ) IV ( null ) ; Ppa-obi-3 ( tu405 ) ; csuIs01 [Ppa-obi-1p::gfp; Ppa-egl-20p::rfp; PS312 gDNA]X; rlh49 ( csuIs01; tu374 ) ‘Ppa-obi-1p::gfp; Ppa-egl-4’; rlh50 ( csuIs01; tu92 ) ‘Ppa-obi-1p::gfp; roller’; rlh51 ( csuIs01; tu404 ) ‘Ppa-obi-1p::gfp; Ppa-obi-1’ .","C . elegans: Bristol ( N2 ) wild type , nrf-5 ( sa513 ) , csuEx02 [C06G1 . 1p::gfp; Cel-myo-2::DsRED]; csuEx03 [C06G1 . 1p::gfp; pRF4 roller] , csuEx29 [T02B11 . 3p::Ppa-obi-1:gfp; Cel-myo-2::DsRED] , and csuEx30 [F16F9 . 3p::Ppa-obi-1:gfp; Cel-myo-2::DsRED] .","The whole-genome sequenced strain vc20357 was outcrossed to him-5 ( e1467 ) three times and the C06G1 . 1 locus was sequenced to confirm that the slim body phenotype is linked to the missense gk306193 mutation ( D48N ) .","We X-ray mutagenized wild-type P . pacificus PS312 ( California ) at 325 Roentgen for 25 min with radioactive beryllium and picked 40 Po onto individual plates .","We subsequently picked 1067 F2s from 400 F1s and screened 713 viable F3 populations for insensitivity to 150 nl of oriental beetle pheromone , Z-7-tetradecen-2-one ( 99% pure; Bedoukian Research , CT ) after 1 hr treatment with 1 mM exogenous 8-Bromo-cGMP ( Sigma–Aldrich , MO ) ( Hong et al . , 2008a ) .","100 mM nicotinic acid ( Sigma–Aldrich , MO ) was dissolved in water .","Population chemotaxis assays were conducted for ∼16 hr at 23°C , and individual candidate F3 from plates showing insensitivity were rescued onto fresh plates by mouth pipetting .","We repeated the cGMP-treated chemotaxis assay from the progeny of the candidate mutant lines two more times with at least two duplicate assay plates per line .","Only those lines that failed the third round of chemotaxis assay were considered Oriental Beetle pheromone Insensitive obi alleles .","Ppa-obi-1 maintained long body morphology and obi-3 displayed more coiled body posture after 3× outcrossing to wild-type PS312 and selecting the ZTDO insensitive phenotype .","Because both the chemotaxis and morphological phenotypes co-segregate after 5× outcross , we subsequently used the long body phenotype as a visible marker for Ppa-obi-1 during positional mapping .","Population chemotaxis assays were performed as previously described based on modified protocol for C . elegans ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .","Well-fed J4 and adult worms were washed in M9 buffer twice before assay .","For a more consistent response , C . elegans was washed in spring water rather than M9 buffer before the ZTDO avoidance assay .","For the exogenous cGMP treatment , each washed population was split for mock or 1 hr treatment with 1 mM 8-Bromo-cGMP ( in water ) .","For each condition , at least 10 worms had to reach the odor sources in each assay and 12 assays ( sample size N ) were performed over three sessions .","Chemotaxis assays with transgenic obi-1 rescue worms were scored by rfp expression in the tail .","20 live worms or eggs from non-starved cultures were placed on NGM agar plates with fresh OP50 .","For consistency , eggs were synchronized by picking ∼50 adults onto OP50 and allowing them to lay eggs for 3 hr .","The eggs were then transferred to different plates and counted .","10 µl of ZTDO or ETDA diluted in ethanol was added to the lid of the plate , immediately sealed with Parafilm , and incubated at room temperature .","Larvae were scored 3 hr later or the next day by counting the number of paralyzed and active worms .","A worm was scored as paralyzed if it did not move at least one pharynx length after tapping on the outside of the plate .","Eggs were scored 2 days later by counting the number of unhatched eggs .","For dauer larvae exit assays on NGM plates containing none , 0 . 001% or 0 . 01% ZTDO ( vol/vol ) , 25 dauer larvae from the same 2- to 3-week old starved plate were picked onto each type of plate with OP50 and scored for arrested/active dauer larvae , J3 larvae , or J4 larvae after 2 days at ∼23°C .","A single library was paired-end sequenced twice in separate flow cell lanes with 2 × 76 bp that yielded 61 million and 65 million reads after preprocessing ( quality control , adapter removal ) .","More than 90% of all reads could be mapped to the P . pacificus genome ( www . pristionchus . org ) with BWA ( version 0 . 5 . 9 ) for each library .","We then employed ACCUSA for SNP detection ( Froehler and Dieterich , 2010 ) .","Briefly , we performed a head-to-head comparison of the mutant Ppa-obi-1 and wild-type PS312 mapped libraries .","The algorithm represents read stacks ( base calls and qualities thereof ) as Dirichlet distributions .","For any given genome position , we require a read coverage of at least three in both samples .","The smaller read stack serves as reference distribution from which the larger stack is drawn .","A probability density for this draw is computed , and a SNP is called if the log-density is lower than −30 .","Stable transgene expression in P . pacificus requires co-injection of endogenous genomic DNA and co-injection markers containing compatible cohesive ends with the target gene as previously described ( Schlager et al . , 2009; Cinkornpumin and Hong , 2011 ) .","A 4 . 3 kb Ppa-obi-1 promoter fragment was amplified from PS312 gDNA using RHL121/RHL136 ( primary ) and RHL124/RHL149 ( secondary ) primers .","The Ppa-obi-1 promoter was then fused with the gfp- Ppa-rpl-23-3′UTR by PCR ( Hobert , 2002 ) .","PS312 animals were injected with a DNA mix containing a final concentration of 10 ng/μl of the SpeI/SalI digested 4 . 3 kb Ppa-obi-1 promoter::NLS-gfp-SalI fusion PCR product from three independent reactions; 10 ng/μl SpeI digested Ppa-egl-20p::rfp linearized plasmid; and 60 ng/μl SpeI/SalI digested PS312 genomic DNA .","Individual F1s expressing Ppa-egl-20p::rfp in the hermaphrodite tail were selected for Ppa-obi-1 expression analysis .","Two stable transgenic lines were initially generated with similar expression patterns .","We used the stably transmitted reporter line csuIs01[Ppa-obi-1p::gfp; Ppa-egl-20p::rfp; PS312 gDNA]X for detailed expression analyses .","csuIs01 is likely to be the result of a spontaneous integration into the X chromosome based on the observation that transgenic males expressing the co-injection rfp marker crossed to Ppa-unc-1 ( ChrIV ) hermaphrodites gave rise to all F1 males lacking rfp expression but all F1 hermaphrodites expressing rfp .","More than 50 individuals at all developmental stages were analyzed .","Ventral views of the vulva expression were obtained from csuIs01 in the prl-1 roller background .","The same 4 . 3 kb Ppa-obi-1 promoter was used to drive the full length 1 . 6 kb cDNA fused to GFP by injecting into PS312 and crossing into the Ppa-obi-1 ( tu404 ) background to generate csuEx27[Ppa-obi-1p::Ppa-obi-1 cDNA:gfp; Ppa-egl-20::rfp; Ppa-obi-1 ( tu404 ) gDNA] .","For unknown reasons , the N-terminally fused gfp coding region did not express GFP .","To create the C06G1 . 1p::gfp reporter gene construct , we PCR amplified a 3 . 2 kb upstream sequence from the C06G1 . 1 ATG using RHL169 and RHL187 and fused the promoter with GFP:Ppa-rpl-23 3′ UTR by fusion PCR , using RHL181 and RHL154 primers to obtain a 4183 bp amplicon .","Independently amplified C06G1 . 1p::gfp fragments were co-injected with either pRF4 rol-6 ( su1006 ) or myo-2p::DsRED vectors along with the pBluescript vector as carrier DNA ( 50 ng/μl each ) .","This reporter likely encompasses C06G1 . 1's maximum regulatory region because the 3 . 2 kb promoter fragment extends ∼300 bp upstream of the next gene on the opposite strand ( C06G1 . 14 ) , and C06G1 . 1 does not contain any intron larger than 1 kb that may act as potential intronic enhancers .","Selection for DsRED animals in the reporter strain csuEx02 [C06G1 . 1p::gfp; Cel-myo-2::DsRED] results in 90% GFP+/DsRED+ progeny and equal DsRED+ expression in pharyngeal muscle cells , suggesting stable mitotic transmission of the transgene .","All five F1 transgenic lines showed similar expression profile and intensity in post-hatching stages , but strong body epidermal expression was detected only in the csuEx03 [C06G1 . 1p::gfp; pRF4; pBST] line .","Images were obtained using a Leica DM6000 fluorescent microscope and Leica SP5 confocal microscope .","To create the T02B11 . 3p::Ppa-obi-1:gfp amphid sheath expressing translational fusion construct , an ∼2 . 1 kb T02B11 . 3 promoter fragment was amplified from C . elegans gDNA using RHL476/RHL490 ( primary reaction ) .","The Cel-T02B11 . 3 promoter is then fused to the same 1 . 6 kb Ppa-obi-1 cDNA rescue construct by PCR using RHL477/RHL483 ( secondary ) to generate a 3 . 7 kb fusion .","This construct and the GFP-unc-54-3′UTR fragment , amplified from the pPD95 . 75 vector using RHL480/RHL481 ( primary ) was fused using RHL501/RHL482 ( tertiary ) to create the complete construct .","A similar scheme was used to generate F16F9 . 3p::Ppa-obi-1:gfp using RHL498/RHL500 ( promoter ) .","C . elegans N2 were injected with a mixture of purified T02B11 . 3p::Ppa-obi-1:gfp ( 25 ng/μl ) or F16F9 . 3p::Ppa-obi-1:gfp , myo-2::DsRED ( 25 ng/μl ) and pBluescript carrier DNA ( 100 ng/μl ) .","One stable line from each construct was characterized for ZTDO chemotaxis and paralysis .","The GenBank accession for Ppa-obi-1 mRNA is JQ946290 .","Primer sequences are listed in Table 2 . 10 . 7554/eLife . 03229 . 016Table 2 . PrimersDOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 016PrimerPrimer sequence ( 5′→3′ ) Template targetRHL101TGAACGGGCTTTTAATCTGGPpa-obi-1 gDNARHL102GTGTCGAGATAGTCGGGCATPpa-obi-1 gDNARHL104ATGACTGCTCCAAAGAAGPpa-obi-1 cDNARHL107ATTTGCCCTTCGTTCCACPpa-obi-1 for RNAiRHL108CTGTCTCAGTATGCCGAATGPpa-obi-1 for RNAiRHL109ATTCAGCCGTGTTACAAATCPpa-obi-1 cDNARHL110CTGCATGTGTTCCGGTCTCGPpa-obi-1 cDNARHL113AAACCTCTGACACATGCAGCPpa-obi-1 cDNARHL121AGTTTCAAAGTTGCATGGPpa-obi-1 promoterRHL124CTTCTTTGGAGCAGTCATGTAGCGATAATCAGGAGTgfp; Ppa-obi-1 promoterRHL132ATGACTGCTCCAAAGAAGgfpRHL136CAACCGTGTGCAGTAGAACCPpa-obi-1 5′ RACERHL149ACTATCCTATCATCGGAAGCPpa-obi-1 promoterRHL151AGGGCATTGACAAATCATCGPpa-obi-1 cDNARHL154CACGACGTTGTAAAACGACGPpa-obi-1 cDNARHL169CGCGCTAAACCAATTCCGCCC06G1 . 1 promoterRHL181GTTGAGAGAAGAGGTGGAGTCCC06G1 . 1 promoterRHL187CTTCTTTGGAGCAGTCATCAGATCTGAAAATTTGACACATTGgfp; C06G1 . 1 promoterRHL267GGAACTAACATTAATGTCACC06G1 . 1 RNAiRHL268GAGAAGTGCATAGTTGATGC06G1 . 1 RNAiRHL477GCGCTACAGGGGATTTTGTCT02B11 . 3 promoterRHL480TGAAAGATGCAAGTAAAGGAGAAGAACTTTTCp95 . 75 GFPRHL481AAGGGCCCGTACGGCCGACTAp95 . 75 GFPRHL482GTAGGAAACAGTTATGTTTGGp95 . 75 GFPRHL484CTTCAGAAAATGATCAGGAGTCTGGTTCobi-1 cDNARHL487AGTGAAAAGTTCTTCTCCTTTACTTGCATCTTTCACGGAGTCobi-1 cDNA; gfpRHL490AACGAGAACCAGACTCCTGATCATTTTCTGAAGAAAGTTGAAAAACT02B11 . 3 promoter; obi-1 cDNARHL498GCGATAAGATCGGTCAATCTGAGF16F9 . 3 promoterRHL499GCCAGTAAGGGCTAGTAAGTGF16F9 . 3 promoterRHL500AACGAGAACCAGACTCCTGATCATATTTTGTTTCTTACTGTCTTGF16F9 . 3 promoterRHL501ATGGGTACGTTTCTGGGTATAGT02B11 . 3 promoter; obi-1 cDNAVR86GACTCCTGATTATCGCTACGPpa-obi-1 cDNAVR87AGATAGTCAAACTATGCATCPpa-obi-1 cDNAVR88CTAATAATCTACACTAAATGPpa-obi-1 cDNAAG11112CTCGGAGGAGGAACTGGATCPpa-beta-tubulin RT-qPCRAG11113GACCGTGTCAGAGACCTTAGPpa-beta-tubulin RT-qPCRRH12548GCAGGAACATATATTCGCAGPpa-egl-4 RT-qPCRRH12549TGTCACGGAACGTTTTGTACPpa-egl-4 RT-qPCRSL1GGTTTAATTACCCAAGTTTGAGSL1 for 5′ RACESSLP21FACTGGTGCTCATCGCAAGAMapping markerSSLP21RCCTTCTGTTTTCCTACCCCCMapping markerSSLP7FTCTTGCATAACACCGAACAAAMapping markerSSLP7RAGGGCGTTACGTGAATAAGCMapping markerSSLP17FCAATGCAGTAGCCGAATCATMapping markerSSLP17RCAATATTGGCCTTCACCCTGMapping markerS447FTGGGGATAGCGAAAATCATCMapping markerS447RCGCATCGTTATTCACGAAATTCMapping markerLetters in bold are based on different templates for fusion PCR .","Body measurements were obtained as previously described ( Hong et al . , 2008a ) .","In brief , we used a Leica DM6000 and LAS application to measure body dimensions at 24-hr intervals starting at mid-J4/L4 stage .","To determine hatching rate , we picked single J4-stage PS312 hermaphrodites onto fresh OP50 plates and transferred the nematodes every 24 hr three times at 20°C .","The total number of eggs present on the plates following each transfer and the resulting number of 3-day old animals for each worm during the 3-day period were used to calculate the percentage of hatching and brood size .","The bacterial feeding strain containing the C06G1 . 1 fragment 1 was previously described in a large-scale C . elegans RNAi screen ( Simmer et al . , 2003 ) .","C06G1 . 1 fragment 2 was made using RHL267 and RHL268 primers to amplify an ∼400 bp fragment of C06G1 . 1 , digesting this product with BglII and PstI , and ligating the 190 bp fragment into the BglII/PstI sites of pL4440 .","In vitro synthesis of dsRNA from 1 μg of T7 amplified PCR template from the RNAi vector or the pL4440 negative control was accomplished using the Ambion in vitro synthesis kit ( Grand Island , NY ) .","RNAi by bacterial feeding of C06G1 . 1 was performed by scoring the progeny of L4 stage rrf-3 ( pk1426 ) animals raised on RNAi or L4440 control plates containing 1 mM IPTG and 50 μg/ml carbenicillin .","Means , SEM ( error bars ) , and p values for two-tailed t test were performed by Microsoft Excel .","One-way ANOVA with multiple comparisons post-hoc testing were performed using Prism statistical software .","Sample sizes represent total technical replicates from two or more biological replicates ."]],"string":"[\n [\n \"An understanding of ecological interactions and evolutionary history can be beneficial to the understanding of the cellular and developmental processes of an organism .\",\n \"For example , the genes responsible for fruit and bacterial odor preferences in Drosophila melanogaster and Caenorhabditis elegans are most informative when considering the natural ecologies in rotting fruits and vegetation ( Bargmann et al . , 1993; Bargmann , 2006; Hallem and Carlson , 2006; Kiontke and Sudhaus , 2006 ) .\",\n \"In Pristionchus pacificus , an emerging model organism for the study of development and behavior ( Hong and Sommer , 2006b ) , a systematic effort to identify the natural ecology of Pristionchus nematodes revealed that P . pacificus host preferences include the oriental beetle ( Exomala orientalis ) found in Japan and northeastern United States ( Herrmann et al . , 2007 ) .\",\n \"Unlike several fructivorous Caenorhabditis species , Pristionchus species are considered to be necromenic nematodes that have species-specific beetle host preferences and feed on the microorganisms that emerge from the beetle carcass ( Herrmann et al . , 2006a , 2006b; Brown et al . , 2011 ) .\",\n \"Pristionchus species are members of the Diplogasteridae family of nematodes whose common ancestor with the Rhabditidae family ( e . g . , C . elegans ) diverged ∼300 million years ago ( Dieterich et al . , 2008 ) .\",\n \"Given that beetles ( Coleoptera ) represent a group with the most described species , the possibility that most beetle species can harbor specific entomophilic nematode associations underscores the vast number of nematode species remaining to be described .\",\n \"Although biologists have been aware of such species-specific insect–nematode associations for some time , the genetic basis for such interactions remains largely unknown .\",\n \"Previous studies suggest that the beetle host specificity of Pristionchus species is in part due to nematode attraction to various plant volatile compounds released during beetle feeding , as well as to beetle aggregation and sex pheromones during mating ( Hong and Sommer , 2006a; Hong et al . , 2008b , 2008a ) .\",\n \"For example , Pristionchus maupasi associates primarily with the European May beetle Melolontha spp .\",\n \"and is attracted to the blend of the green leaf alcohol from plants and the phenol pheromone from beetles ( Hong et al . , 2008b ) .\",\n \"Thus , insect and plant odors can synergize to provide nematodes with information on host presence , sex , and maturity .\",\n \"By contrast , C . elegans attractants such as 2 , 3-butanedione ( diacetyl ) , 2 , 3-pentanedione , and isoamyl alcohol are significantly less attractive to P . pacificus ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .\",\n \"This piggybacking of intra-species communication for inter-species interactions between Pristionchus and beetles can also evolve rapidly at the population level , as exhibited by the natural variation of P . pacificus odor preference for insect pheromones ( Hong et al . , 2008a; McGaughran et al . , 2013b ) .\",\n \"In particular , P . pacificus Washington strain ( PS1843 ) is attracted to the lepidopteran sex pheromone E-11-tetradecenyl acetate ( ETDA ) and the oriental beetle sex pheromone z-7-tetradece-2-one ( ZTDO ) ( Herrmann et al . , 2007 ) , but the wild-type reference strain from California ( PS312 ) requires exogenous cGMP treatment to become attracted to these insect pheromones ( Hong et al . , 2008a ) .\",\n \"These natural variations in insect pheromone attraction , combined with regional-specific P . pacificus host preferences , may offer support for the geographic mosaic theory of co-evolution ( Thompson , 2005 ) .\",\n \"Although the distinct chemosensory profiles of P . pacificus populations suggest insect pheromone attraction to be an important and rapidly evolving trait , other studies on insect–nematode interactions show that there are other potential roles host chemicals can play in coordinating host–parasite development and nematode dispersal behavior .\",\n \"For example , the pinewood nematode Bursaphelenchus xylophilus is intimately associated with its Monochamus beetle vector , and fatty acid ethyl esters exuded during beetle eclosion promote the formation of the dispersal fourth larval stage B . xylophilus ( Zhao et al . , 2013 ) .\",\n \"Similarly , the species-specific phoretic association between the burrower bug Parastrachia japonensis and the nematode Caenorhabditis japonica may be mediated by the beetle cuticular compounds that not only attract C . japonica but also assist infestation by inhibiting C . japonica dispersal ( Okumura et al . , 2013 ) .\",\n \"P . pacificus populations have been found on various beetle hosts worldwide , but the association with the oriental beetle was the first to be identified and the only one whose sex pheromone is a strong attractant ( Herrmann et al . , 2007; McGaughran et al . , 2013b ) .\",\n \"Therefore , the oriental beetle sex pheromone represents the single most ecologically relevant semiochemical by which we could repeatedly interrogate the P . pacificus genes involved for host sensing and other unexplored developmental roles .\",\n \"In this study , we show that although the beetle pheromone , ZTDO , is attractive to P . pacificus adults , the beetle host pheromone may moderate nematode infection by arresting embryonic development , inducing larval paralysis , and inhibiting dauer exit .\",\n \"We leveraged the ability to use exogenous cGMP to potentiate the California reference strain for ZTDO attraction to find additional genes important for ZTDO sensitivity by performing an unbiased forward genetic screen for mutants with altered responses to ZTDO .\",\n \"We found that a putative lipid-binding protein Ppa-OBI-1 mediates both beetle pheromone attraction as well as a previously unknown role of the beetle pheromone to regulate P . pacificus development .\",\n \"Our results suggest that the host pheromone may play a complex role in reducing infection or coordinating development for the dispersal cycle of the necromenic lifestyle .\"\n ],\n [\n \"P . pacificus and C . elegans have diametrically opposed odor preference profiles .\",\n \"Pristionchus species are generally attracted to plant volatiles , insect pheromones , and cuticular hydrocarbons , but each Pristionchus species can have its own chemosensory profile reflective of its preferred beetle hosts ( Hong and Sommer , 2006a; McGaughran et al . , 2013a ) .\",\n \"P . pacificus populations found on the oriental beetle E . orientalis in Japan and northeastern United States are attracted to the sex pheromone of the beetle , z-7-tetradecen-2-one ( ZTDO ) ( Zhang et al . , 1994; Herrmann et al . , 2007 ) .\",\n \"In contrast , the compost-dwelling C . elegans is less attracted to or even repelled by plant and insect odors , including ZTDO , but is strongly attracted to small compounds released by microbes ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .\",\n \"To identify genes required to mediate ZTDO attraction in a pheromone-insensitive reference strain , California PS312 , we performed a behavior-based genetic screen for mutants that are insensitive toward ZTDO following a brief 1-hr treatment with the cell-permeable 8-br-cGMP .\",\n \"We isolated two Oriental Beetle pheromone Insensitive mutants , Ppa-obi-1 ( tu404 ) and Ppa-obi-3 ( tu405 ) .\",\n \"The chemosensory phenotype of Ppa-obi-1 and Ppa-obi-3 is strongly specific for ZTDO and does not affect the chemoattraction towards other insect pheromones ETDA and myristate nor plant volatiles β-caryophyllene and nicotine ( Figure 1 ) .\",\n \"Odor avoidance in obi mutants towards 1-octanol , a repellant common between P . pacificus and C . elegans , also resembles wild type .\",\n \"These results indicate that both obi mutants are specifically defective in sensing a beetle pheromone rather than more generally defective in chemosensation .\",\n \"Ppa-obi-1 ( tu404 ) and Ppa-obi-3 ( tu405 ) are non-allelic recessive mutants that complement each other in their physical and behavioral phenotypes .\",\n \"We proceeded to characterize Ppa-obi-1 ( tu404 ) based on its altered response to ZTDO attraction and its unexpected function in protecting larvae . 10 . 7554/eLife . 03229 . 003Figure 1 . Chemosensory behavior of oriental beetle pheromone insensitive mutants . Ppa-obi-1 and Ppa-obi-3 mutant animals are specifically defective for sensing the host oriental beetle sex pheromone , Z-7-tetradece-2-one .\",\n \"The ZTDO structure is depicted below the beetle .\",\n \"In contrast , obi mutants' response is wild type towards another cGMP-dependent attractant , E-11-tetradecenyl acetate ( ETDA , a lepidopteran pheromone ) , as well as myristate ( methyl tetradecanoate , an insect allomone ) , plant volatiles beta-caryophyllene and nicotinic acid , and the repellent 1-octanol .\",\n \"N ≥ 15 replicates in ≥3 trials .\",\n \"Significant differences to wild type are indicated by p values ( Dunnett's test ) and error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 003 Necromeny occupies a midpoint in the continuum of nematode–insect interactions that span from mutualism to pathogenicity .\",\n \"To address possible developmental effects of long-term exposure to ZTDO , we attempted to grow worms in the presence of the ZTDO by placing eggs onto OP50 bacteria in plates containing 10 µl of 0 . 5% ZTDO suspended under the plate lids , which is approximately a third of the amount used in chemotaxis assays .\",\n \"Surprisingly , very few of the synchronized eggs from wild type and Ppa-obi-1 ( tu404 ) hatched after 36 hr , compared to ∼94% of the untreated eggs ( Figure 2A ) .\",\n \"ZTDO therefore may function not just as a volatile attractant but also as an antagonistic host-derived developmental regulator . 10 . 7554/eLife . 03229 . 004Figure 2 . The oriental beetle pheromone ZTDO inhibits P . pacificus development and induces paralysis .\",\n \"( A ) Ppa-obi-1 mutant is hypersensitive to 0 . 10% ZTDO compared to wild type PS312 ( California ) .\",\n \"However , PS312 is more sensitive to 0 . 5% ZTDO than another strain , PS1843 ( Washington ) .\",\n \"PS312 ( n = 372 , 82 , 90 , 320 ) ; PS1843 ( n = 309 , 88 , 92 , 237 ) ; Ppa-obi-1 ( n = 332 , 69 , 71 , 389 ) ; C . elegans wild-type N2 ( n = 316 , 63 , 84 , 174 ) .\",\n \"( B ) Both wild-type PS312 and Ppa-obi-1 mutant J2 larvae are more sensitive to ZTDO than PS1843 .\",\n \"PS312 ( n = 154 , 115 ) ; PS1843 ( n = 110 , 102 ) ; Ppa-obi-1 ( n = 104 , 100 ) ; C . elegans wild-type N2 ( n = 23 , 73 ) .\",\n \"( C ) Ppa-obi-1 J4 larvae is uniquely sensitive to ZTDO compared to PS312 ( p < 0 . 0001 ) .\",\n \"PS312 ( n = 209 , 290 ) ; PS1843 ( n = 102 , 99 ) ; obi-1 ( n = 244 , 237 ) ; C . elegans wild-type N2 ( n = 69 , 109 ) .\",\n \"( D ) 90 min exposure of a Ppa-obi-1 ( tu404 ) J4 larvae to ethanol control compared to ( E ) 0 . 50% ZTDO resulted in a rigid fully paralyzed posture .\",\n \"( F ) Adults are not paralyzed by ZTDO .\",\n \"PS312 ( n = 108 , 113 ) ; PS1843 ( n = 114 , 107 ) ; Ppa-obi-1 ( n = 109 , 109 ) ; C . elegans wild-type N2 ( n = 62 , 72 ) .\",\n \"( G ) 0 . 01% ZTDO prevents resumption of post-dauer development in approximately 40% of wild-type animals and nearly all Ppa-obi-1 ( tu404 ) mutants .\",\n \"This ZTDO inhibition of dauer exit is dependent on the cGMP-dependent PKG , Ppa-egl-4 .\",\n \"PS312 ( n = 384 , 654 , 267 ) ; PS1843 ( n = 195 , 139 , 101 ) ; Ppa-obi-1 ( n = 339 , 343 , 146 ) ; Ppa-egl-4 ( tu374 ) ( n = 123 , 181 , 77 ) ; Ppa-obi-1;Ppa-egl-4 ( n = 132 , 144 , 63 ) ; N2 ( n = 131 , 109 , 115 ) .\",\n \"( H ) Exposure to 0 . 50% ETDA moth pheromone from Spodoptera littoralis does not paralyze Ppa-obi-1 mutant and wild-type J4 larvae .\",\n \"PS312 ( n = 40 , 103 ) ; PS1843 ( n = 40 , 64 ) ; Ppa-obi-1 ( n = 40 , 193 ) .\",\n \"p values indicate Dunnett's multiple comparisons test to wild-type PS312 .\",\n \"***p < 0 . 001; **p < 0 . 01 indicate difference between ethanol control and ZTDO exposed groups ( pairwise unpaired t test ) .\",\n \"Error bars are SEM and n is the number of nematodes tested . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 004 Detailed examination revealed that Ppa-obi-1 ( tu404 ) eggs had significantly lower hatching rate in 0 . 10% ZTDO compared to wild-type PS312 .\",\n \"Interestingly , the Washington strain that has natural cGMP-independent attraction to ZTDO was more resistant to 0 . 25–0 . 50% ZTDO than the ZTDO-insensitive California strain .\",\n \"Although ZTDO exposure had no noticeable effect on egg laying output by adult hermaphrodites , eggs continuously exposed to ZTDO for 2 days at an early stage remain arrested at the pre-comma globular embryonic stage , before gastrulation .\",\n \"The embryocidal effect of ZTDO was concentration-dependent and species-specific , since wild-type C . elegans eggs were completely unaffected ( Figure 2A ) .\",\n \"Ppa-obi-1 ( tu404 ) hypersensitivity to the developmental and paralyzing effects of ZTDO also continued into the subsequent J2 and J4 larval stages ( Figure 2B , C ) .\",\n \"Both wild-type PS312 and Ppa-obi-1 ( tu404 ) J2 larvae exhibited ZTDO-induced paralysis , while the Washington strain was significantly more resistant to paralysis .\",\n \"However , by the J4 larval stage , only Ppa-obi-1 ( tu404 ) was sensitive to ZTDO compared to both wild-type P . pacificus strains ( Figure 2D–E ) .\",\n \"Ppa-obi-1 ( tu404 ) J4 larvae exposed to ZTDO displayed initial agitation , followed by full body paralysis between 90 and 120 min .\",\n \"In contrast , wild-type C . elegans J4 larvae displayed only minute sensitivity to ZTDO .\",\n \"To determine if the Ppa-obi-1 ( tu404 ) mutation renders it susceptible to paralysis from other insect pheromones , we also tested the moth pheromone ETDA and found that the J4 larvae of Ppa-obi-1 ( tu404 ) and the wild-type strains were not affected ( Figure 2H ) .\",\n \"All strains , including Ppa-obi-1 ( tu404 ) , were insensitive to ZTDO-induced paralysis when they reach adulthood ( Figure 2F ) , the dominant stage used in our chemotaxis assays .\",\n \"Taken together , our findings show that ZTDO is capable of arresting wild-type P . pacificus embryogenesis and severely paralyzing J2 larvae , indicating that ZTDO is a potential host-specific developmental regulator .\",\n \"We speculate that wild-type Ppa-OBI-1 may serve to protect P . pacificus embryos and reproductive larvae against the volatile anesthetic-like effects of ZTDO from the adult beetle host .\",\n \"The dauer larva ( DL ) is an alternative , non-reproductive , long-lasting stage in nematodes capable of withstanding starvation , as well as the infective stage in parasitic nematodes ( Lee , 2002 ) .\",\n \"To explore the possibility that ZTDO also acts to restrain the dispersive stage of P . pacificus development after hatching , we asked if ZTDO can also regulate DL exit in the presence of food using culturing plates containing 0 . 001–0 . 01% ZTDO and bacteria .\",\n \"Nearly all untreated wild-type P . pacificus DL developed into J4 larvae after 2 days on food , whereas ∼40% of the wild-type worms remained as active DL on 0 . 01% ZTDO with OP50 food that would have otherwise promoted reproductive development ( Figure 2G ) .\",\n \"However for Ppa-obi-1 DL , this dauer exit defect is exacerbated by ZTDO due to paralysis .\",\n \"Approximately 80% of the developmentally blocked Ppa-obi-1 ( tu404 ) DL show immobility on 0 . 001% ZTDO , and nearly 95% of the Ppa-obi-1 ( tu404 ) DL became paralyzed on 0 . 01% ZTDO .\",\n \"Although DL is the primary infective stage in many entomophilic nematodes found on live beetles ( Weller et al . , 2010 ) , we were unable to assess DL's chemotaxis response to ZTDO because of its immobilizing effect on DL .\",\n \"The negative regulation of DL exit by ZTDO is specific to P . pacificus and barely affected C . elegans even at 0 . 01% ZTDO .\",\n \"These results show that 0 . 01% ZTDO can block active wild-type P . pacificus DL from progressing to the reproductive stage without causing paralysis , but 0 . 001–0 . 01% ZTDO significantly reduced dauer exit in Ppa-obi-1 DL by primarily inducing paralysis and death .\",\n \"Since Ppa-obi-1 ( tu404 ) has a defect in DL exit that is further enhanced by exposure to ZTDO , we asked if DL arrest in Ppa-obi-1 ( tu404 ) by ZTDO is similarly dependent on Ppa-EGL-4 function .\",\n \"Egl-4 mutants are defective in a cGMP-dependent protein kinase in C . elegans , which affect dauer formation as well as chemosensation ( Daniels et al . , 2000; L'Etoile et al . , 2002 ) .\",\n \"We found that while the Ppa-egl-4 ( tu374 ) loss-of-function mutation did not significantly affect the ability to exit the dauer stage , the Ppa-egl-4 ( tu374 ) mutation in the Ppa-obi-1 ( tu404 ) background did partially suppress the inhibitory effect of ZTDO , such that Ppa-egl-4;Ppa-obi-1 post-dauer development on 0 . 001% ZTDO is higher than Ppa-obi-1 ( tu404 ) ( Figure 2G ) .\",\n \"The Ppa-egl-4 ( tu374 ) mutation did not , however , reduce the ZTDO inhibition of DL exit in the presence of a higher amount of 0 . 01% ZTDO .\",\n \"This outcome suggests that the susceptibility of Ppa-obi-1 ( tu404 ) DL to ZTDO is partially dependent on Ppa-egl-4 .\",\n \"While the loss of Ppa-obi-1 alone can cause reduced rate of DL exit on food independent of ZTDO , the aggravated DL arrest by ZTDO in Ppa-obi-1 ( tu404 ) is partially mediated by Ppa-egl-4 .\",\n \"Although wild-type P . pacificus adult animals are attracted to ZTDO , the host beetle sex pheromone regulates P . pacificus development by inhibiting embryogenesis , restricting DL exit , and inducing paralysis in larval stages .\",\n \"This ZTDO sensitivity is the most acute at the embryonic stage and is likely to be buffered by wild-type Ppa-OBI-1 in later developmental stages as the strongest hypersensitivity to ZTDO was observed in the J4 stage of the Ppa-obi-1 ( tu404 ) mutant .\",\n \"By contrast , the PS1843 Washington strain that is naturally attracted to ZTDO is more resistant to ZTDO in non-dauer stages compared to the PS312 California wild type .\",\n \"Further studies in other natural populations are needed to determine the correlation between ZTDO attraction and resistance to ZTDO-induced paralysis in P . pacificus .\",\n \"In addition to the specific chemosensory defect in Ppa-obi-1 ( tu404 ) mutants , we also noticed a linked phenotype in its body morphology .\",\n \"Detailed measurements indicate that Ppa-obi-1 ( tu404 ) animals are significantly longer and thinner than wild type ( Figure 3 ) , resulting in a slim body phenotype with adult Ppa-obi-1 ( tu404 ) hermaphrodites ∼20% longer than wild-type animals .\",\n \"The slim body phenotype may also contribute to Ppa-obi-1 ( tu404 ) 's moderate egg-laying defect ( egl; Table 1 ) and reduction in hatching rate similar to Ppa-egl-4 ( tu374 ) mutants , possibly due to defects in vulva formation and cuticle development .\",\n \"Using molecular markers and the slim phenotype , we were able to delineate the Ppa-obi-1 lesion to an ∼138 kb region on Supercontig 1 of chromosome I between markers SSLP21 and SSLP17 ( Figure 4A ) .\",\n \"We subsequently identified all polymorphic sites between PS312 and Ppa-obi-1 ( tu404 ) in the fine mapping interval by Illumina whole genome sequencing and focused on disruptions of predicted open reading frames .\",\n \"We found a single base pair deletion in a predicted coding region orthologous to C06G1 . 1 in C . elegans and confirmed this tu404 deletion by PCR amplification and Sanger sequencing of both wild type and Ppa-obi-1 ( tu404 ) genomic and cDNA .\",\n \"The tu404 allele contains a single base pair deletion in exon 9 .\",\n \"The resulting frame shift and nonsense mutation are predicted to stop translation half way into the reading frame to result in a hypomorphic allele .\",\n \"In the two Ppa-obi-1 splice forms , the longer transcript contains a VKDDDPR peptide that may be the result of an alternative splice donor site .\",\n \"Both splice variants were isolated from the California and Washington strains in equal abundance by sub-cloning RT-PCR products ( data not shown ) , and no differences in nucleotide sequence were found in the coding region of these two strains .\",\n \"Therefore , splice form variation in Ppa-OBI-1 is unlikely to contribute to the difference in insect pheromone attraction between these two strains .\",\n \"The region encoding for VKDDDPR is also absent in all the Ppa-obi-1 orthologs we have examined in available databases ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 03229 . 005Figure 3 . Ppa-obi-1 mutants are long and slim .\",\n \"( A and B )\",\n \"Wild-type J4 larva and young adult .\",\n \"( C and D )\",\n \"Ppa-obi-1 J4 larva and young adult .\",\n \"( E and F )\",\n \"The body lengths and widths of wild-type ( dotted red line ) versus Ppa-obi-1 mutants ( solid black line ) recorded over 4 days post J4 stage ( μm ) ; n = 15–30 animals for each time point .\",\n \"Error bars are SEM .\",\n \"Scale bars denote 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00510 . 7554/eLife . 03229 . 006Table 1 . Ppa-obi-1 affects hatching rate and brood sizeDOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 006Hatching rate ( % ) Brood size ( 3 days ) nWild-type ( PS312 ) 95 ± 3156 ± 1618Ppa-obi-1 ( tu404 ) 85 ± 3103 ± 171510 . 7554/eLife . 03229 . 007Figure 4 . Ppa-obi-1 gene structure and protein comparisons .\",\n \"( A ) The order of mapping markers is depicted on top ( not to scale ) .\",\n \"The Ppa-obi-1 mRNA is SL1 trans-spliced and contains 19 exons spanning ∼19 kb .\",\n \"The 50 bp 5′ UTR and 341 bp 3′ UTR are indicated in light blue .\",\n \"The tu404 allele contains a single base pair deletion in exon 9 that results in a frameshift , converting a codon to a stop codon ( inverted triangles ) .\",\n \"The red dot represents the splice variant .\",\n \"( B ) A diagram of the secretion and two BPI lipid-binding domains ( PFAM and SMART ) .\",\n \"( C–E )\",\n \"Transgenic rescue of the Ppa-obi-1 ( tu404 ) .\",\n \"( C ) A 4 . 3 kb promoter driving Ppa-obi-1 cDNA completely rescued cGMP-dependent ZTDO attraction in Ppa-obi-1 ( tu404 ) mutants .\",\n \"PS312 ( N = 21 ) ; obi-1 ( N = 15 ) ; obi-1 rescue ( N = 12 ) .\",\n \"The Ppa-obi-1 transgenic rescue ( D ) restored the body length defect in Ppa-obi-1 ( tu404 ) mutants , PS312 ( n = 15 ) ; obi-1 ( n = 15 ) ; obi-1 rescue ( n = 19 ) as well as ( E ) significantly reduced ZTDO-induced paralysis in Ppa-obi-1 ( tu404 ) J4 larvae .\",\n \"PS312 ( N = 11 ) ; obi-1 rescue rfp+ ( N = 12 ) ; obi-1 rescue rfp− ( N = 5 ) .\",\n \"**p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 ( Tukey's multiple comparisons test ) .\",\n \"Error bars represent SEM .\",\n \"N is the number of assays performed and n is the number of individual worms scored for each time point . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00710 . 7554/eLife . 03229 . 008Figure 4—figure supplement 1 . An alignment of putative Ppa-OBI-1 protein orthologs . Caenorhabditis elegans ( C06G1 . 1 ) , C . briggsae ( CBP03891 ) , C . remanei ( RP35181 ) , C . japonica ( JA26805 ) , Brugia malayi ( BM32564 ) , and a human Bactericidal Permeability Increasing protein ( NP_001716 ) .\",\n \"The predicted protein secretion signal peptides for Ppa-OBI-1 ( PP33427 ) and C06G1 . 1 ( CE27054 ) are 17–18 amino acids long at the N terminus ( red bar ) .\",\n \"The longest predicted protein splice forms are used in the alignment , with orthologs in Caenorhabditis species containing ‘GS’ ( red boxed ) and Ppa-OBI-1 in P . pacificus containing ‘VKDDDPR’ peptides ( red boxed with a red dot ) .\",\n \"The single base pair deletion in Ppa-obi-1 ( tu404 ) converts the leucine codon to a stop codon ( inverted red triangle ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00810 . 7554/eLife . 03229 . 009Figure 4—figure supplement 2 . A maximum likelihood relationship of lipid-binding proteins in P . pacificus and C . elegans . The ‘PP’ and ‘CE’ prefixes denote P . pacificus and C . elegans proteins , respectively ( www . wormbase . org ) .\",\n \"10 orthologous protein pairs are circled .\",\n \"Ppa-OBI-1 ( PP33427 ) and the 12 other paralogs are also represented in the P . pacificus proteome ( Pristionchus . org ) .\",\n \"A human BPI protein ( GenBank NP_001716 . 2 ) was used as outgroup .\",\n \"Bootstrap values are indicated along branches . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 00910 . 7554/eLife . 03229 . 010Figure 4—figure supplement 3 . Chemosensory behavior of C . elegans mutants C06G1 . 1 and nrf-5 . A mutation in C06G1 . 1 ( vc20357 ) , the ortholog of Ppa-obi-1 , does not alter chemotaxis behavior towards 10% ZTDO , 1% 2 , 3-pentanedione , 1% 2 , 3-butanedione ( diacetyl ) , and 1% isoamyl alcohol .\",\n \"However , nrf-5 ( sa513 ) mutants have reduced attraction to 1% 2 , 3-butanedione and 1% isoamyl alcohol .\",\n \"nrf-5 ( sa513 ) was not tested for response to 2 , 3-pentanedione .\",\n \"Significant differences to wild-type are indicated by p values ( two sampled t test ) .\",\n \"N ≥ 10 replicates .\",\n \"Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01010 . 7554/eLife . 03229 . 011Figure 4—figure supplement 4 . Reduction of C06G1 . 1 function produce a slimmer body phenotype .\",\n \"( A ) RNAi using two dsRNA fragments of C06G1 . 1 fed to C . elegans rrf-3 ( pk1426 ) resulted in a longer and thinner body proportion .\",\n \"( B ) Genetic mutation in C06G1 . 1 ( VC20357 ) resulted in significantly slimmer body proportion ( increase length/width ) similar to RNAi against C06G1 . 1 and the Ppa-obi-1 ( tu404 ) mutant .\",\n \"n ≥ 13 animals .\",\n \"Note that y-axes do not begin at zero .\",\n \"Significant differences to wild-type are indicated by p values ( two sampled t-test ) .\",\n \"Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 011 To confirm that Ppa-obi-1 is indeed responsible for the lack of ZTDO attraction in adults and for hypersensitivity to ZTDO-induced paralysis in the J4 larvae , we made transgenic lines containing a 4 . 3 kb Ppa-obi-1 promoter driving the Ppa-obi-1 full-length cDNA ( Schlager et al . , 2009 ) .\",\n \"The introduction of the wild-type Ppa-obi-1 transgene restored the cGMP-dependent ZTDO attraction in Ppa-obi-1 ( tu404 ) adults compared to Ppa-obi-1 ( tu404 ) adults and sibling animals that did not segregate for the rescue construct ( Figure 4C ) .\",\n \"Furthermore , the Ppa-obi-1 rescue transgene significantly shortened the body length of Ppa-obi-1 ( tu404 ) adults ( Figure 4D ) , confirming that both the ZTDO chemosensory and the long body length phenotype used for subsequent positional mapping were due to the same mutation in Ppa-obi-1 ( tu404 ) .\",\n \"Lastly , we tested the ability of the wild-type Ppa-obi-1 transgene to reduce the ZTDO hypersensitivity and found that it significantly reduced the percentage of ZTDO-induced paralysis in Ppa-obi-1 ( tu404 ) J4 larvae ( Figure 4E ) .\",\n \"Ppa-obi-1 therefore mediates ZTDO chemosensation , body dimensions , and ZTDO susceptibility .\",\n \"Sequence analyses of coding regions show that Ppa-obi-1 and C06G1 . 1 encode for proteins that represent a class of highly conserved but functionally diverse group of secreted lipid-transfer/lipid-binding proteins present in almost all metazoans except Drosophila ( Beamer et al . , 1998 ) , including lipopolysaccharide-binding proteins ( LBP ) ( Tobias et al . , 1997 ) , bactericidal permeability-increasing proteins ( BPI ) ( Weiss et al . , 1978 ) , phospholipid transfer proteins ( PLTP ) ( Gautier and Lagrost , 2011 ) , and cholesteryl ester transfer proteins in mammals ( CETP ) ( Kawano et al . , 2000 ) .\",\n \"The LBP/BPI/CETP/PTLP family in humans is known to play a role in immunological responses to bacterial pathogens and mediate the transfer of cholesteryl ester and triglycerides in the blood plasma ( Bruce et al . , 1998; Kawano et al . , 2000; Schultz and Weiss , 2007 ) .\",\n \"Ppa-OBI-1 and C06G1 . 1 each contain two predicted lipid-binding domains as well as a predicted secretion peptide sequence in the N-terminal regions ( PFAM and SMART; SignalP 4 . 0 Server ) ( Figure 4—figure supplement 1 ) .\",\n \"To identify all members of the LBP family in the sequenced genome and transcriptome of P . pacificus , we performed reciprocal BLASTP searches ( WormBase WS221 ) , which indicated 12 members predicted to be encoded in the P . pacificus genome .\",\n \"Maximum likelihood phylogenetic analysis of the OBI-1/C06G1 . 1 LBP protein family indicates 10 closely related orthologous pairs in the two species .\",\n \"Interestingly , the lengths of the branches suggest that all the orthologs between P . pacificus and C . elegans share similar divergence times , with the exception of the OBI-1/C06G1 . 1 pair that seemed to have undergone faster divergence relative to other paralogs ( Figure 4—figure supplement 2 ) .\",\n \"Consistent with their roles in immune response in mammals , a member of the LBP family in C . elegans , F44A2 . 3 , was found to be up-regulated in response to several species of pathogenic bacteria ( Wong et al . , 2007 ) .\",\n \"Another C . elegans LBP member , NRF-5 , is partially necessary for nose contraction in response to the anti-depressant fluoxetine ( Prozac ) ( Choy et al . , 2006 ) , and for mediating phosphatidylserine transfer from apoptotic cells to engulfing cells during cell corpse removal ( Zhang et al . , 2012 ) .\",\n \"We found that nrf-5 ( sa513 ) mutants are also defective in attraction to 2 , 3-butanedione ( diacetyl ) , and isoamyl alcohol , although the C06G1 . 1 ( vc20357 ) allele with a missense mutation did not show chemosensory defects ( Figure 4—figure supplement 3 ) .\",\n \"Thus LBP members may have a conserved previously uncharacterized role in chemosensation in diverse nematodes .\",\n \"To determine if C06G1 . 1 can also regulate body morphology in C . elegans , we performed C06G1 . 1 RNAi by feeding .\",\n \"C06G1 . 1 RNAi was previously observed to generate a long body phenotype in a large-scale RNAi screen in C . elegans ( Simmer et al . , 2003 ) .\",\n \"We used this fragment ( Fragment 1 ) , along with another C06G1 . 1 fragment more proximal to the 3′ end ( Fragment 2 ) , to quantify the effect of C06G1 . 1 RNAi in the enhanced RNAi rrf-3 ( pk1426 ) strain .\",\n \"Although C06G1 . 1 RNAi did not significantly change the absolute body length of adult worms , we found RNAi against C06G1 . 1 using both fragments resulted in a significantly slimmer body proportion ( length/width ratio ) similar to the Ppa-obi-1 ( tu404 ) mutant ( Figure 4—figure supplement 4 ) .\",\n \"A missense mutation in C06G1 . 1 ( VC20357 ) also resulted in a similar slim body phenotype .\",\n \"Thus both Ppa-OBI-1 and its C . elegans ortholog also share a conserved function in regulating body proportions .\",\n \"To characterize which tissue types Ppa-OBI-1 functions in , we sought to determine the Ppa-obi-1 expression pattern using a Ppa-obi-1p::gfp transcriptional reporter gene containing a 4 . 3 kb promoter fragment upstream of the Ppa-obi-1 ATG initiation codon .\",\n \"Our analysis was based on a stably integrated transgenic line csuIs01 and parsimonious inferences to homologous C . elegans cell positions and known cellular functions .\",\n \"Detailed analyses of all developmental stages show that the earliest Ppa-obi-1 expression occurs in at least one cell in the pre-comma embryonic stage but soon disappears until weak expression reappears in the hyp7 and seam cells in J4 larvae ( Figure 5 ) .\",\n \"The most robust and diverse Ppa-obi-1 expression was found in mid-J4 hermaphrodites , which is consistent with real-time quantitative PCR results indicating that transcript levels in J2 and J3 larvae are less than in J4 larvae ( data not shown ) .\",\n \"In the mid-J4 stage , Ppa-obi-1 is strongly expressed in the major hypodermal syncytium and the lateral hypodermal cells that cover most of the body ( hyp7 and seam cells ) as well as the ventral tail ( hyp 8 and 9 ) ( Figure 5 ) .\",\n \"In contrast to young adult C . elegans , which have 16 seam cells on each lateral surface ( H0–H3 , V1–V6 , T descendants; The WormAtlas ) , we counted only 15 seam cells per side in P . pacificus J4 larvae ( n = 20 ) .\",\n \"Ppa-obi-1 is also strongly expressed in the P ( 5–7 ) .\",\n \"p cell descendants that form the vulva lumen ( vulC , D , E ) during the mid-J4 stage until just prior to vulval eversion as well as in the vulva muscles ( likely vm1 , 2 ) ( Figure 5B inset; Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 03229 . 012Figure 5 . Ppa-obi-1 and C06G1 . 1 expression in their respective nematode species .\",\n \"( A–D )\",\n \"An integrated Ppa-obi-1 promoter::gfp transgene ( csuIs01 ) in P . pacificus .\",\n \"( A ) Lateral view of a single animal showing prominent expression in an amphid sheath cell , seam cells , and the syncytial hypodermal cell ( hyp7 ) in a J4 hermaphrodite .\",\n \"( B ) Medial view of a transgenic animal with expression in the putative dorsal and ventral inner labial ( IL ) or outer labial ( OL ) neuronal support cells ( socket or sheath ) , the duct and excretory cells on the ventral side , the amphid sheath cell on the dorsal side ( arrow head ) , and the vulval muscles .\",\n \"The inset shows a ventral view of the young adult vulva in another animal .\",\n \"Autofluorescence is visible in the intestine .\",\n \"( C ) A DiI-labeled neuron ( red arrow ) anterior to a Ppa-obi-1p::gfp expressing amphid sheath cell ( white arrow ) is dorsal to an excretory cell ( arrow head ) .\",\n \"A putative IL or OL socket or sheath cell ( arrow ) is also visible .\",\n \"( D ) DIC and fluorescent overlay image of expression in early P . pacificus embryo .\",\n \"( E–I )\",\n \"C06G1 . 1 promoter::gfp transgenes ( csuEx02 , csuEx03 ) in C . elegans .\",\n \"( E ) Lateral view of a seam cell expression in a L3 hermaphrodite .\",\n \"( F ) Medial view of putative inner or outer labial neuron support cells ( arrow ) and excretory cells ( arrowhead ) .\",\n \"No amphid sheath shows gfp expression .\",\n \"( G ) Ventral view of head region shows prominent expression in excretory cells and canal ( arrow ) .\",\n \"( H ) Ventral view of young adult vulva .\",\n \"( I ) Embryonic expression in C . elegans embryos appears later , stronger , and in more cells than in Ppa-obi-1 .\",\n \"Anterior is left and the scale bars represent 50 μm .\",\n \"Each panel is representative of ≥50 animals . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01210 . 7554/eLife . 03229 . 013Figure 5—figure supplement 1 . Ppa-obi-1 is expressed in putative amphid socket cells , amphid sheath cells , and vulval cells . Dorsal and ventral views of Ppa-obi-1p::gfp expression in roller prl-1 ( tu92 ) background with merged DIC and fluorescence ( A , C , E ) and corresponding fluorescence images ( B , D , F ) .\",\n \"( A and B )\",\n \"Dorso-lateral view of head region shows possible Ppa-obi-1 expression in a socket cell ( top ) , an IL sheath cell ( bottom ) , two seam cells ( asterisks ) , and two amphid sheath cells ( arrows ) .\",\n \"( C and D )\",\n \"Ventral view of a mid-J4 larvae vulva shows Ppa-obi-1p::gfp expression in P ( 5–7 ) .\",\n \"p vulval epidermal cell descendants , a putative vulval muscle ( triangle ) , and a hyp7 cell ( asterisk ) .\",\n \"( E and F )\",\n \"Ventral view of a young adult vulva ( top left ) shows Ppa-obi-1p::gfp expression in P ( 5–7 ) .\",\n \"p vulval epidermal cell descendents and four vulval muscles ( triangles ) and a lateral view of a mid-J4 vulva ( bottom right ) in another animal .\",\n \"Anterior is left and the scale bars represent 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 01310 . 7554/eLife . 03229 . 014Figure 5—figure supplement 2 . Targeted misexpression of Ppa-obi-1 in C . elegans . L2 larvae show amphid sheath cell expression using ( A ) Cel-T02B11 . 3 promoter::Ppa-obi-1:gfp ( csuEx29 ) and ( B ) Cel-F16F9 . 3 promoter::Ppa-obi-1:gfp ( csuEx30 ) .\",\n \"The co-injection marker myo-2p::DsRED is visible in the anterior pharyngeal bulb due to bleeding over into the GFP spectrum .\",\n \"Arrows indicate amphid sheath cell bodies .\",\n \"( C ) Chemotaxis response to the ZTDO beetle pheromone remains unchanged towards 2% or 10% ZTDO .\",\n \"( D ) ZTDO-induced paralysis in L4 larvae shows that transgenic Cel-F16F9 . 3p::Ppa-obi-1::gfp are better protected than wild-type animals in a 16 hr assay .\",\n \"N2 ( N = 8 ) , T02B11 . 3p::Ppa-obi-1 ( N = 6 ) , F16F9 . 3p::Ppa-obi-1 ( N = 6 ) .\",\n \"Anterior is left and the scale bar represents 25 μm .\",\n \"p value indicates Dunnett's multiple comparisons test to wild-type N2 .\",\n \"Error bars are SEM and N is the number of assays performed . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 014 Most notably , Ppa-obi-1 expression was observed in the two bilaterally symmetrical specialized epithelial cells that envelope the chemosensory neurons , known as the amphid sheath cells ( Figure 5A–B , arrows ) .\",\n \"The amphid sheath cells are support cells for the sensory amphid neurons and completely enclose the ciliated endings of the AWA , AWB , and AWC chemosensory neurons in C . elegans .\",\n \"Ppa-obi-1p::gfp expression in the putative amphid sheath cells lie dorsal–lateral and posterior to the DiI-labeled amphid neurons ASK , ADL , and ASI ( Figure 5C ) .\",\n \"DiI staining of amphid neurons in Ppa-obi-1 mutants is superficially wild type , suggesting that Ppa-OBI-1 is unlikely to be involved in the development of the amphid neurons ( data not shown ) .\",\n \"We also noticed expression in four unknown cell types anterior to the metacarpus ( anterior pharyngeal bulb ) , which could be putative neuronal support cells for the inner labial or outer labial quadrant sensilla ( IL or OLQ ) ( Figure 5—figure supplement 1 ) .\",\n \"Ppa-obi-1 is also expressed in putative duct and excretory cells on the ventral anterior side that form part of the excretory system ( Figure 5B , arrowhead ) .\",\n \"Based on conserved DiI labeling of P . pacificus amphid neurons and cell positions , the Ppa-obi-1 expression profile and secretion peptide sequence suggest that Ppa-OBI-1 is likely an extracellular protein involved in sensory neuron support , epithelial cell development , and osmoregulation .\",\n \"Although Ppa-OBI-1 and its ortholog C06G1 . 1 in C . elegans share 61% amino acid identity , P . pacificus and C . elegans have opposite responses to the pheromone ZTDO .\",\n \"While small changes in sequence can lead to large changes in protein function , the absence of expression of the C . elegans obi-1 ortholog in amphid sheath cells suggests that changes in gene expression pattern may also play a role in determining ZTDO sensitivity .\",\n \"We therefore sought to determine if the Ppa-obi-1 ortholog C06G1 . 1 have shared tissue expression with Ppa-obi-1 .\",\n \"We examined endogenous C06G1 . 1p::gfp expression in C . elegans in five independent transgenic lines with extrachromosomal arrays and found strong mid-L4 larvae expression in the seam cells , two tail hypodermal cells , four cells in the excretory duct system ( duct , excretory , gland cells ) , vulval muscles , and possible IL or OLQ sheath or socket neuronal support cells similar to the expression pattern observed for Ppa-obi-1 ( Figure 5E–H ) .\",\n \"In contrast to the earliest Ppa-obi-1 expression in pre-comma stage , C06G1 . 1 expression is detectable during late embryogenesis in putative seam cells ( Figure 5I ) .\",\n \"This seam cell expression is earlier than Ppa-obi-1p::gfp in the L1 stage , and is maintained until the young adult stage .\",\n \"A reason for this earlier seam cell expression could be that the equivalent P . pacificus J1 larvae remain inside the egg while C . elegans eggs hatch as L1 larvae ( Fürst von Lieven , 2005 ) .\",\n \"Like Ppa-obi-1 , C06G1 . 1 expression is largely absent in all tissue types when the transgenic animals become 1-day old adult hermaphrodites , coinciding with the fusion of seam cells into a syncytium .\",\n \"This observation indicates that C06G1 . 1 expression in the excretory and epithelial system may be coordinated and involved in seam cell development .\",\n \"Our expression analysis of obi-1 orthologs in the their respective nematode species revealed that the most striking difference is the lack of expression in the amphid sheath cells in C . elegans , suggesting that unlike Ppa-obi-1 , C06G1 . 1 is not likely to function directly with chemosensory neurons in C . elegans .\",\n \"In addition to differences in gene expression pattern , the possibility that there is a ZTDO receptor that interacts with Ppa-OBI-1 in P . pacificus but not in C . elegans may also help to explain how Ppa-obi-1 orthologues confer different ZTDO sensitivities in the two species during certain developmental stages .\",\n \"To explore yet another possibility that changes in both protein function and expression may lead to differences in ZTDO sensitivity , we expressed the Ppa-obi-1:cDNA under the C . elegans amphid sheath cell-specific promoters T02B11 . 3 and F16F9 . 3 in C . elegans ( Bacaj et al . , 2008; Kramer et al . , 2010; Oikonomou et al . , 2011; Procko et al . , 2011 ) .\",\n \"Both T02B11 . 3p::Ppa-obi-1:gfp ( csuEx29 ) and F16F9 . 3p::Ppa-obi-1:gfp ( csuEx30 ) transgenic animals showed strong GFP expression in the amphid sheath cells ( Figure 5—figure supplement 2 ) as well as phasmid sheath cells in the tail region ( not shown ) .\",\n \"While the targeted misexpression of Ppa-obi-1 in C . elegans did not alter the strong avoidance behavior towards either 2% or 10% ZTDO , Ppa-obi-1 expression in C . elegans did reduce the paralytic effects of ZTDO ( Figure 5—figure supplement 2 ) .\",\n \"Because wild-type C . elegans N2 is not susceptible to ZTDO-induced paralysis at 0 . 5% ZTDO that P . pacificus is , we increased the stringency of the assay by using 10% ZTDO , incubating for 16 hr , and exposing the animals in the absence of OP50 food .\",\n \"Under this enhanced ZTDO paralysis assay , we found that F16F9 . 3p::Ppa-obi-1:gfp ( csuEx30 ) transgenic L4 larvae but not T02B11 . 3p::Ppa-obi-1:gfp ( csuEx29 ) exhibited a small but statistically significant increase in protection from ZTDO-induced paralysis compared to wild-type N2 ( Figure 5—figure supplement 2C , D ) .\",\n \"Because we could not detect apparent differences in GFP expression patterns between the two amphid promoters , the ability of F16F9 . 3p::Ppa-obi-1:gfp to reduce ZTDO paralysis may be due to subtle transient expression differences .\",\n \"These results suggest that while the expression of the Ppa-OBI-1 protein in the C . elegans amphid sheath cells is not sufficient to reduce avoidance to ZTDO , it can contribute to the obi-1-dependent protection from ZTDO-induced paralysis .\"\n ],\n [\n \"Sensitivity to ZTDO-induced paralysis/arrest is Ppa-obi-1-dependent predominantly during the J4 larval stage and , to a lesser degree , in the embryos and dauers .\",\n \"Given the diversity of tissues expressing Ppa-obi-1 in the J4 larvae , it is not apparent which tissues require Ppa-obi-1 expression for proper chemosensation and if the same tissues are involved with protection from ZTDO-induced paralysis in wild-type animals ( Figure 6 ) .\",\n \"Our gene expression studies show that despite coding highly similar proteins , P . pacificus Ppa-obi-1 is uniquely expressed in the amphid sheath cells compared to its C . elegans ortholog C06G1 . 1 .\",\n \"Based on this species-specific expression pattern , we are tempted to speculate that these chemosensory glial cells , the IL and amphid sheath cells , play a dominant role in mediating ZTDO sensitivity .\",\n \"However , additional experiments will be needed to test the possibility that epidermal cells such as the vulva , hypodermis , and seam cells , or cells in the excretory system may also mediate ZTDO sensing . 10 . 7554/eLife . 03229 . 015Figure 6 . Summary and model of the oriental beetle pheromone ZTDO on P . pacificus development and lifecycle .\",\n \"( Top )\",\n \"Peak Ppa-obi-1 expression in the wild-type J4 larvae coincides with the least sensitive developmental stage to the paralytic effects of the beetle pheromone ZTDO ( at 0 . 5% ) .\",\n \"Ppa-obi-1 mutants are most sensitive to ZTDO-induced paralysis in the J4 stage .\",\n \"Our working model on Ppa-OBI-1 functions in the J4 larvae hypothesizes that Ppa-OBI-1 proteins in the extracellular space , such as the lumen of the amphid sheath cell , may be required to remove ZTDO bound to odor receptors on neurons .\",\n \"( Bottom )\",\n \"This working model shows both P . pacificus adults and dauer larvae from non-beetle populations associate with the host oriental beetle through attraction to the beetle's sex pheromone ZTDO , whereby the progeny born on the beetle are arrested as eggs or dauer larvae .\",\n \"Death of the host allows microorganism to grow , which then encourages dauer larvae to exit and resume reproductive development .\",\n \"Several iterations of asynchronous nematode populations likely complete each dispersal cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 015 Given the growing evidence that the amphid sheath cells in C . elegans are critical for proper neuronal development and function of the 12 pairs of chemosensory amphid neurons ( Shaham , 2006 ) , we propose the following models on how the Ppa-OBI-1 protein can act in the sheath cells to directly or indirectly modulate ZTDO chemosensation .\",\n \"In C . elegans , many proteins have enriched expression in the amphid sheath , such as DAF-6 and the secreted venom allergen-like VAP-1 , and these secretions are corroborated by dense populations of vesicles in amphid sheath cells under electron microscopy ( Bacaj et al . , 2008; Doroquez et al . , 2014 ) .\",\n \"However , it is not yet known which amphid proteins , if any , are required in processing odors using behavioral assays .\",\n \"Interestingly , DAF-6 is a Patched-related protein with expression pattern that overlaps with Ppa-obi-1 expression ( amphid lumen , excretory canal , vulva lumen ) and is required for chemotaxis and dauer formation ( Perens and Shaham , 2005 ) .\",\n \"Similarly , Ppa-obi-1 expression in the putative sheath or socket cells of the inner labial neurons may also be involved in ZTDO sensing , as recent ultrastructural electron micrograph reconstructions indicate that BAG ( gas sensing ) and FLP ( nociceptive ) neurons in C . elegans associate closely with the socket cells of the inner labial sensillae ( Doroquez et al . , 2014 ) .\",\n \"One possible mode of action for ZTDO may be its ability to change cell membrane permeability , thereby indirectly affecting the functions of the glia-associated neurons .\",\n \"Hence , Ppa-OBI-1 may function to counter the cell membrane permeability changes brought about by ZTDO by interacting with cell membrane lipids .\",\n \"Alternatively , Ppa-OBI-1 could mediate pheromone binding directly .\",\n \"In lepidopterans such as the wild silk moth Antheraea polyphemus and the cotton leafworm Spodoptera littoralis , pheromone-degrading and odorant-degrading enzymes ( PDEs and ODEs ) are esterases specifically expressed in the male antennal sensillar lymph analogous to the amphid lumen ( Ishida and Leal , 2005; Durand et al . , 2011 ) .\",\n \"The termination of the signal by removing odors that have already triggered a response is an important process for preventing odor adaptation and increasing odor sensitivity .\",\n \"Thus , it is possible that Ppa-OBI-1 acts as a solubilizing factor for the lipid pheromones in the excretion-filled amphid sheath lumen ( Figure 6 ) .\",\n \"The weak ability of amphid sheath-specific expression of Ppa-OBI-1 to confer ZTDO protection in C . elegans suggests that both expression pattern and protein function could be important factors in ZTDO sensing in P . pacificus .\",\n \"Future work will be necessary to determine if Ppa-obi-1 expression restricted to the amphid sheath is sufficient to rescue the ZTDO attraction defect in P . pacificus and if the amphid sheath cells are required to mediate ZTDO sensing .\",\n \"Given that Ppa-obi-1-independent susceptibility to ZTDO occurs during the J2 larval stage when Ppa-obi-1 is expressed in the seam and hypodermal cells rather than in the amphid sheath that is only active in the J4 larvae , ZTDO-induced paralysis likely acts through the extracellular matrix of the nematode cuticle .\",\n \"P . pacificus adults , whose cuticle may be compositionally different from the larval cuticle , are not susceptible to ZTDO .\",\n \"However in P . pacificus embryos , the susceptibility of ZTDO may be due to ZTDO limiting gas or ion exchange with the environment rather than direct interaction between ZTDO and cellular components , given that nematode egg shells are considered to have very limited permeability accessible only to small molecular weight solutes and gas molecules ( Chitwood and Chitwood , 1974 ) .\",\n \"Since ZTDO exposure does not lead to embryonic arrest and paralysis in C . elegans , ZTDO must also interact with other non-conserved P . pacificus-specific molecules that have yet to be identified .\",\n \"There are very few known nematocides from natural compounds .\",\n \"One rare example comes from the oyster mushroom Pleurotus ostreatus , which secretes droplets that can immobilize rhabditid nematodes in an hour , though its effects on embryos have not been investigated ( Barron and Thorn , 1987; Stadler et al . , 1994 ) .\",\n \"Future work on the different susceptibility to ZTDO may help to answer if there is a wider pattern of negative correlation between ZTDO attraction and ZTDO-induced paralysis in other natural isolates , as observed in the California and Washington strains .\",\n \"Such antagonistic traits may reveal possible co-evolution between host finding and host tolerance in necromenic associations .\",\n \"We further speculate that in the natural ecology of P . pacificus , ZTDO may enable the beetle host to limit the nematode number and to thus synchronize the developmental stage of infective P . pacificus by arresting embryogenesis and lethally paralyzing J2 larvae .\",\n \"The unexpected role of a P . pacificus attractant to act as a developmental regulator against the younger larval stages , particularly the dauer larvae ( DL ) , suggests that the ZTDO-resistant adult stage is a potential dispersal stage .\",\n \"This attenuated antagonistic interaction also revealed that nematode larvae and adults may be susceptible to distinct selective pressures .\",\n \"P . pacificus DL does not disperse readily , likely due to the secretion of the wax ester nematoil that enables P . pacificus to form dauer towers when potential hosts are nearby ( Penkov et al . , 2014 ) .\",\n \"Therefore under natural conditions , lower concentrations of ZTDO than the concentrations we have tested may serve as a close-range host dwelling strategy by inhibiting DL dispersal to form dauer aggregates .\",\n \"The developmental role of ZTDO may be part of a mechanism to limit nematode dispersal until the beetles mature .\",\n \"In these respects , the promotion of a non-DL dispersal stage and inhibition of DL dispersal by ZTDO resemble other known host-derived compounds found in the entomophilic nematode B . xylophilus and the phoretic nematode C . japonica ( Okumura et al . , 2013; Zhao et al . , 2013 ) .\",\n \"Attraction and paralysis by ZTDO may have co-evolved in the necromenic association to allow successive populations of founder P . pacificus DL and adults to infect beetles , culminating in arrested DL remaining on sexually mature beetles during the dispersal cycle .\",\n \"The pheromone arrested DL state may also help to account the observation that Pristionchus nematodes emerge several days later than rhabditids from the same species of host beetles .\",\n \"Our findings that a lipid-binding protein has a role in coordinating chemosensation , body proportions , and protection from a host pheromone encourages fresh inquiries on additional genes involved in ZTDO sensory signaling and its mechanism of action .\"\n ],\n [\n \"All nematodes were raised on NGM media containing OP50 E . coli at ∼23°C as previously described ( Hong and Sommer , 2006a; Hong et al . , 2008a ) .\",\n \"The following strains were used: P . pacificus: California ( PS312 ) wild type , Washington ( PS1843 ) , Ppa-obi-1 ( tu404 ) I; Ppa-egl-4 ( tu374 ) IV ( null ) ; Ppa-obi-3 ( tu405 ) ; csuIs01 [Ppa-obi-1p::gfp; Ppa-egl-20p::rfp; PS312 gDNA]X; rlh49 ( csuIs01; tu374 ) ‘Ppa-obi-1p::gfp; Ppa-egl-4’; rlh50 ( csuIs01; tu92 ) ‘Ppa-obi-1p::gfp; roller’; rlh51 ( csuIs01; tu404 ) ‘Ppa-obi-1p::gfp; Ppa-obi-1’ .\",\n \"C . elegans: Bristol ( N2 ) wild type , nrf-5 ( sa513 ) , csuEx02 [C06G1 . 1p::gfp; Cel-myo-2::DsRED]; csuEx03 [C06G1 . 1p::gfp; pRF4 roller] , csuEx29 [T02B11 . 3p::Ppa-obi-1:gfp; Cel-myo-2::DsRED] , and csuEx30 [F16F9 . 3p::Ppa-obi-1:gfp; Cel-myo-2::DsRED] .\",\n \"The whole-genome sequenced strain vc20357 was outcrossed to him-5 ( e1467 ) three times and the C06G1 . 1 locus was sequenced to confirm that the slim body phenotype is linked to the missense gk306193 mutation ( D48N ) .\",\n \"We X-ray mutagenized wild-type P . pacificus PS312 ( California ) at 325 Roentgen for 25 min with radioactive beryllium and picked 40 Po onto individual plates .\",\n \"We subsequently picked 1067 F2s from 400 F1s and screened 713 viable F3 populations for insensitivity to 150 nl of oriental beetle pheromone , Z-7-tetradecen-2-one ( 99% pure; Bedoukian Research , CT ) after 1 hr treatment with 1 mM exogenous 8-Bromo-cGMP ( Sigma–Aldrich , MO ) ( Hong et al . , 2008a ) .\",\n \"100 mM nicotinic acid ( Sigma–Aldrich , MO ) was dissolved in water .\",\n \"Population chemotaxis assays were conducted for ∼16 hr at 23°C , and individual candidate F3 from plates showing insensitivity were rescued onto fresh plates by mouth pipetting .\",\n \"We repeated the cGMP-treated chemotaxis assay from the progeny of the candidate mutant lines two more times with at least two duplicate assay plates per line .\",\n \"Only those lines that failed the third round of chemotaxis assay were considered Oriental Beetle pheromone Insensitive obi alleles .\",\n \"Ppa-obi-1 maintained long body morphology and obi-3 displayed more coiled body posture after 3× outcrossing to wild-type PS312 and selecting the ZTDO insensitive phenotype .\",\n \"Because both the chemotaxis and morphological phenotypes co-segregate after 5× outcross , we subsequently used the long body phenotype as a visible marker for Ppa-obi-1 during positional mapping .\",\n \"Population chemotaxis assays were performed as previously described based on modified protocol for C . elegans ( Bargmann et al . , 1993; Hong and Sommer , 2006a ) .\",\n \"Well-fed J4 and adult worms were washed in M9 buffer twice before assay .\",\n \"For a more consistent response , C . elegans was washed in spring water rather than M9 buffer before the ZTDO avoidance assay .\",\n \"For the exogenous cGMP treatment , each washed population was split for mock or 1 hr treatment with 1 mM 8-Bromo-cGMP ( in water ) .\",\n \"For each condition , at least 10 worms had to reach the odor sources in each assay and 12 assays ( sample size N ) were performed over three sessions .\",\n \"Chemotaxis assays with transgenic obi-1 rescue worms were scored by rfp expression in the tail .\",\n \"20 live worms or eggs from non-starved cultures were placed on NGM agar plates with fresh OP50 .\",\n \"For consistency , eggs were synchronized by picking ∼50 adults onto OP50 and allowing them to lay eggs for 3 hr .\",\n \"The eggs were then transferred to different plates and counted .\",\n \"10 µl of ZTDO or ETDA diluted in ethanol was added to the lid of the plate , immediately sealed with Parafilm , and incubated at room temperature .\",\n \"Larvae were scored 3 hr later or the next day by counting the number of paralyzed and active worms .\",\n \"A worm was scored as paralyzed if it did not move at least one pharynx length after tapping on the outside of the plate .\",\n \"Eggs were scored 2 days later by counting the number of unhatched eggs .\",\n \"For dauer larvae exit assays on NGM plates containing none , 0 . 001% or 0 . 01% ZTDO ( vol/vol ) , 25 dauer larvae from the same 2- to 3-week old starved plate were picked onto each type of plate with OP50 and scored for arrested/active dauer larvae , J3 larvae , or J4 larvae after 2 days at ∼23°C .\",\n \"A single library was paired-end sequenced twice in separate flow cell lanes with 2 × 76 bp that yielded 61 million and 65 million reads after preprocessing ( quality control , adapter removal ) .\",\n \"More than 90% of all reads could be mapped to the P . pacificus genome ( www . pristionchus . org ) with BWA ( version 0 . 5 . 9 ) for each library .\",\n \"We then employed ACCUSA for SNP detection ( Froehler and Dieterich , 2010 ) .\",\n \"Briefly , we performed a head-to-head comparison of the mutant Ppa-obi-1 and wild-type PS312 mapped libraries .\",\n \"The algorithm represents read stacks ( base calls and qualities thereof ) as Dirichlet distributions .\",\n \"For any given genome position , we require a read coverage of at least three in both samples .\",\n \"The smaller read stack serves as reference distribution from which the larger stack is drawn .\",\n \"A probability density for this draw is computed , and a SNP is called if the log-density is lower than −30 .\",\n \"Stable transgene expression in P . pacificus requires co-injection of endogenous genomic DNA and co-injection markers containing compatible cohesive ends with the target gene as previously described ( Schlager et al . , 2009; Cinkornpumin and Hong , 2011 ) .\",\n \"A 4 . 3 kb Ppa-obi-1 promoter fragment was amplified from PS312 gDNA using RHL121/RHL136 ( primary ) and RHL124/RHL149 ( secondary ) primers .\",\n \"The Ppa-obi-1 promoter was then fused with the gfp- Ppa-rpl-23-3′UTR by PCR ( Hobert , 2002 ) .\",\n \"PS312 animals were injected with a DNA mix containing a final concentration of 10 ng/μl of the SpeI/SalI digested 4 . 3 kb Ppa-obi-1 promoter::NLS-gfp-SalI fusion PCR product from three independent reactions; 10 ng/μl SpeI digested Ppa-egl-20p::rfp linearized plasmid; and 60 ng/μl SpeI/SalI digested PS312 genomic DNA .\",\n \"Individual F1s expressing Ppa-egl-20p::rfp in the hermaphrodite tail were selected for Ppa-obi-1 expression analysis .\",\n \"Two stable transgenic lines were initially generated with similar expression patterns .\",\n \"We used the stably transmitted reporter line csuIs01[Ppa-obi-1p::gfp; Ppa-egl-20p::rfp; PS312 gDNA]X for detailed expression analyses .\",\n \"csuIs01 is likely to be the result of a spontaneous integration into the X chromosome based on the observation that transgenic males expressing the co-injection rfp marker crossed to Ppa-unc-1 ( ChrIV ) hermaphrodites gave rise to all F1 males lacking rfp expression but all F1 hermaphrodites expressing rfp .\",\n \"More than 50 individuals at all developmental stages were analyzed .\",\n \"Ventral views of the vulva expression were obtained from csuIs01 in the prl-1 roller background .\",\n \"The same 4 . 3 kb Ppa-obi-1 promoter was used to drive the full length 1 . 6 kb cDNA fused to GFP by injecting into PS312 and crossing into the Ppa-obi-1 ( tu404 ) background to generate csuEx27[Ppa-obi-1p::Ppa-obi-1 cDNA:gfp; Ppa-egl-20::rfp; Ppa-obi-1 ( tu404 ) gDNA] .\",\n \"For unknown reasons , the N-terminally fused gfp coding region did not express GFP .\",\n \"To create the C06G1 . 1p::gfp reporter gene construct , we PCR amplified a 3 . 2 kb upstream sequence from the C06G1 . 1 ATG using RHL169 and RHL187 and fused the promoter with GFP:Ppa-rpl-23 3′ UTR by fusion PCR , using RHL181 and RHL154 primers to obtain a 4183 bp amplicon .\",\n \"Independently amplified C06G1 . 1p::gfp fragments were co-injected with either pRF4 rol-6 ( su1006 ) or myo-2p::DsRED vectors along with the pBluescript vector as carrier DNA ( 50 ng/μl each ) .\",\n \"This reporter likely encompasses C06G1 . 1's maximum regulatory region because the 3 . 2 kb promoter fragment extends ∼300 bp upstream of the next gene on the opposite strand ( C06G1 . 14 ) , and C06G1 . 1 does not contain any intron larger than 1 kb that may act as potential intronic enhancers .\",\n \"Selection for DsRED animals in the reporter strain csuEx02 [C06G1 . 1p::gfp; Cel-myo-2::DsRED] results in 90% GFP+/DsRED+ progeny and equal DsRED+ expression in pharyngeal muscle cells , suggesting stable mitotic transmission of the transgene .\",\n \"All five F1 transgenic lines showed similar expression profile and intensity in post-hatching stages , but strong body epidermal expression was detected only in the csuEx03 [C06G1 . 1p::gfp; pRF4; pBST] line .\",\n \"Images were obtained using a Leica DM6000 fluorescent microscope and Leica SP5 confocal microscope .\",\n \"To create the T02B11 . 3p::Ppa-obi-1:gfp amphid sheath expressing translational fusion construct , an ∼2 . 1 kb T02B11 . 3 promoter fragment was amplified from C . elegans gDNA using RHL476/RHL490 ( primary reaction ) .\",\n \"The Cel-T02B11 . 3 promoter is then fused to the same 1 . 6 kb Ppa-obi-1 cDNA rescue construct by PCR using RHL477/RHL483 ( secondary ) to generate a 3 . 7 kb fusion .\",\n \"This construct and the GFP-unc-54-3′UTR fragment , amplified from the pPD95 . 75 vector using RHL480/RHL481 ( primary ) was fused using RHL501/RHL482 ( tertiary ) to create the complete construct .\",\n \"A similar scheme was used to generate F16F9 . 3p::Ppa-obi-1:gfp using RHL498/RHL500 ( promoter ) .\",\n \"C . elegans N2 were injected with a mixture of purified T02B11 . 3p::Ppa-obi-1:gfp ( 25 ng/μl ) or F16F9 . 3p::Ppa-obi-1:gfp , myo-2::DsRED ( 25 ng/μl ) and pBluescript carrier DNA ( 100 ng/μl ) .\",\n \"One stable line from each construct was characterized for ZTDO chemotaxis and paralysis .\",\n \"The GenBank accession for Ppa-obi-1 mRNA is JQ946290 .\",\n \"Primer sequences are listed in Table 2 . 10 . 7554/eLife . 03229 . 016Table 2 . PrimersDOI: http://dx . doi . org/10 . 7554/eLife . 03229 . 016PrimerPrimer sequence ( 5′→3′ ) Template targetRHL101TGAACGGGCTTTTAATCTGGPpa-obi-1 gDNARHL102GTGTCGAGATAGTCGGGCATPpa-obi-1 gDNARHL104ATGACTGCTCCAAAGAAGPpa-obi-1 cDNARHL107ATTTGCCCTTCGTTCCACPpa-obi-1 for RNAiRHL108CTGTCTCAGTATGCCGAATGPpa-obi-1 for RNAiRHL109ATTCAGCCGTGTTACAAATCPpa-obi-1 cDNARHL110CTGCATGTGTTCCGGTCTCGPpa-obi-1 cDNARHL113AAACCTCTGACACATGCAGCPpa-obi-1 cDNARHL121AGTTTCAAAGTTGCATGGPpa-obi-1 promoterRHL124CTTCTTTGGAGCAGTCATGTAGCGATAATCAGGAGTgfp; Ppa-obi-1 promoterRHL132ATGACTGCTCCAAAGAAGgfpRHL136CAACCGTGTGCAGTAGAACCPpa-obi-1 5′ RACERHL149ACTATCCTATCATCGGAAGCPpa-obi-1 promoterRHL151AGGGCATTGACAAATCATCGPpa-obi-1 cDNARHL154CACGACGTTGTAAAACGACGPpa-obi-1 cDNARHL169CGCGCTAAACCAATTCCGCCC06G1 . 1 promoterRHL181GTTGAGAGAAGAGGTGGAGTCCC06G1 . 1 promoterRHL187CTTCTTTGGAGCAGTCATCAGATCTGAAAATTTGACACATTGgfp; C06G1 . 1 promoterRHL267GGAACTAACATTAATGTCACC06G1 . 1 RNAiRHL268GAGAAGTGCATAGTTGATGC06G1 . 1 RNAiRHL477GCGCTACAGGGGATTTTGTCT02B11 . 3 promoterRHL480TGAAAGATGCAAGTAAAGGAGAAGAACTTTTCp95 . 75 GFPRHL481AAGGGCCCGTACGGCCGACTAp95 . 75 GFPRHL482GTAGGAAACAGTTATGTTTGGp95 . 75 GFPRHL484CTTCAGAAAATGATCAGGAGTCTGGTTCobi-1 cDNARHL487AGTGAAAAGTTCTTCTCCTTTACTTGCATCTTTCACGGAGTCobi-1 cDNA; gfpRHL490AACGAGAACCAGACTCCTGATCATTTTCTGAAGAAAGTTGAAAAACT02B11 . 3 promoter; obi-1 cDNARHL498GCGATAAGATCGGTCAATCTGAGF16F9 . 3 promoterRHL499GCCAGTAAGGGCTAGTAAGTGF16F9 . 3 promoterRHL500AACGAGAACCAGACTCCTGATCATATTTTGTTTCTTACTGTCTTGF16F9 . 3 promoterRHL501ATGGGTACGTTTCTGGGTATAGT02B11 . 3 promoter; obi-1 cDNAVR86GACTCCTGATTATCGCTACGPpa-obi-1 cDNAVR87AGATAGTCAAACTATGCATCPpa-obi-1 cDNAVR88CTAATAATCTACACTAAATGPpa-obi-1 cDNAAG11112CTCGGAGGAGGAACTGGATCPpa-beta-tubulin RT-qPCRAG11113GACCGTGTCAGAGACCTTAGPpa-beta-tubulin RT-qPCRRH12548GCAGGAACATATATTCGCAGPpa-egl-4 RT-qPCRRH12549TGTCACGGAACGTTTTGTACPpa-egl-4 RT-qPCRSL1GGTTTAATTACCCAAGTTTGAGSL1 for 5′ RACESSLP21FACTGGTGCTCATCGCAAGAMapping markerSSLP21RCCTTCTGTTTTCCTACCCCCMapping markerSSLP7FTCTTGCATAACACCGAACAAAMapping markerSSLP7RAGGGCGTTACGTGAATAAGCMapping markerSSLP17FCAATGCAGTAGCCGAATCATMapping markerSSLP17RCAATATTGGCCTTCACCCTGMapping markerS447FTGGGGATAGCGAAAATCATCMapping markerS447RCGCATCGTTATTCACGAAATTCMapping markerLetters in bold are based on different templates for fusion PCR .\",\n \"Body measurements were obtained as previously described ( Hong et al . , 2008a ) .\",\n \"In brief , we used a Leica DM6000 and LAS application to measure body dimensions at 24-hr intervals starting at mid-J4/L4 stage .\",\n \"To determine hatching rate , we picked single J4-stage PS312 hermaphrodites onto fresh OP50 plates and transferred the nematodes every 24 hr three times at 20°C .\",\n \"The total number of eggs present on the plates following each transfer and the resulting number of 3-day old animals for each worm during the 3-day period were used to calculate the percentage of hatching and brood size .\",\n \"The bacterial feeding strain containing the C06G1 . 1 fragment 1 was previously described in a large-scale C . elegans RNAi screen ( Simmer et al . , 2003 ) .\",\n \"C06G1 . 1 fragment 2 was made using RHL267 and RHL268 primers to amplify an ∼400 bp fragment of C06G1 . 1 , digesting this product with BglII and PstI , and ligating the 190 bp fragment into the BglII/PstI sites of pL4440 .\",\n \"In vitro synthesis of dsRNA from 1 μg of T7 amplified PCR template from the RNAi vector or the pL4440 negative control was accomplished using the Ambion in vitro synthesis kit ( Grand Island , NY ) .\",\n \"RNAi by bacterial feeding of C06G1 . 1 was performed by scoring the progeny of L4 stage rrf-3 ( pk1426 ) animals raised on RNAi or L4440 control plates containing 1 mM IPTG and 50 μg/ml carbenicillin .\",\n \"Means , SEM ( error bars ) , and p values for two-tailed t test were performed by Microsoft Excel .\",\n \"One-way ANOVA with multiple comparisons post-hoc testing were performed using Prism statistical software .\",\n \"Sample sizes represent total technical replicates from two or more biological replicates .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Nematodes and insects are the two most speciose animal phyla and nematode–insect associations encompass widespread biological interactions .","To dissect the chemical signals and the genes mediating this association , we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus .","We found that while the beetle pheromone is attractive to P . pacificus adults , the pheromone arrests embryo development , paralyzes J2 larva , and inhibits exit of dauer larvae .","To uncover the mechanism that regulates insect pheromone sensitivity , a newly identified mutant , Ppa-obi-1 , is used to reveal the molecular links between altered attraction towards the beetle pheromone , as well as hypersensitivity to its paralyzing effects .","Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types , including the amphid neuron sheath and excretory cells .","Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny ."],"string":"[\n \"Nematodes and insects are the two most speciose animal phyla and nematode–insect associations encompass widespread biological interactions .\",\n \"To dissect the chemical signals and the genes mediating this association , we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus .\",\n \"We found that while the beetle pheromone is attractive to P . pacificus adults , the pheromone arrests embryo development , paralyzes J2 larva , and inhibits exit of dauer larvae .\",\n \"To uncover the mechanism that regulates insect pheromone sensitivity , a newly identified mutant , Ppa-obi-1 , is used to reveal the molecular links between altered attraction towards the beetle pheromone , as well as hypersensitivity to its paralyzing effects .\",\n \"Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types , including the amphid neuron sheath and excretory cells .\",\n \"Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["The nematode worm Pristionchus pacificus can live as a parasite inside the oriental beetle , where it waits for the beetle to die so it can feed off the bacteria that live on the beetle's decomposing carcass .","This ecologically important interaction is called necromeny .","P . pacificus is attracted to a new host by a sex pheromone produced by the beetle , but the genes and biological mechanisms that enable this interaction to occur are not understood in much detail .","To identify the genetic basis of this interaction , Cinkornpumin et al . identified and examined a mutant form of P . pacificus that cannot sense the beetle sex pheromone .","This revealed that although this pheromone attracts the adult nematodes , it stops P . pacificus embryos developing and can paralyze larvae .","Cinkornpumin et al . suggest that the pheromone has likely evolved this ability in order to counteract the spread of the nematodes .","This result implies that being invaded by P . pacificus makes life more difficult for the beetles than was previously thought .","Further investigation of the gene damaged in the P . pacificus mutants revealed that it encodes a protein that may bind to molecules called lipids , which are needed to form cell membranes and are used in cell signaling .","As well as helping the nematodes to detect the sex pheromone , the lipid-binding protein also appears to help protect the worms from the pheromone's detrimental effects .","Cinkornpumin et al . observed that the gene for the lipid-binding protein is activated in several tissues , including the cells that form a sheath around some of the nerves that detect chemical signals .","Whether this tissue is responsible for the chemical-sensing abilities of the lipid-binding protein , and whether these same tissues are responsible for protecting the nematodes from the damaging effects of the pheromone , remains to be discovered ."],"string":"[\n \"The nematode worm Pristionchus pacificus can live as a parasite inside the oriental beetle , where it waits for the beetle to die so it can feed off the bacteria that live on the beetle's decomposing carcass .\",\n \"This ecologically important interaction is called necromeny .\",\n \"P . pacificus is attracted to a new host by a sex pheromone produced by the beetle , but the genes and biological mechanisms that enable this interaction to occur are not understood in much detail .\",\n \"To identify the genetic basis of this interaction , Cinkornpumin et al . identified and examined a mutant form of P . pacificus that cannot sense the beetle sex pheromone .\",\n \"This revealed that although this pheromone attracts the adult nematodes , it stops P . pacificus embryos developing and can paralyze larvae .\",\n \"Cinkornpumin et al . suggest that the pheromone has likely evolved this ability in order to counteract the spread of the nematodes .\",\n \"This result implies that being invaded by P . pacificus makes life more difficult for the beetles than was previously thought .\",\n \"Further investigation of the gene damaged in the P . pacificus mutants revealed that it encodes a protein that may bind to molecules called lipids , which are needed to form cell membranes and are used in cell signaling .\",\n \"As well as helping the nematodes to detect the sex pheromone , the lipid-binding protein also appears to help protect the worms from the pheromone's detrimental effects .\",\n \"Cinkornpumin et al . observed that the gene for the lipid-binding protein is activated in several tissues , including the cells that form a sheath around some of the nerves that detect chemical signals .\",\n \"Whether this tissue is responsible for the chemical-sensing abilities of the lipid-binding protein , and whether these same tissues are responsible for protecting the nematodes from the damaging effects of the pheromone , remains to be discovered .\"\n]","isConversational":false},"year":{"kind":"string","value":"2014"}}},{"rowIdx":290,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["microbiology and infectious disease"],"string":"[\n \"microbiology and infectious disease\"\n]","isConversational":false},"title":{"kind":"string","value":"MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis"},"id":{"kind":"string","value":"elife-32471-v3"},"sections":{"kind":"list like","value":[["Although many bacteria are rod shaped , the cellular mechanisms that construct and replicate this geometry have remained largely unknown .","Bacterial shape is determined by the cell wall sacculus , a giant , encapsulating macromolecule that serves to resist internal osmotic pressure .","One of the primary components of the cell wall is peptidoglycan ( PG ) , which is created by the polymerization of single glycan strands linked by peptide crossbridges .","Studies of isolated cell walls from rod-shaped bacteria suggest material is generally oriented circumferentially around the rod , perpendicular to the long axis of the cell ( Gan et al . , 2008; Hayhurst et al . , 2008; Verwer et al . , 1980 ) , or in thick cables in others ( Hayhurst et al . , 2008 ) .","This mostly circumferential , hoop-like organization of cell wall material allows the cell wall to better resist the internal osmotic pressure , as this pressure causes a stress twice as large in the circumferential direction ( on the rod sidewalls ) than in the axial direction ( on the poles ) ( Amir and Nelson , 2012; Chang and Huang , 2014 ) .","This organization of material confers a mechanical anisotropy to the cell wall , causing it to stretch more along its length than across its width for a given stress; this anisotropy may assist rod-shaped cells in preferentially elongating along their length ( Baskin , 2005; Chang and Huang , 2014 ) .","Concordantly , atomic force microscopy ( AFM ) has shown that Escherichia coli sacculi are 2–3 times more elastic along their length than across their width ( Yao et al . , 1999 ) .","This rod-reinforcing circumferential organization is also observed in the cell walls of plants; hypocotyl and root axis cells rapidly elongate as rods by depositing cellulose fibrils in circumferential bands around their width , resulting not only in a similar dispersive rod-like growth , but also a similar anisotropic response to stress ( Baskin , 2005 ) .","The organized deposition of cellulose arises from cortical microtubules self-organizing into a radial array oriented around the rod width , and this orients the directional motions of the cellulose synthases to insert material in circumferential bands ( Paredez et al . , 2006 ) .","In contrast to our understanding of the self-organization underlying rod-shaped growth in plants , how bacteria construct a circumferential organization of glycan strands is not known .","This organization may arise via the actions of a small number of genes essential for the formation and maintenance of rod shape .","Collectively termed the Rod complex , ( or elongasome ) these include MreB , MreC , MreD ( encoded by the mreBCD operon ) ( Wachi et al . , 1989 ) , RodZ ( Alyahya et al . , 2009; Bendezú et al . , 2009 ) , and the glycosyltransferase/transpeptidase enzyme pair RodA/Pbp2 ( Cho et al . , 2016 ) .","These components are conserved across a wide range of rod shaped bacteria , and mostly absent in cocci ( Alyahya et al . , 2009; Chastanet and Carballido-Lopez , 2012 ) , leading many to speculate they function as the central determinants of rod shape ( Carballido-Lopez , 2006; Jones et al . , 2001 ) .","The spatial coordination of RodA/Pbp2-mediated PG synthesis is conferred by MreB , an actin homolog ( Jones et al . , 2001; van den Ent et al . , 2001 ) .","MreB polymerizes onto membranes as antiparallel double filaments , which have been observed to bend liposome membranes inward ( Figure 1A ) ( Salje et al . , 2011; van den Ent et al . , 2014 ) .","Loss or depolymerization of MreB causes rod-shaped cells to grow as spheres ( Gitai et al . , 2005; Jones et al . , 2001; Bendezú et al . , 2009 ) .","B . subtilis contains 3 MreB paralogs ( MreB , Mbl , and MreBH ) that have been shown to co-polymerize into mixed filaments in vitro , and always colocalize in vivo ( Defeu Soufo and Graumann , 2004; Soufo and Graumann , 2010; Dempwolff et al . , 2011 ) .","MreB filaments move circumferentially around the width of rod-shaped cells ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011; van Teeffelen et al . , 2011 ) .","Super-resolution imaging has demonstrated that MreB filaments always translocate along their length , moving in the direction of their orientation ( Olshausen et al . , 2013 ) .","MreB filaments move in concert with MreC , MreD , and RodA/Pbp2 ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011 ) , and loss of any one component stops the motion of the others .","The directional motion of MreB filaments and associated Rod complexes depends on , and thus likely reflects , the insertion of new cell wall , as this motion halts upon the addition of cell wall synthesis-inhibiting antibiotics ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011; van Teeffelen et al . , 2011 ) , or specific inactivation or depletion of Pbp2 ( Garner et al . , 2011; van Teeffelen et al . , 2011 ) or RodA ( Cho et al . , 2016 ) .","It is not known how MreB and the rest of the Rod complex construct rod-shaped cells .","As the motions of the Rod complexes reflect the insertion of new cell wall , their circumferential motions could deposit glycans in the hoop-like organization required to both build and reinforce rod shape .","Therefore , we worked to understand the origin of this circumferential organization , seeking to determine what orients the motions of MreB and associated enzymes around the rod width in Bacillus subtilis ."],["The mechanism by which MreB filaments and associated PG synthases orient their motion around the rod circumference is not known .","Each filament-synthase complex is disconnected from the others , moving independently of proximal neighbors ( Garner et al . , 2011 ) .","The organized , circumferential motion of these independent filament-synthase complexes could arise in two ways: ( 1 ) A templated organization , where cell wall synthetic complexes move along an existing pattern of ordered glycan strands in the cell wall as they insert new material into it ( Höltje , 1998 ) , or ( 2 ) A template-independent organization , where each synthetic complex has an intrinsic mechanism that orients its motion and resultant PG synthesis around the rod circumference .","To explore the extent of order within the motions of the Rod complex , we analyzed the trajectories of GFP-Mbl and GFP-MreB with respect to the cell body using total internal reflection fluorescence microscopy ( TIRFM ) ( Figure 1B ) .","Overall , these motions are circumferentially oriented , but not perfectly aligned , a characteristic reflected by the broad distribution of angles that GFP-MreB , its homologs , and the other components of the Rod complex move relative to the long axis of the cell ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011 ) .","However , examination of TIRFM time lapse movies revealed that both MreB and Mbl trajectories close in time ( within the period of one revolution ) frequently cross ( Figure 1C , Figure 1—video 1 ) , making it unlikely that MreB filaments move along a perfectly ordered template .","As MreB movement reflects the insertion of new glycan strands , these motions indicate that the siacculus is built from somewhat disorganized , yet predominantly circumferential strands .","This conclusion is in agreement with X-ray diffraction ( Balyuzi et al . , 1972; Labischinski et al . , 1979 ) and cryoelectron microscopy studies of E . colisacculi ( Gan et al . , 2008 ) which found that , while glycans are oriented circumferentially around the rod width on average , they are not ordered , running at variable angles to each other .","Similarly , atomic force microscopy of B . subtilis sacculi has observed a generally oriented , but unaligned arrangement of 50 nm thick cables oriented roughly perpendicular to the long axis ( Hayhurst et al . , 2008 ) .","Thus , both the motions of MreB and ultrastructural studies indicate the sacculus is not highly ordered , making it unlikely that it can serve as a self-propagating spatial template for rod shape .","Furthermore , given that preexisting cell wall is not necessary for the regeneration of rod shape from wall-less B . subtilis L-forms ( Kawai et al . , 2014 ) , it is likely that both oriented MreB motion and rod shape can arise without an ordered template .","As it appeared that organized MreB motion does not arise from patterns in the cell wall , we hypothesized there was an intrinsic mechanism orienting the motion of each MreB filament-cell wall synthetic complex .","To test this hypothesis , we examined MreB motions as we changed the shape of cells from rods to spheres .","As the internal osmotic pressure and stiffness of B . subtilis resists external mechanical perturbations to its shape ( Renner et al . , 2013 ) , we first altered the shape of cells by controlling the level of wall teichoic acids ( WTAs ) .","WTAs are negatively charged cell wall polymers believed to increase the rigidity of the sacculus ( Matias and Beveridge , 2005 ) , a process that could occur via their coordination of extracellular Mg2+ ( Thomas and Rice , 2014; Kern et al . , 2010 ) , or modulation of hydrolase activity ( Atilano et al . , 2010 ) .","Knockouts of tagO , the first gene in the WTA synthesis pathway , create large , slow-growing , round cells that still synthesize PG , building extremely thick and irregular cell walls ( D'Elia et al . , 2006 ) .","We placed tagO under xylose-inducible control and grew cells at different induction levels .","As expected , at high TagO inductions , cells displayed normal widths .","As we reduced TagO levels , rods became gradually wider ( Figure 1D–E ) until , beneath a given induction , cells were no longer able to maintain rod shape , growing as spheres ( or clumps of spheres ) with no identifiable long axis .","At intermediate induction levels , we observed a transition region between the two states , with cells growing as steady state populations of interconnected rods and spheres ( Figure 1D ) .","In agreement with models that ( A ) WTAs work with PG to bind Mg2+ ( Thomas and Rice , 2014; Kern et al . , 2010 ) , and ( B ) are required for cell wall rigidity ( Matias and Beveridge , 2005 ) , both the cell width and the amount of TagO induction determining the rod/sphere transition could be modulated by Mg2+ levels ( Figure 1F , Figure 1—figure supplement 1B ) .","Likewise , decreasing extracellular Mg2+ or tagO induction resulted in increasingly curved cell contours ( Figure 1—figure supplement 1B ) , suggesting the wall was becoming more flexible .","By tracking the motion of GFP-MreB filaments in these differing cell shapes , we found that motion is always oriented in rods , moving predominantly circumferentially at all induction levels above the rod/sphere transition .","However , in round cells ( those induced beneath the rod/sphere transition point or in tagO knockouts ) MreB filaments continued to move directionally , but their motions were isotropic , moving in all directions ( Figure 2A , Movie Figure 2—video 1 ) .","To quantify the relative alignment of MreB under each condition , we calculated the angle between trajectory pairs less than 1 μm apart ( Figure 2B , Figure 2—figure supplement 1A ) .","This analysis revealed that MreB motions are more aligned when cells are rods: above the rod/sphere transition , trajectories have a median angle difference of 26°; while at low TagO inductions , where cells are round , the angle difference increases to 42° , close to that of randomly oriented trajectories ( 45° ) .","To verify that the loss of oriented MreB motion was due to the changes in cell shape , and not from some other effect of reduced WTA levels , we created round cells by alternate means .","Depletion of both elongation PG transpeptidases ( Pbp2a and PbpH ) causes rod-shaped cells to become wider over time as they convert to spheres ( Garner et al . , 2011 ) .","We used this gradual transition of rods into spheres to examine both the width and overall shape dependence of MreB motion .","At initial points of depletion ( 1–2 hr ) the rods widened but maintained circumferential MreB motion .","At 2 . 5 hr of PbpA depletion , cells were a mix of spheres and rods of differing widths .","These cells displayed the same pattern of MreB orientation observed with tagO depletions: round cells contained unoriented MreB , while nearby rod-shaped cells showed circumferential motion .","Identical behavior was observed for GFP-Mbl during PbpA depletions ( Figure 2C , Movie Figure 2—video 2 ) .","Quantitation of trajectories from all cells ( both rods and spheres ) at each time point of depletion indicated an increase in the median angle between trajectories as the population grew wider and rounder over time ( Figure 2D , Figure 2—figure supplement 1B ) .","In E . coli , the angle of mutant MreB filaments relative to the long axis has been reported to increase with cell width ( Ouzounov et al . , 2016 ) .","To test if the angle of MreB movement changes with respect to cell width in B . subtilis , we calculated the angle of each trajectory to the long axis for all cells in our data with an identifiable width axis .","At the same time , we also measured the curvature of each cell to determine how the overall shape of the cell affected the orientation of motion .","This revealed that MreB motion in rods remained equivalently oriented over a wide range of rod widths , up to ~2 μm ( Figure 2E , Figure 2—figure supplement 1C–E ) .","Beyond a 2 μm width , cells began to lose their rod shape as they became more spherical , and the predominantly circumferential orientation of MreB motion was lost ( Figure 2E , Figure 2—figure supplement 1E ) .","This suggested that oriented MreB motion does not sense or rely on a specific cell radius; rather the orientation relies on differences between the two principal curvatures of the membrane .","It appears that the motion of MreB filaments is oriented along the direction of greatest principal curvature: In rods , there is zero curvature along the rod length , and high curvature around the rod circumference , along which filaments orient .","In contrast , in round cells where MreB motion is isotropic , the two principal curvatures are equal ( Figure 2F ) .","To further verify that MreB filaments orient in response to overall cell shape , we externally imposed rod shape on cells with unoriented MreB motion .","We loaded TagO-induced cells into long 1 . 5 ×1 . 5 μm microfluidic chambers , then reduced TagO expression to levels insufficient to produce rods in liquid culture ( Figure 3A , Figure 3—figure supplement 1A ) .","After TagO depletion , cells expanded to fill the chamber indicating that WTA-depletion caused shape changes just as in bulk culture ( Figure 3A , Figure 3—figure supplement 1A ) .","Within these chambers , cells grew as rods , but at a wider width ( 1 . 5 μm ) than wild-type cells , set by the chamber .","When cells grew out of the chamber they swelled just as in bulk culture , showing confinement was required for rod shape at this induction level ( Figure 3B , Figure 3—figure supplement 1A ) .","In the TagO-depleted cells confined into rod shapes , MreB moved circumferentially ( Figure 3C , Figure 3—video 1 ) , confirming that MreB orients in response to the cells having rod shape .","This experiment demonstrates that the isotropic MreB motion observed in round cells arises from the lack of rod shape , and not from some other effect of our genetic perturbations .","This experiment also showed another unexpected result: the doubling time of free ( unconstrained ) cells induced at similar TagO levels is long ( 53 ± 10 min ) , but confining them into rod shape restored their doubling time ( 44 ± 4 min ) toward wild-type times ( 39 ± 9 min ) ( Figure 3—figure supplement 1B ) .","We next attempted to minimize any contribution to MreB filament orientation from ( A ) the directional motion of filaments , and ( B ) any pre-existing order within the sacculus .","To accomplish this , we examined filament orientation in protoplasts ( cells that had their cell wall enzymatically removed ) confined into different shapes , using highly expressed GFP-MreB to assay long filaments , and GFP-Mbl to assay short filaments .","We protoplasted cells in osmotically stabilized media ( Wyrick and Rogers , 1973 ) , then grew them under agar pads containing micro-patterned cross shapes .","Cells in the center of these crosses ( ~5 μm diameter ) were forced to grow as spheres , whereas cells in the arms were constrained to grow into rods of various widths ranging from 2 to 5 μm ( Figure 3D ) .","As reported previously ( Domínguez-Escobar et al . , 2011 ) , MreB filaments within protoplasts did not move directionally ( Figure 4—video 1 ) , likely because the cell wall provides the fixed surface along which the PG synthesis enzymes move .","Within the protoplasts confined into the smallest rod shapes ( 2 μm ) , filaments oriented at a distribution of angles predominantly perpendicular to the cell length ( Figure 3E–F ) .","The angular distributions of short GFP-Mbl filaments and longer GFP-MreB filaments were similar to each other , and also similar to the distribution of","1 ) filament trajectories observed in intact , wild-type cells and","2 ) filament trajectories of TagO depleted cells confined into the mother machine ( Figure 3Fi ) .","As we increased the width of the imposed rod shape from 2 to 5 μm , filaments remained predominantly oriented in all cases ( Figure 3Fii ) , but their mean deviation from 90° increased as the rod width increased ( 34° at 2 μm , 35° at 4 μm , and 41° at 5 μm ) .","In contrast to confinement in rods , both short and long filaments in spherically confined protoplasts remained unoriented ( Figure 3E ) .","Together , these data demonstrate that MreB filaments orient to point around the rod width even in the absence of pre-existing cell wall or directional motion , as long as the cell has a rod shape .","These experiments also demonstrate that MreB filaments will align even in wider rods , where the difference in principal curvatures is smaller than in wild-type cells , but that , as the difference in principal curvatures decreases , filament alignment becomes more disordered .","To test if MreB filaments are themselves sufficient to align along the predominant direction of membrane curvature , we assembled purified T . maritima MreB within liposomes and visualized it using cryoelectron microscopy and tomography .","While controlling the final concentration of protein encapsulated within liposomes ≤1 μm is difficult , we were able to assemble MreB inside liposomes at high concentrations .","At these concentrations , MreB filaments tubulated liposomes , creating rod-like shapes ( Figure 4A , Figure 4—figure supplement 1A–B , Figure 4—video 1 ) .","In tubulated regions , MreB filaments could be traced around the circumference of the liposome tube , while filaments in spherical regions were found in all possible orientations ( Figure 4A ) .","At the highest concentrations , tubulated liposomes contained closely packed filament bundles , allowing us to observe a regular patterning of the canonical double filaments of MreB ( Figure 4B ) .","Purified wild-type MreB did not bind to the outside surface of small liposomes contained within larger ones ( Figure 4A ) , indicating that MreB filaments preferentially polymerize on inward ( negative ) curvatures , akin to the inner leaflet of the bacterial membrane .","In the absence of MreB , liposomes are spherical , with no deformations ( Figure 4—figure supplement 1C ) .","Together , this data suggests that MreB filaments themselves are sufficient to align along the predominant direction of membrane curvature , as observed here with laterally associated filaments .","We note that the experimental limitations of the liposomal system , combined with the tendency of MreB filaments to self-associate make it difficult for us to acquire and study the alignment of single filaments in vitro .","Also , it remains to be determined if membrane-associated MreB filaments exist as bundles or isolated filaments in vivo .","The above observations demonstrate that MreB filaments sense and align along the direction of greatest principal curvature , that is , the more curved inner surface of the rod circumference .","The ultrastructure of MreB filaments provides a possible mechanism: MreB filaments are bent ( Salje et al . , 2011 ) , with the membrane-interacting surface on the outer face of the bend ( Figure 4C ) .","This bent conformation could cause filaments to preferentially orient along the curved rod circumference , rather than the flat rod length , to maximize the burial of hydrophobic moieties into the membrane , a mechanism suggested by previous theory ( Wang and Wingreen , 2013 ) .","As the curvature of MreB filaments bound to liposomes is much greater ( ~200 nm diameter [van den Ent et al . , 2014] ) than that of B . subtilis cells ( ~900 nm diameter ) , we performed analytical calculations to model how highly curved MreB filaments would align within a cell with a less curved surface ( Figure 4D–G , Appendix 1 ) .","As many of the biochemical and physical parameters of MreB are still unknown , we first assumed a fixed set of parameters , and later verified that our results were robust over a large parameter range .","We initially assumed a membrane interaction energy of 10 kT per monomer ( calculated from residues involved in membrane associations [Salje et al . , 2011] ) , and a similar Young’s modulus to actin ( 2 GPa ) .","We modeled filaments as elastic beams made of two protofilaments .","In addition , we used the Helfrich free energy to model the energetics of membrane deformation , and accounted for the work done against osmotic pressure due to changes in volume ( Appendix 1 ) .","These calculations indicate that the total energy is minimized when filaments orient along the direction of maximal curvature ( Figure 4F ) and that , importantly , the energy penalty for incorrectly-oriented filaments is much greater than the energy of thermal fluctuations .","Interestingly , this modeling indicates a decrease in energetic preference for the preferred filament orientation as the radius of the cell is increased ( Figure 4F ) , a prediction in qualitative agreement with our observations of alignment in protoplasts .","Furthermore , our calculations indicate that orientation is robust over a large , biologically relevant range of parameters , including the membrane binding energy , filament length , and filament Young’s modulus ( Figure 4G ) .","These calculations predict that filaments should orient circumferentially both if the membrane deforms to the filament ( at low osmotic pressures or if filaments are stiff ) ( Salje et al . , 2011 ) , or if filaments deform to the membrane ( at high osmotic pressures or if filaments are flexible ) ( Figure 4E ) .","Our experimental data demonstrates MreB filament alignment across a range of pressures: high within cells , low to none within liposomes , and a pressure between the two within osmotically-stabilized protoplasts .","In the absence of osmotic pressure , MreB filaments deform liposomes since it is energetically more favorable to bend the membranes than to bend the filaments , as observed in our in vitro data ( Figure 4A , Figure 4—figure supplement 1 ) .","However , in live cells , our modeling predicts that MreB filaments cannot deform the inner membrane due to the large osmotic pressure , and instead deform to match the greatest principal membrane curvature .","Hence filaments create curvature in liposomes and sense it in cells .","Together , the above data demonstrate that MreB filaments are sufficient to preferentially orient along the direction of greatest principal membrane curvature .","In rod-shaped cells , this direction is along the rod circumference .","As filaments move along their length , their orientation constrains the spatial activity of the PG synthetic enzymes such that new cell wall is inserted in a mostly circumferential direction ( Hayhurst et al . , 2008 ) to reinforce rod shape ( Chang and Huang , 2014; Yao et al . , 1999 ) .","While the ability of MreB filaments to orient in pre-existing rods can help explain how rod shape is maintained , we also wanted to understand how MreB filaments facilitate the de novo formation of rod shape .","To explore this , we observed how cells interconvert between spheres and rods .","We first examined how rod shape fails , by growing our TagO-inducible strain at induction levels that produced rods and then reducing the Mg2+ concentration to induce them to convert to spheres .","This transition revealed that rods convert into round cells by continuous swelling: once a rod begins to widen , it continues to do so until reaching a fully spherical state with no reversion during the process ( Figure 5A ) .","Similar rod to sphere transitions could be attained by holding Mg2+ constant while reducing TagO expression .","Likewise , cells grown at intermediate TagO induction levels ( 8–12 mM ) grew as steady state populations of interconnected rods and spheres , indicating that cells underwent repeated cycles of rod shape formation followed by reversion to spheres ( Figure 1D , F ) .","These results indicate that rod shape can be maintained only as long as the cell wall is sufficiently rigid to resist the internal osmotic pressure .","We next examined how rod shape forms from round cells .","As the recovery of protoplasted B . subtilis is so infrequent that it has never been directly visualized ( Mercier et al . , 2013 ) , we assayed how round cells with preexisting cell walls convert back into rods , using three systems: ( 1 ) re-inducing WTA expression within TagO-depleted , spherical cells , ( 2 ) holding TagO expression beneath the rod/sphere transition and increasing Mg2+ levels , and ( 3 ) re-inducing Pbp2a expression in spherical , Pbp2a-depleted cells .","In all three cases , rods reformed in a discrete , local manner; spheres did not form into rods by progressively shrinking along one axis , but rather , rods abruptly emerged from one point on the cell , growing more rapidly than the parent sphere ( Figure 5B , Figure 5—video 1 and Figure 5—video 2 ) .","This morphology is similar to the initial outgrowth of germinating B . subtilis spores ( Pandey et al . , 2013 ) .","We occasionally observed another mode of recovery , occurring when round cells were constrained , or divided into , ovoid or near-rod shapes .","Once these near-rod shaped cells formed , they immediately began rapid , rod-like elongation along their long axis ( Figure 5—figure supplement 1A ) .","We focused on two salient features of the rod shape recoveries: ( 1 ) rod shape forms locally , most often at one point on the cell surface , and ( 2 ) once a rod-like region is formed , it appears self-reinforcing , both propagating rod shape and growing faster than adjacent or attached non-rod shaped cells .","We first wanted to understand how rod shape initiates de novo from spherical cell surfaces .","By examining the initial time points of recoveries , we found that rods begin as small outward bulges: local regions of outward ( positive Gaussian ) curvature flanked by regions of inward ( negative Gaussian ) curvature ( Figure 5C ) .","These initial outward bulges showed a width distribution similar to that of the later emerging rods ( Figure 5D ) .","Once these bulges formed , they immediately began rapid elongation into nascent rods , which would then thin down to wild type width over time .","Bulge formation and rod recovery were independent of cell division , as cells depleted of FtsZ still recovered rod shape ( Figure 5—figure supplement 1B ) .","Rather , these bulges appeared to arise randomly , evidenced by the fact that different cells produced rods at different times during WTA or Pbp2a repletion .","We conclude that the appearance of a local outward bulge can act as the nucleating event of rod shape formation .","As emerging rods appeared to grow faster than adjacent round cells , we tested if the doubling times of rod-shaped cells were faster than those of non-rods by measuring the doubling times in our inducible TagO strain at different induction levels using both OD600 measurements and single cell microscopy under steady state conditions ( Figure 3—figure supplement 1B ) .","This revealed a sharp transition in doubling time that matched the conditions of the rod/sphere transition: growth is slow when cells are spheres , yet greatly increases when cells are rods ( Figure 3—figure supplement 1B , Figure 5—figure supplement 1C ) .","Furthermore , the doubling times of recovering rods was similar to that of rods at steady state ( Figure 3—figure supplement 1B ) .","We believe the lower doubling time of rods is likely due to cell shape and not another effect , such as the lack of WTAs , as ( 1 ) the doubling time of TagO-depleted cells confined in the microfluidic chambers matched that of wild type cells; and ( 2 ) both the doubling times and the boundary of the rod/sphere transition could be equivalently shifted by changing the Mg2+ concentration ( Figure 1F , Figure 1—figure supplement 1 , Figure 5—figure supplement 1C , Figure 5—video 2 ) .","Combined , these results indicate that rod shape creates local , self-reinforcing regions that are poised for more rapid growth; once any small region of the cell approximates a rod shape , growth of the rod-like region is amplified , growing faster than other regions , and thereby outcompeting non-rod growth at the population level .","We next sought to determine what features distinguished rods from round cells .","As the elongation of rod-shaped cells requires a sufficiently rigid cell wall ( Figures 1D–E and 5A ) , the localized , self-reinforcing formation of rods in our system could arise from either of the two major cell wall components , PG and WTAs: ( 1 ) the PG strands could be arranged such that they better reinforce rod shape ( Amir and Nelson , 2012; Chang and Huang , 2014 ) , or ( 2 ) WTAs could be preferentially incorporated into the emerging rods to stabilize them .","To assay the orientation of newly inserted cell wall , we imaged the motions of MreB as we induced TagO-depleted cells to recover into rods .","This revealed that oriented MreB motion correlates with local shape: emerging rods displayed oriented MreB motion even at the initial points of their formation , while attached round parent cells displayed unaligned motion ( Figure 6A , Figure 6—video 1 ) .","This demonstrates that oriented MreB motion correlates with local geometry and does not arise from a global , cell spanning change .","We next examined the overall cellular distribution of MreB in recovering cells with confocal microscopy .","This revealed that , immediately prior to rod emergence , MreB transiently accumulated in a bright ring oriented perpendicular to the direction of rod emergence , most often occurring at the interface of the bulge and the round cells ( Figure 6B , Figure 5—figure supplement 1D ) .","To observe whether both spheres and rods inserted new PG during the process of rod shape recovery , we used fluorescent D-amino-acids ( FDAAs ) , which crosslink into newly inserted cell wall .","We grew TagO-depleted cells in a microfluidic device in the presence of HADA , then switched the media to contain Cy3B-ADA as we re-induced TagO expression .","During rod emergence , the old cell wall signal ( HADA ) remained in the sphere , while the emerging rod was almost entirely composed of new ( Cy3B-ADA ) material , confirming the discrete nature of rod shape recovery .","However , the attached spheres also incorporated Cy3B-ADA , indicating PG synthesis occurs in both rods and spheres during recovery ( Figure 5—figure supplement 1E ) .","The local reinforcement of rod shape in recovering cells could arise from preferential incorporation of the cell wall rigidifying WTAs .","As the WTA ligases have been reported to interact with MreB ( Kawai et al . , 2011 ) , we tested if rod shape correlated with increased WTA accumulation in emerging rods .","To test this , we labeled recovering cells with fluorescently labeled lectins that specifically bind to WTAs ( Figure 6—figure supplement 1A ) .","Following TagO reinduction , WTAs in recovering cells were ( Figure 6—figure supplement 1B ) , equally present in the cell walls of both rods and spheres ( Figure 6C ) .","To test if the WTA ligases move with MreB , we created GFP fusions to these proteins at their native locus and examined their dynamics with TIRFM .","We were unable to observe any of the circumferential motions expected if the WTA ligases moved with MreB; instead they appeared to be rapidly diffusing on the membrane ( Figure 6—figure supplement 1C , Figure 6—video 2 , Appendix 2 ) .","For further confirmation , we tracked the single molecule motions of these ligases , using JF549 labeled HaloTag fusions expressed at the native locus and promoter .","We did not observe any directional motions of the ligases that would indicate they move or associate with MreB and Pbp2A ( Figure 6—figure supplement 2D–E , Figure 6—video 3 ) .","In summary , these data gives new insights into what properties of the cell wall can be modulated to create and stabilize rod shape: rod shape is not formed by preferential localization of teichoic acids to rods , and both spheres and rods incorporate PG before and during rod shape recovery , in line with reports that PG synthesis is unchanged by the inhibition of WTA synthesis ( Pooley et al . , 1993 ) .","Rather , the only differences we detected between rod shaped and round cells were increased growth rates and oriented MreB motion .","We note that as WTAs have been shown to affect hydrolase activity ( Kasahara et al . , 2016 ) , their depletion may cause other rod-shape inhibiting PG abnormalities that we cannot observe ."],["To construct regular , micron-spanning shapes made of covalently crosslinked material , nature must devise strategies for coordinating the activities of disperse , nanometer-scale protein complexes .","This work reveals that the role of MreB in creating rod shape is to locally sense and subsequently reinforce differences in principal curvatures .","The local , short-range feedback between differences in curvature , MreB orientation , and shape-reinforcing cell wall synthesis could provide a robust , self-organizing mechanism for the stable maintenance and rapid reestablishment of rod shape , allowing the local activity of short MreB filaments to guide the emergence of a shape many times their size ."],["All B . subtilis strains were prepared for experimentation as follows: strains were streaked from −80°C freezer stocks onto lysogeny broth ( LB ) agar plates .","Following >12 hr of growth at 37°C , single colonies were transferred to serially diluted overnight bulk liquid cultures in LB supplemented with 20 mM magnesium chloride , placed on a roller drum agitating at 60 rpm , and grown at 25°C .","After >12 hr growth to OD600 <0 . 6 , these starter cultures were transferred to or inoculated into subsequent growth conditions .","All strains with tagO under inducible control were grown overnight in the presence of 30 mM xylose unless otherwise noted .","For the experiments in Figure 6C and Figure 3—figure supplement 1B , BEG300 cells were inoculated in the indicated medium ( LB with 20 mM MgCl2 unless otherwise stated ) from logarithmic phase overnights; ‘rods’ were grown from a low dilution with 30 mM xylose , and ‘spheres’ were grown with 0 mM xylose .","For bulk culture doubling time measurements , doubling times were calculated from the slope of a graph of time vs . dilution for a succession of serial dilutions of a given strain .","Time , the dependent variable , was taken as the time for a given dilution to pass the OD cutoff of OD600 = 0 . 20 .","In place of technical replicates , a large number of replicates were performed on a continuous gradient of xylose induction , showing a consistent trend between the extreme values depicted in the figure .","Single cell measurements were made in three ways .","Phase contrast images were collected on a Nikon ( Tokyo , Japan ) Ti microscope equipped with a 6 . 5 μm-pixel Hamamatsu ( Hamamatsu City , Japan ) CMOS camera and a Nikon 100x NA 1 . 45 objective .","Cells were collected by centrifugation at 6000 x g for 2 min and re-suspended in the original growth medium .","Unless otherwise specified , cells were then placed on No . 1 . 5 cover glass , 24 × 60 mm , under a 1 mm thick agar pad ( 2–3% agar ) containing LB supplemented with 20 mM magnesium chloride .","Unless otherwise noted , all cells were imaged at 37°C on a heated stage .","Images were collected on a Nikon TI microscope with a 6 . 5 μm-pixel CMOS camera and a Nikon 100x NA 1 . 45 objective .","Cells of strain BEG300 were grown overnight in LB supplemented with 30 mM xylose , 20 mM magnesium chloride , 1 μg/mL erythromycin , and 25 μg/mL lincomycin at 25°C at the specified xylose concentrations .","11 μM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) was added to induce GFP-MreB and the cells were shifted to 37°C and allowed to grow for 2 hr before imaging .","Cells of strain BEG202 ( ΔtagO ) with GFP-Mbl under a xylose-inducible promoter were grown overnight at 25°C in LB supplemented with 20 mM magnesium chloride and 0 . 125 mM xylose , and shifted to 37°C for 2 hr before imaging .","Cells were placed on cleaned glass coverslips thickness No . 1 . 5 , as described in the next section .","3–6% agar pads were prepared in LB supplemented with 20 mM magnesium chloride , 11 μM IPTG and the desired concentration of xylose .","Images were collected for 3 min at 1 or 2 s intervals , as specified .","30–50 cells were imaged in a day , and the experiments were repeated on at least one other day to test for technical variation .","Coverslips were sonicated in 1 M KOH for 15 min , followed by five washes with water .","Coverslips were washed twice with 100% ethanol , and then sonicated in 100% ethanol , followed by one more wash in 100% ethanol .","They were stored in ethanol and dried for 10 min before use .","Images were collected on a Nikon TI microscope with a Hamamatsu ImagEM ( EM-CCD ) camera ( effective pixel size 160 nm ) and Nikon 100x NA 1 . 45 TIRF objective .","Z stacks were obtained at 0 . 2 μm slices .","Total image depth was 3 μm .","Only the top 3 slices of the cell were used in maximum intensity projections in Figure 3E .","Cells were prepared as in ‘Imaging – MreB particle tracking’ above .","Cells were placed under an agar pad in a MatTek ( Ashland , Massachusetts ) dish for imaging .","Images were collected on an DeltaVision OMX SR Blaze system in SIM TIRF mode , using an Edge 5 . 5 sCMOS camera ( PCO ) and a 60x objective .","75msec exposures from a 488 nm diode laser were used for each rotation .","Spherical aberration was minimized using immersion oil matching .","Raw images were reconstructed using Softworx ( Applied Precision ) .","All image processing unless otherwise specified was performed in FIJI ( Schindelin et al . , 2012 ) .","Images used for particle tracking were unaltered , except for trimming five pixels from the edges of some videos to remove edge artifacts detected by the tracking software .","Phase contrast images and fluorescent images of protoplasts were adjusted for contrast .","Phase contrast images presented in the manuscript collected from cells in the custom microfluidic device , which did not undergo quantitative processing , were gamma-adjusted ( γ = 1 . 5 ) to compensate for changes in brightness occurring at the device’s feature borders; such processing was not used for growth quantification .","Images were background-subtracted for viewing purposes; unaltered images were used for quantitative processing in all cases .","The custom microfluidic setup used to confine cells in Figure 3A–C , Figure 3—figure supplement 1A , and Figure 3—video 1 was previously described in Norman et al . ( 2013 ) .","Briefly , a polydimethylsiloxane slab with surface features was bonded to a 22 × 60 mm glass coverslip by oxygen plasma treatment followed by heating to 65°C for >1 hr .","The features in our setup differed from those described in Norman et al . ( 2013 ) , particularly in the omission of a second , wider layer in the cell chambers , which enhanced growth at timescales beyond that of our experiments .","Syringes containing growth medium were connected to the microfluidic features using Tygon tubing stainless steel dispensing needles ( McMaster Carr Supply Company , Elmhurst , Illinois ) .","Medium was supplied to cells at a constant rate of 2–5 μL/min using automatic syringe pumps .","Imaging was carried out using phase contrast microscopy as described above .","For the microfluidics experiments in Figures 5 and 6 and Figure 5—video 1 ( top ) , Figure 5—video 2 , and Figure 6—video 1 , the CellASIC platform from Merck Millipore ( Billerica , Massachusetts ) was used with B04A plates .","The cell confinement experiment in Figure 3A–C was conducted by first loading cells into the chamber: BEG300 cells were grown to stationary phase ( OD6003 . 0–5 . 0 ) in LB supplemented with 20 mM magnesium chloride , passed through a 5 μm filter , and concentrated 100-fold before loading in the custom-made microfluidic device .","Both phase contrast and fluorescent imaging were performed as described in the ‘Imaging’ section above .","For observing MreB movement , MreB-GFP expression was induced with 50 μM IPTG upon loading into the microfluidic chamber , and cells were imaged every 2 s with a camera exposure time of 300 ms . The biological phenomena depicted in Figure 3A–C , Figure 3—figure supplement 1A , and Figure 3—video","1 . are representative data drawn from three experiments for phase contrast data and two experiments for fluorescent data .","Biological replicates , providing information on cell-to-cell clonal variation , for the purposes of this experiment should be considered individual channels , which are seeded by one or a few cells at the start of the experiment .","Cells of strains bJS18 ( GFP-Mbl ) and bEG300 ( GFP-MreB ) were grown overnight at 25°C in the osmoprotective SMM media ( LB supplemented with 20 mM magnesium chloride , 17 mM maleic acid , 500 mM sucrose , brought to a pH of 7 . 0 ) with maximum xylose induction ( 30 mM ) ; cells were shifted to 37°C in the morning .","For strain bEG300 , the SMM media was supplemented with 8 mM xylose ( for intermediate TagO induction ) .","Following 2 hr of growth , 10 mg/mL of freshly suspended lysozyme was added to the cultures with OD600 >0 . 2 .","After growing for 1–2 hr in lysozyme , the cells were spun and concentrated .","6% agar pads made in LB-SMM were made using a polydimethylsiloxane ( PDMS ) mold with crosses ( 2 , 4 and 5 μm arms and 5 μm center ) .","The cells were placed on the agar pad for 2 min , allowing the cells to settle in the crosses .","The pad was then placed in a MatTek ( Ashland , Massachusetts ) dish for imaging .","10–20 biological replicates were imaged per day and the experiment was replicated on 3 days .","TagO depletions in Figure 2A were conducted using strain BEG300 in liquid culture .","Cells were prepared as overnights , as described above , then grown at the specified xylose concentration at 37°C in LB with 20 mM magnesium chloride for 4 hr .","The cells were then imaged as described above in the ‘Imaging – MreB particle tracking’ section .","Pbp2A depletions shown in Figure 2 were conducted in liquid culture using strain BRB785 and BRB786 with an IPTG‐inducible Pbp2A fusion at the native locus with the redundant transpeptidase PbpH deleted .","This strain was grown overnight in the presence of 2 mM IPTG , and then inoculated into CH media containing 2 mM IPTG , 0 . 015% xylose , and 20 mM magnesium chloride to stabilize the cells against lysis .","At an OD600 of 0 . 6 , cells were spun down in a tabletop centrifuge and washed three times in CH media lacking IPTG .","Cells were placed under agar pads containing 20 mM magnesium chloride , and spinning disk confocal images were taken every 5 s on a Nikon Ti microscope with a 100 × 1 . 49 TIRF objective and a Hamamatsu ImagEM C9100-13 EM-CCD camera ( effective pixel size of 160 nm ) .","Depletions shown in Figure 5A were conducted using strain BEG300 .","Cells were prepared as overnights in LB with 1 mM magnesium chloride and 12 mM xylose .","In the morning , they were washed in LB with 12 mM xylose and no magnesium and placed under a 3% agar pad with the same medium .","Phase contrast images were collected every 5 min using a Photometrics ( Tucson , Arizona ) CoolSNAP HQ2 CCD camera .","The experiment was replicated on 2 days .","Repletions of TagO or Pbp2a on pads , as shown in Figure 5B and Figure 5—video 1 ( bottom ) , were performed with strains BEG300 and BRB785 respectively .","Cells were grown as overnights , as described above , then depleted at 37°C for >4 hr in LB with 20 mM magnesium chloride and collected by centrifugation at 6000 x g for 2 min .","The cells were re-suspended in LB supplemented with 20 mM magnesium chloride and 1 mM IPTG ( BRB785 ) and 30 mM xylose ( BEG300 ) , placed under 5% agarose pads on coverslips with thickness No . 1 . 5 for imaging .","Phase contrast images were collected every 5 min using a Photometrics CoolSNAP HQ2 CCD camera .","For the repletions shown in Figures 5B–C and 6A , Figure 5—figure supplement 1 , and Figure 5—video 1 ( top ) and 2 , performed in the CellASIC microfluidic device in a B04A plate , BCW82 and BEG300 cells were grown to OD6001 . 2–1 . 5 in LB supplemented with 20 mM magnesium chloride , centrifuged to pellet large clumps for 3 min at <500 x g , and the supernatant loaded into the plate .","Growth medium was supplied at 5–6 PSI .","Cells were grown for at least an additional 30 min before the addition of inducer to the growth medium .","Phase contrast images were collected every 10 min .","Fluorescent images were collected on the imaging setup described in the ‘Imaging – MreB Particle Tracking’ section above: GFP-MreB was induced upon loading into the microfluidic chamber with 1 mM IPTG , and MreB dynamics were observed for 3 min after every 10 min , using 300 ms camera exposures taken every 2 s .","The experiment was repeated six times , with the number of biological replicates per experiment limited by chance variation in the loading procedure , but on the order of one to four per experiment .","For the repletions shown in Figure 6B and Figure 5—figure supplement 1D , the same procedure was used , but with imaging performed on the spinning disk confocal microscope described in ‘Imaging – Spinning Disk Confocal’ .","Z-stacks were collected with a range of 3 μm around the focal plane and 0 . 2 μm steps .","The MreB localization experiments were done using strain bEG300 with full induction of GFP-MreB ( 1 mM IPTG ) and recovering cells were imaged using the spinning disk microscope , collecting Z-stacks as described before .","Where indicated , instead of visualizing MreB dynamics , fluorescent D-amino acids ( Kuru et al . , 2012 ) ( 7 μM ) were added to the growth medium in the CellASIC device: HADA during depletions of TagO ( 0 mM xylose ) and Cy3B-ADA during repletion of TagO ( 30 mM xylose ) .","Cells were washed with LB supplemented with 20 mM magnesium chloride containing no D-amino acids for 1–2 min before imaging .","To test if rod shape recovery occurs in the absence of cell division , three strains were tested ( BAB327 , BAB343 and BAB388 ) .","Cells of BAB327 and BAB388 were grown in CH media with 25 mM magnesium chloride in the absence of xylose at 37°C until OD600 ~0 . 5 and diluted 10-fold in fresh media .","After 2 hr of growth , IPTG was added to a final concentration of 1 mM ( MinCD and FtsA , respectively ) and cells were incubated for an extra 1 hr .","Cells were imaged on a spinning disk confocal under pads with 1 mM IPTG and 60 mM xylose ( for TagO repletion ) .","Phase-contrast and fluorescent images were acquired at 10 min intervals for a total of 8 hr .","Cells of BAB343 were grown in LB supplemented with 20 mM , magnesium chloride in the absence of xylose at 25°C overnight .","The next day , after 2 hr of growth in the same media at 37°C , IPTG was added to a final concentration of 1 mM ( MciZ ) and cells were incubated for an extra 1 hr .","Cells were imaged on a spinning disk confocal under pads with 1 mM IPTG and 30 mM xylose ( for TagO repletion ) .","Phase-contrast and fluorescent images were acquired at 10 min intervals for a total of 4 hr .","For Figure 5—video 2 , cells of BCW51 were grown overnight at 25°C in LB supplemented with 8 mM xylose , 20 mM magnesium chloride , 1 μg/ml erythromycin and 25 μg/ml lincomycin ( MLS ) .","Cells were shifted to 37°C for 2 hr and loaded into the CellASIC B04A plate at OD600 ~ 0 . 6 .","At the start of imaging , magnesium was depleted by flowing in LB supplemented only with 8 mM xylose and MLS at 3 psi .","Images were collected every 20 min over a 4 hr period .","Magnesium was resupplied to the cells by changing to LB supplemented with 8 mM xylose , 20 mM magnesium chloride , and MLS .","Imaging was continued every 20 min for an additional 4 hr . 5–10 cells were imaged and the experiment was done once .","Cells were grown overnight at 25°C in LB supplemented with 30 mM xylose , 20 mM magnesium chloride , 1 μg/mL erythromycin and 25 μg/mL lincomycin .","In the morning they were collected at OD600 ~ 0 . 2 , spun in a tabletop centrifuge at 9000 rpm for 3 min and washed in LB supplemented with various xylose ( 0–30 mM ) and magnesium chloride ( 0–20 mM ) levels .","25-fold serial dilutions into LB supplemented with the same xylose and magnesium chloride concentrations were made and allowed to grow at 37°C for 4 hr .","Cells at OD600 ~ 0 . 2 were concentrated by spinning in a tabletop centrifuge at 9000 rpm for 3 min .","They were placed on a coverslip thickness No . 1 . 5 under 3% agarose pads made in LB supplemented with the same concentrations of xylose and magnesium chloride .","Images were collected using the imaging setup described in the ‘Imaging – phase contrast microscopy’ section above , as well as with a Photometrics CoolSNAP HQ2 CCD camera .","The magnification and pixel size were the same in both setups .","The experiment was replicated on two days and the data pooled together .","The MATLAB based software uTrack was used for particle tracking ( Jaqaman et al . , 2008 ) .","We used the comet detection algorithm to detect filaments ( difference of Gaussian: one pixel low-pass to 4–6 pixels high pass , watershed segmentation parameters: minimum threshold 3–5 standard deviations with a step size of 1 pixel ) which , at our MreB induction levels gave better localization of the resultant asymmetric particles over algorithms that search for symmetric Gaussians .","Visual inspection of detected particles confirmed that most of the particles and none of the noise were being detected .","A minimum Brownian search radius of 0 . 1–0 . 2 pixels and a maximum of 1–2 pixels was applied to link particles with at least five successive frames .","Directed motion propagation was applied , with no joins between gaps allowed .","Tracks were visualized using the FIJI plug-in TrackMate ( Tinevez et al . , 2017 ) .","For sphere to rod transitions and cells confined in microfluidic channels , movies were processed by subtracting every 8th frame from each frame to remove stationary spots using the FIJI plugin StackDifference before tracking .","The tracking was done as described earlier in this section .","Strains containing fluorescent fusions to TagT , TagU , and TagV were grown as described in the ‘Overnight culture growth’ section but in CH medium instead of LB .","Cells were grown for 3 hr at 37°C before imaging , then collected by centrifugation at 6000 x g for 2 min and re-suspended in CH .","Cells were then placed on a glass coverslip thickness No . 1 . 5 under an agar pad thickness 1 mm made from CH and 1 . 5% agarose .","Timelapse images were collected with TIRF illumination , using continuous 100 ms 488 nm exposures .","Epifluorescent illuminated images were collected from a single exposure , while maximal intensity projections were formed from a series of continuous 100 ms TIRF exposures .","Technical replicates were not collected , but each strain was imaged under a variety of appropriate imaging conditions to establish that the phenomena observed were not an artifact of the experimental setup .","For single-molecule experiments imaging HaloTag-15aa-TagU ( bAB196 ) , HaloTag-15aa-TagV ( bAB197 ) , HaloTag-15aa-TagT ( bAB198 ) , Pbp2A-30aa-HaloTag ( BSY201 ) and MreB-30aa-HaloTag ( BYS40 ) , cells were grown at 37°C in CH medium from fresh colonies until reaching mid-exponential phase ( OD600 = 0 . 5 ) , ten-fold diluted back in fresh CH medium and grown one more round until OD600 = 0 . 5 .","Cultures were then incubated for 15 min with 25 nM ( HaloTag-15aa-TagU/V/T and Pbp2A-30aa-HaloTag ) and 25 pM ( MreB-30aa-HaloTag ) of HaloTag-JF549 ligand .","Cells were spun for 1 min at 4000 xg , 10-fold concentrated and imaged under 2% agarose pads .","Images were collected on a Nikon TI microscope equipped with an EMCCD camera , together with a Nikon 100x NA 1 . 45 objective .","Exposure times were 0 . 25 s , and illumination was accomplished using a 561 nm laser .","BEG300 cells were grown from overnights as described in ‘Overnight culture growth’ but in CH medium instead of LB .","Cells were then grown at 37°C for 4 hr without xylose to deplete WTAs , then induced with 30 mM xylose for 1 . 5 hr to re-induce WTA expression .","Cells were then moved to 25°C for at least 30 min and incubated with 25 μg/mL Concanavalin A conjugated to Alexa Fluor 647 .","Cells were collected by centrifugation at 6000 x g for 2 min , washed with CH medium , then re-suspended in fresh CH medium .","Cells were then placed on a glass coverslip thickness No . 1 . 5 under an agar pad thickness 1 mm made from CH medium and 1 . 5% agarose .","For PY79 and BCW61 controls , lectin-Alexa Fluor conjugate concentration was 200 μg/mL .","Separate technical replicates were collected with the microscopes described in ‘Imaging – MreB Particle Tracking’ and ‘Imaging – Spinning Disk Confocal’ .","For quantitative analysis , the latter setup was used .","Quantification was performed in FIJI .","Five individual cells were selected arbitrarily and pooled for quantitative analysis .","The contours of each cell were manually traced , and intensity along these contours measured , then corrected for the mean fluorescent background .","The per cell average was calculated from the mean of each pixel in the contour less background ( calculated from an empty field ) .","The per strain average was taken as the mean of the per cell average .","The output of uTrack is the position coordinates of tracks over frames .","We fit a line through these coordinates using orthogonal least squares regression to minimize the perpendicular distance of the points from the line of best fit .","We used principal component analysis for orthogonal regression using custom written MATLAB code .","The R2 values we obtain range from 0 . 5 to","1 . We calculated mean track positions , angles and displacement using the line of best fit for all tracks .","We also calculated the mean square displacement versus time of individual tracks and fit these curves to the quadratic equation MSD ( t ) = 4Dt+ ( Vt ) 2 , using nonlinear least squares fitting .","As later times have fewer points and are noisier , we fit the first 80% of the data for each track .","We determined α by fitting a straight line to the logMSDt vs . log ( t ) curve .","The goodness of fit was evaluated by determining the R2 value .","We selected tracks for linearity and directional motion , based on the following cutoffs: R2 >0 . 9 , displacement >0 . 2 μm , , velocity >1e−9 μm/s , and R2 of the linear fit of log ( MSD ( t ) ) vs . log ( t ) >0 . 6 .","The MATLAB-based software Morphometrics ( Ursell et al . , 2017 ) was used to segment phase contrast images of cells .","We used the phase contrast setting for rod-shaped and intermediate states and the peripheral fluorescence setting for spherical states , because in this latter condition , peripheral fluorescence empirically did a better job of fitting cell outlines .","The cell contours obtained were visually inspected and any erroneous contours were removed by custom written MATLAB code .","Track angles were calculated with respect to the cell midline as defined by the Morphometrics ‘Calculate Pill Mesh’ feature , which identifies the midline based on a unique discretization of the cell shape determined from its Voronoi diagram .","The difference between the track angle and midline angle was then calculated .","Since the track angles θt and midline angles θm both ranged from −90° to 90° , the range of angle differences ∆θ= θt-θm was −180° to 180° .","We changed the range to 0 to 180° by the transformation: Δθ=180+ Δθ if Δθ<0 , and 0 to 90° by the transformation: Δθ=180− Δθ if Δθ>90 .","The mean deviation from 90° ( σ90 ) for each distribution was calculated using the following formula , where xi is each angle in the distribution and N is the total number of angles:σ90= ∑i=1N ( xi-90 ) 2N Custom written MATLAB code was used to calculate the normalized dot product ( DP ) of track pairs along with the distance ( d ) between their mean positions x- and y- as follows: DPij= cos ( θi −θj ) , dij= ( x−i−x−j ) 2+ ( y−i−yj− ) 2 To eliminate out-of-cell tracks we only considered those that had three other tracks within a 5 μm radius of their mean position .","The dot product of track pairs ( DP ) and distance ( d ) between them was stored in data files , along with all the previous information for each individual track ( R2 , velocities , angles , mean positions , displacement etc ) .","The files were then parsed using the cutoffs described in the ‘Data analysis – selecting directional tracks’ section .","The tracks were binned based on the distance and the mean dot product calculated for each distance range as follows: DP-= 1N∑i>jNcos ( θi -θj ) A cutoff of 3 μm was chosen as the maximum binning distance , which is the average length of a cell .","A data file containing simulated tracks was created by a custom written MATLAB script , which generates random angles distributed randomly on a 100 × 100 μm area .","Each track has R2 = 0 . 95 , velocity = 25 nm/s and displacement = 1 μm .","The same analysis code was run on these simulated tracks to generate track pairs with dot product and distance stored in a new data file .","The data file was parsed using the same cutoffs as the real data and the mean dot product for each distance range calculated .","The total numbers of trajectories within the simulation were much higher than the actual data ( 2–10 times higher ) .","Pill meshes were created using Morphometrics ( Ursell et al . , 2017 ) , which calculates the coordinates of line segments perpendicular to the cell long axis .","For cell widths at various steady state TagO and Mg2+ levels , the distance of these line segments was calculated using a custom written MATLAB script ( Hussain , 2017; copy archived at https://github . com/elifesciences-publications/hussain-2017-elife ) and the maximum width along the length of the cell was taken as the cell width .","When measuring cell width nearest to a track ( for calculating track angle as a function of cell width ) , the mean width of the 10 nearest contour points from the track was calculated using a custom written MATLAB script .","Cell widths of emerging bulges and rods from round cells were measured manually in FIJI .","Our ability to segment individual spherical cells was limited by their nonuniform contrast , perhaps arising from the nonuniform thickness of these cells in the Z dimension; consequently , Morphometrics-based width measurements in these cells was limited , especially in cells exceeding 2 μm in diameter .","Sidewall curvature of cells was extracted from the pill mesh obtained from Morphometrics .","The curvature values are calculated from three successive contour points and smoothed over two pixels .","The mean curvature of 3 nearest points to each track were calculated from both sides of the cell contour and called the mean curvature .","Principal curvature ratio was calculated by dividing the sidewall curvature with the curvature in the radial direction ( calculated from cell width assuming the cell is radially symmetric ) .","For radial curvature we used the following expression , where rcell is half the cell width:κ2=1rcell A value close to one indicates the two principal curvatures are similar and the cells are round .","Phase contrast images were used to show rod shape emergence from local bulges .","Edges were enhanced in FIJI and contrast adjusted to give bright cell outlines in the images .","The stack was then colored in time using temporal color code function in FIJI .","To create the curvature plot , the phase contrast images were run through Morphometrics which calculates the curvature at each contour point along the cell outline .","The contour points of interest were selected and plotted using a custom written MATLAB script , which colored each point according to its local curvature as calculated by Morphometrics .","To provide a good resolution for positive curvatures , we rescaled the color map such that negative curvatures were colored blue and positive curvatures were scaled by their curvature value .","Data from agar pads experiments was analyzed using custom written MATLAB code .","Data from cellASIC experiments was analyzed in Morphometrics to get areas for each cell .","For doubling times during sphere to rod transitions , the data was collected by manually measuring the areas of the sphere and rod regions of the same cell in FIJI .","In all cases , the area of each cell per frame was calculated and the log plot of area vs time was fit to a line .","The doubling time was calculated using the slope of this line .","N = 15 cells ( transitioning from spheres to rods ) .","Cell contours ( Figure 1—figure supplement 1 ) were used to calculate tangent angles using the equation: θi=tan-1yi+1-yixi+1-xi .","The correlation between angles was calculated using the cosine of the angle difference binned as a function of number of points ( n ) between the angles: Gn= 1N∑i=1Ncos ( θi+n-θi ) .","The number of points was converted to contour length using the pixel size of the camera to get the final correlation function: Gl= 1N∑i=1Ncos ( θi+l-θi ) .","Tracks were generated using the TrackMate plugin in Fiji ( 2017 ) .","Particles were detected with the Laplacian of Gaussians ( LoG ) detector , with a 0 . 4 μm spot diameter .","Tracks were generated using the Simple LAP Tracker , with a 0 . 1 μm linking max distance and no frame gaps allowed .","Tracks were exported into MATLAB for further processing .","Mean squared displacement ( MSD ) was calculated for all tracks as a function of time delay ( t ) .","For visualization of HaloTag tracks overlaid on phase images , we analyzed all tracks 10 frames or longer .","For each track , the scaling exponent ( α ) was calculated by fitting MSD ( t ) =C ( t∧ α ) +4 ( σ∧2 ) using nonlinear least-squares fitting with constant C and where σ is the localization error ( Monnier et al . , 2012 ) .","For analysis of alpha value and velocity frequencies tracks were first filtered using masks generated from phase images acquired after single-molecule imaging .","We then included all tracks between 10 and 120 frames in length .","α values were calculated by linear fitting log ( MSD ) versus log ( t ) .","Velocity ( v ) was calculated by fitting MSD ( t ) =4D ( t ) + ( v*t ) ∧2 + 4 ( σ∧2 ) using nonlinear least-squares fitting , where D is the diffusion constant ( Monnier et al . , 2012 ) .","For the graphs in Figure 6—figure supplement 2 , only plotted are molecules that moved in a consistent manner during their lifetime [>0 . 95 r2 fit to log ( MSD ) versus log ( t ) ] .","Analysis of alpha value and velocity frequencies for MreB filaments was the same as for HaloTag , except we included all tracks between 10 and 60 frames in length and filtration by phase image masks was not necessary .","Full length , un-tagged Thermotoga maritima MreB was purified as described previously ( Salje et al . , 2011 ) .","The protein was encapsulated inside unilamellar liposomes following a previously published protocol ( Szwedziak et al . , 2014 ) .","For this , 50 μL of E . coli total lipid extract , dissolved in chloroform at 10 mg/mL , was dried in a glass vial under a stream of nitrogen gas and left overnight under vacuum to remove traces of the solvent .","The resulting thin lipid film was hydrated with 50 μL of TEN100 8 . 0 ( 50 mM Tris/HCl , 100 mM NaCl , 1 mM EDTA , 1 mM NaN3 , pH 8 . 0 ) , supplemented with 20 mM CHAPS ( Anatrace , Maumee , Ohio ) , and shaken vigorously at 800 rpm using a benchtop micro centrifuge tube shaker for 2 hr .","The lipid-detergent solution was then sonicated for 1 min in a water bath sonicator .","Subsequently , 50 μL of MreB protein solution at 30 μM , supplemented with 0 . 5 mM magnesium ATP ( Jena Bioscience , Germany ) was added and left for 30 min at room temperature .","Next , the mixture was gradually diluted within 10–20 min to 600 μL with TEN100 8 . 0 plus 0 . 5 mM magnesium ATP ( without detergent ) to trigger spontaneous liposome formation .","2 . 5 μL of the solution was mixed with 0 . 2 μL 10 nm IgG immunogold conjugate ( TAAB , UK ) and plunge-frozen onto Quantifoil R2/2 carbon grid , using a Vitrobot automated freeze plunger ( FEI Company , Hillsboro , Oregon ) into liquid ethane .","2D electron cryomicroscopy images were taken on an FEI Polara TEM ( FEI Company ) operating at 300 kV with a 4k × 4 k Falcon II direct electron detector ( FEI Company ) at a pixel size of 1 . 8 Å .","For electron cryotomography , samples were imaged using an FEI Titan Krios TEM ( FEI Company ) operating at 300 kV , equipped with a Gatan imaging filter set at zero-loss peak with a slit-width of 20 eV .","A 4k × 4 k post-GIF K2 Summit direct electron detector ( Gatan , a subsidiary of Roper Technologies , Lakewood Ranch , Florida ) was used for data acquisition with SerialEM software ( 2005 ) at a pixel size of 3 . 8 Å at the specimen level .","Specimens were tilted from −60˚ to +60˚ with uniform 1˚ increments .","The defocus was set to between 8 and 10 μm , and the total dose for each tilt series was around 120–150 e/Å2 .","Tomographic reconstructions from tilt series were calculated using RAPTOR ( 2008 ) and the IMOD tomography reconstruction package followed by SIRT reconstruction with the TOMO3D package ( 1996; 2011a ) .","Movies showing liposomes were prepared with Chimera and PyMOL ( 2002; 2004 ) .","Imaging of E . coli strain RM478 was conducted as in ‘Overnight culture growth’ , save the cells were grown in LB or M63-Glucose .","When indicated , cells were grown and imaged under 2% agar pads made in the same media .","For the media down shift ( LB at 37°C to M63-Glucose at 25°C ) , cells were first grown in LB at 37°C in a drum roller , then placed under agar pads made of M63-Glucose at 25°C and allowed to equilibrate 15 min prior to imaging .","Imaging was conducted as in ‘Imaging – MreB particle tracking’ using a Nikon TI-E with TIRF illumination .","All strains used in this study are available in Supplementary file","1 . All primers used in this study are available in Supplementary file","2 . BCW51 [ycgO::Pxyl-tagO , tagO::erm , amyE::sfGFP-mreB , sinR::phleo] was generated by transforming BEG300 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers Sinr_up_F and Sinr_up_R and template PY79 genomic DNA; ( 2 ) PCR with primers oJM028 and oJM029 and template plasmid pWX478a ( containing phleo ) ; ( 3 ) PCR with primers Sinr_DOWN_R and Sinr_DOWN_F and template genomic DNA .","BCW61 [tagE::erm] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW054 and oCW055 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM028 and oCW057 and template plasmid pWX467a containing cat; ( 3 ) PCR with primers oCW058 and oCW059 and template PY79 genomic DNA .","BCW72 [yvhJ::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW139 and oCW141 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW142 and oCW143 and template PY79 genomic DNA .","BCW77 [ywtF::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW159 and oCW161 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW164 and oCW165 and template PY79 genomic DNA .","BCW78 [ywtF::msfGFP-ywtF] was generated by transforming BCW77 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW160 and oCW161 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW163 and oCW165 and template PY79 genomic DNA .","BCW79 [yvhJ::msfGFP-yvhJ] was generated by transforming BCW72 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW139 and oCW146 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW143 and oCW145 and template PY79 genomic DNA .","BCW80 [lytR::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW101 and oCW109 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW100 and oCW125 and template PY79 genomic DNA .","BCW81 [lytR::msfGFP-lytR] was generated by transforming BCW72 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW101 and oCW137 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW100 and oCW138 and template PY79 genomic DNA .","BCW82 [tagO::erm , ycgO::PxylA-tagO , amyE::Pspac-gfp-mreB ( spec ) , dacA::kan] was generated by transforming BEG300 with genomic DNA from BGL19 .","BEG202 [tagO::erm amyE::Pxyl-gfp-mbl ( spec ) ] was generated by transforming BEB1451 with genomic DNA from BJS18 .","BEG281 [ycgO::PxylA-tagO] was generated by transforming with a plasmid created via ligating a Gibson assembly into pKM077 .","pKM77 was digested with EcoRI and XhoI .","The assembly was created with two fragments: ( 1 ) PCR with primers oEG85 and oEG86 and template py79 genomic DNA; ( 2 ) PCR with primers oEG87 and oEG88 .","BEG291 [tagO::erm , ycgO::PxylA-tagO] was generated by transforming BEG281 with genomic DNA from BRB4282 .","BEG300 [tagO::erm , ycgO::PxylA-tagO , amyE::Pspac-gfp-mreB ( spec ) ] was generated by transforming BEG291 with genomic DNA from BEG275 .","BMD61 [mbl::mbl-msfGFP ( spec ) ] was generated by transforming py79 with a Gibson assembly consisting of four fragments: ( 1 ) PCR with primers oMD44 and oMD90 and template PY79 genomic DNA; ( 2 ) PCR with primers oMD47 and oMD56 and template synthetic , codon-optimized msfGFP; ( 3 ) PCR with primers oJM028 and oJM029 and template plasmid pWX466a ( containing spec ) ; ( 4 ) PCR with primers oMD48 and oMD50 and template genomic DNA .","bSW99 [amyE::spc-Pspac-mciZ] was generated by transforming PY79 with a Gibson assembly consisting of five fragments: ( 1 ) PCR with primers oMD191 and oMD108 and template PY79 genomic DNA ( containing upstream region of amyE ) ; ( 2 ) PCR with primers oJM29 and oJM28 and template plasmid pWX466a ( containing spec ) ; ( 3 ) PCR with primers oMD234 and oSW76 and template plasmid pBOSE1400 ( a gift from Dr . Briana Burton , containing spec ) ; ( 4 ) PCR with primers oAB307 and oAB291 and template PY79 genomic DNA ( containing mciZ ) ; ( 5 ) PCR with primers oMD196 and oMD197 and template PY79 genomic DNA ( containing downstream region of amyE ) .","bAB343 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Pspac-mciZ , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB185 ( Bisson-Filho et al . , 2017 ) with genomic DNA from bSW99 .","The resultant strain was then transformed with the genomic DNA from BEG291 and selected for Cm resistance .","Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 30 mM of xylose and 25 mM MgCl2 .","bAB327 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Physpank-minCD , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB185 ( Bisson-Filho et al . , 2017 ) with genomic DNA from JB60 ( a gift from Dr . Frederico Gueiros-Filho ) .","The resultant strain was then transformed with the genomic DNA from BEG291 and selected for Cm resistance .","Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 60 mM xylose and 25 mM MgCl2 .","bAB388 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Physpank-ftsA , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB199 ( Bisson-Filho et al . , 2017 ) with genomic DNA from BEG291 and selected for Cm resistance .","Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 60 mM xylose and 25 mM MgCl2 .","bAB196 [tagU::erm-Pxyl-HaloTag-15aa-tagU] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagU with primers oCW155 and oCW109 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagU with primers oCW138 and oCW156 and PY79 template genomic DNA .","bAB197 [tagV::erm-Pxyl-HaloTag-15aa-tagV] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagV with primers oCW140 and oCW141 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagV with primers oCW144 and oCW145 and PY79 template genomic DNA .","bAB198 [tagT::erm-Pxyl-HaloTag-15aa-tagT] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagT with primers oCW162 and oCW159 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagT with primers oCW163 and oCW166 and PY79 template genomic DNA .","bYS09 [mreB::mreB-mNeonGreen] was generated by transforming BMD88 ( Schirner et al . , 2015 ) with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD134 and oMD262 and bMD135 template genomic DNA ( containing the upstream of mreB and 40aa linker ) ; ( 2 ) PCR with primers oYS007 and oYS008 and gBlocks gene fragment containing mNeonGreen; ( 3 ) PCR with primers oMD92 and oMD116 and template PY79 genomic DNA .","bYS40 [mreB::mreB-HaloTag] was generated by transforming BMD88 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD134 and oYS602 and bMD135 template genomic DNA; ( 2 ) PCR with primers oYS603 and oYS604 and template plasmid cdr1086 ( containing HALO ) ; ( 3 ) PCR with primers oMD92 and oMD116 and template PY79 genomic DNA .","bYS201 [HaloTag-Pbp2A::cat] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD083 and oYS136 and bMD98 sfGFP-Pbp2A::cat template genomic DNA ( 2 ) PCR with primers oYS599 and oYS598 and template plasmid cdr1086 ( containing HALO ) ; ( 3 ) PCR with primers oMD069 and oMD082 and template PY79 genomic DNA ."]],"string":"[\n [\n \"Although many bacteria are rod shaped , the cellular mechanisms that construct and replicate this geometry have remained largely unknown .\",\n \"Bacterial shape is determined by the cell wall sacculus , a giant , encapsulating macromolecule that serves to resist internal osmotic pressure .\",\n \"One of the primary components of the cell wall is peptidoglycan ( PG ) , which is created by the polymerization of single glycan strands linked by peptide crossbridges .\",\n \"Studies of isolated cell walls from rod-shaped bacteria suggest material is generally oriented circumferentially around the rod , perpendicular to the long axis of the cell ( Gan et al . , 2008; Hayhurst et al . , 2008; Verwer et al . , 1980 ) , or in thick cables in others ( Hayhurst et al . , 2008 ) .\",\n \"This mostly circumferential , hoop-like organization of cell wall material allows the cell wall to better resist the internal osmotic pressure , as this pressure causes a stress twice as large in the circumferential direction ( on the rod sidewalls ) than in the axial direction ( on the poles ) ( Amir and Nelson , 2012; Chang and Huang , 2014 ) .\",\n \"This organization of material confers a mechanical anisotropy to the cell wall , causing it to stretch more along its length than across its width for a given stress; this anisotropy may assist rod-shaped cells in preferentially elongating along their length ( Baskin , 2005; Chang and Huang , 2014 ) .\",\n \"Concordantly , atomic force microscopy ( AFM ) has shown that Escherichia coli sacculi are 2–3 times more elastic along their length than across their width ( Yao et al . , 1999 ) .\",\n \"This rod-reinforcing circumferential organization is also observed in the cell walls of plants; hypocotyl and root axis cells rapidly elongate as rods by depositing cellulose fibrils in circumferential bands around their width , resulting not only in a similar dispersive rod-like growth , but also a similar anisotropic response to stress ( Baskin , 2005 ) .\",\n \"The organized deposition of cellulose arises from cortical microtubules self-organizing into a radial array oriented around the rod width , and this orients the directional motions of the cellulose synthases to insert material in circumferential bands ( Paredez et al . , 2006 ) .\",\n \"In contrast to our understanding of the self-organization underlying rod-shaped growth in plants , how bacteria construct a circumferential organization of glycan strands is not known .\",\n \"This organization may arise via the actions of a small number of genes essential for the formation and maintenance of rod shape .\",\n \"Collectively termed the Rod complex , ( or elongasome ) these include MreB , MreC , MreD ( encoded by the mreBCD operon ) ( Wachi et al . , 1989 ) , RodZ ( Alyahya et al . , 2009; Bendezú et al . , 2009 ) , and the glycosyltransferase/transpeptidase enzyme pair RodA/Pbp2 ( Cho et al . , 2016 ) .\",\n \"These components are conserved across a wide range of rod shaped bacteria , and mostly absent in cocci ( Alyahya et al . , 2009; Chastanet and Carballido-Lopez , 2012 ) , leading many to speculate they function as the central determinants of rod shape ( Carballido-Lopez , 2006; Jones et al . , 2001 ) .\",\n \"The spatial coordination of RodA/Pbp2-mediated PG synthesis is conferred by MreB , an actin homolog ( Jones et al . , 2001; van den Ent et al . , 2001 ) .\",\n \"MreB polymerizes onto membranes as antiparallel double filaments , which have been observed to bend liposome membranes inward ( Figure 1A ) ( Salje et al . , 2011; van den Ent et al . , 2014 ) .\",\n \"Loss or depolymerization of MreB causes rod-shaped cells to grow as spheres ( Gitai et al . , 2005; Jones et al . , 2001; Bendezú et al . , 2009 ) .\",\n \"B . subtilis contains 3 MreB paralogs ( MreB , Mbl , and MreBH ) that have been shown to co-polymerize into mixed filaments in vitro , and always colocalize in vivo ( Defeu Soufo and Graumann , 2004; Soufo and Graumann , 2010; Dempwolff et al . , 2011 ) .\",\n \"MreB filaments move circumferentially around the width of rod-shaped cells ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011; van Teeffelen et al . , 2011 ) .\",\n \"Super-resolution imaging has demonstrated that MreB filaments always translocate along their length , moving in the direction of their orientation ( Olshausen et al . , 2013 ) .\",\n \"MreB filaments move in concert with MreC , MreD , and RodA/Pbp2 ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011 ) , and loss of any one component stops the motion of the others .\",\n \"The directional motion of MreB filaments and associated Rod complexes depends on , and thus likely reflects , the insertion of new cell wall , as this motion halts upon the addition of cell wall synthesis-inhibiting antibiotics ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011; van Teeffelen et al . , 2011 ) , or specific inactivation or depletion of Pbp2 ( Garner et al . , 2011; van Teeffelen et al . , 2011 ) or RodA ( Cho et al . , 2016 ) .\",\n \"It is not known how MreB and the rest of the Rod complex construct rod-shaped cells .\",\n \"As the motions of the Rod complexes reflect the insertion of new cell wall , their circumferential motions could deposit glycans in the hoop-like organization required to both build and reinforce rod shape .\",\n \"Therefore , we worked to understand the origin of this circumferential organization , seeking to determine what orients the motions of MreB and associated enzymes around the rod width in Bacillus subtilis .\"\n ],\n [\n \"The mechanism by which MreB filaments and associated PG synthases orient their motion around the rod circumference is not known .\",\n \"Each filament-synthase complex is disconnected from the others , moving independently of proximal neighbors ( Garner et al . , 2011 ) .\",\n \"The organized , circumferential motion of these independent filament-synthase complexes could arise in two ways: ( 1 ) A templated organization , where cell wall synthetic complexes move along an existing pattern of ordered glycan strands in the cell wall as they insert new material into it ( Höltje , 1998 ) , or ( 2 ) A template-independent organization , where each synthetic complex has an intrinsic mechanism that orients its motion and resultant PG synthesis around the rod circumference .\",\n \"To explore the extent of order within the motions of the Rod complex , we analyzed the trajectories of GFP-Mbl and GFP-MreB with respect to the cell body using total internal reflection fluorescence microscopy ( TIRFM ) ( Figure 1B ) .\",\n \"Overall , these motions are circumferentially oriented , but not perfectly aligned , a characteristic reflected by the broad distribution of angles that GFP-MreB , its homologs , and the other components of the Rod complex move relative to the long axis of the cell ( Domínguez-Escobar et al . , 2011; Garner et al . , 2011 ) .\",\n \"However , examination of TIRFM time lapse movies revealed that both MreB and Mbl trajectories close in time ( within the period of one revolution ) frequently cross ( Figure 1C , Figure 1—video 1 ) , making it unlikely that MreB filaments move along a perfectly ordered template .\",\n \"As MreB movement reflects the insertion of new glycan strands , these motions indicate that the siacculus is built from somewhat disorganized , yet predominantly circumferential strands .\",\n \"This conclusion is in agreement with X-ray diffraction ( Balyuzi et al . , 1972; Labischinski et al . , 1979 ) and cryoelectron microscopy studies of E . colisacculi ( Gan et al . , 2008 ) which found that , while glycans are oriented circumferentially around the rod width on average , they are not ordered , running at variable angles to each other .\",\n \"Similarly , atomic force microscopy of B . subtilis sacculi has observed a generally oriented , but unaligned arrangement of 50 nm thick cables oriented roughly perpendicular to the long axis ( Hayhurst et al . , 2008 ) .\",\n \"Thus , both the motions of MreB and ultrastructural studies indicate the sacculus is not highly ordered , making it unlikely that it can serve as a self-propagating spatial template for rod shape .\",\n \"Furthermore , given that preexisting cell wall is not necessary for the regeneration of rod shape from wall-less B . subtilis L-forms ( Kawai et al . , 2014 ) , it is likely that both oriented MreB motion and rod shape can arise without an ordered template .\",\n \"As it appeared that organized MreB motion does not arise from patterns in the cell wall , we hypothesized there was an intrinsic mechanism orienting the motion of each MreB filament-cell wall synthetic complex .\",\n \"To test this hypothesis , we examined MreB motions as we changed the shape of cells from rods to spheres .\",\n \"As the internal osmotic pressure and stiffness of B . subtilis resists external mechanical perturbations to its shape ( Renner et al . , 2013 ) , we first altered the shape of cells by controlling the level of wall teichoic acids ( WTAs ) .\",\n \"WTAs are negatively charged cell wall polymers believed to increase the rigidity of the sacculus ( Matias and Beveridge , 2005 ) , a process that could occur via their coordination of extracellular Mg2+ ( Thomas and Rice , 2014; Kern et al . , 2010 ) , or modulation of hydrolase activity ( Atilano et al . , 2010 ) .\",\n \"Knockouts of tagO , the first gene in the WTA synthesis pathway , create large , slow-growing , round cells that still synthesize PG , building extremely thick and irregular cell walls ( D'Elia et al . , 2006 ) .\",\n \"We placed tagO under xylose-inducible control and grew cells at different induction levels .\",\n \"As expected , at high TagO inductions , cells displayed normal widths .\",\n \"As we reduced TagO levels , rods became gradually wider ( Figure 1D–E ) until , beneath a given induction , cells were no longer able to maintain rod shape , growing as spheres ( or clumps of spheres ) with no identifiable long axis .\",\n \"At intermediate induction levels , we observed a transition region between the two states , with cells growing as steady state populations of interconnected rods and spheres ( Figure 1D ) .\",\n \"In agreement with models that ( A ) WTAs work with PG to bind Mg2+ ( Thomas and Rice , 2014; Kern et al . , 2010 ) , and ( B ) are required for cell wall rigidity ( Matias and Beveridge , 2005 ) , both the cell width and the amount of TagO induction determining the rod/sphere transition could be modulated by Mg2+ levels ( Figure 1F , Figure 1—figure supplement 1B ) .\",\n \"Likewise , decreasing extracellular Mg2+ or tagO induction resulted in increasingly curved cell contours ( Figure 1—figure supplement 1B ) , suggesting the wall was becoming more flexible .\",\n \"By tracking the motion of GFP-MreB filaments in these differing cell shapes , we found that motion is always oriented in rods , moving predominantly circumferentially at all induction levels above the rod/sphere transition .\",\n \"However , in round cells ( those induced beneath the rod/sphere transition point or in tagO knockouts ) MreB filaments continued to move directionally , but their motions were isotropic , moving in all directions ( Figure 2A , Movie Figure 2—video 1 ) .\",\n \"To quantify the relative alignment of MreB under each condition , we calculated the angle between trajectory pairs less than 1 μm apart ( Figure 2B , Figure 2—figure supplement 1A ) .\",\n \"This analysis revealed that MreB motions are more aligned when cells are rods: above the rod/sphere transition , trajectories have a median angle difference of 26°; while at low TagO inductions , where cells are round , the angle difference increases to 42° , close to that of randomly oriented trajectories ( 45° ) .\",\n \"To verify that the loss of oriented MreB motion was due to the changes in cell shape , and not from some other effect of reduced WTA levels , we created round cells by alternate means .\",\n \"Depletion of both elongation PG transpeptidases ( Pbp2a and PbpH ) causes rod-shaped cells to become wider over time as they convert to spheres ( Garner et al . , 2011 ) .\",\n \"We used this gradual transition of rods into spheres to examine both the width and overall shape dependence of MreB motion .\",\n \"At initial points of depletion ( 1–2 hr ) the rods widened but maintained circumferential MreB motion .\",\n \"At 2 . 5 hr of PbpA depletion , cells were a mix of spheres and rods of differing widths .\",\n \"These cells displayed the same pattern of MreB orientation observed with tagO depletions: round cells contained unoriented MreB , while nearby rod-shaped cells showed circumferential motion .\",\n \"Identical behavior was observed for GFP-Mbl during PbpA depletions ( Figure 2C , Movie Figure 2—video 2 ) .\",\n \"Quantitation of trajectories from all cells ( both rods and spheres ) at each time point of depletion indicated an increase in the median angle between trajectories as the population grew wider and rounder over time ( Figure 2D , Figure 2—figure supplement 1B ) .\",\n \"In E . coli , the angle of mutant MreB filaments relative to the long axis has been reported to increase with cell width ( Ouzounov et al . , 2016 ) .\",\n \"To test if the angle of MreB movement changes with respect to cell width in B . subtilis , we calculated the angle of each trajectory to the long axis for all cells in our data with an identifiable width axis .\",\n \"At the same time , we also measured the curvature of each cell to determine how the overall shape of the cell affected the orientation of motion .\",\n \"This revealed that MreB motion in rods remained equivalently oriented over a wide range of rod widths , up to ~2 μm ( Figure 2E , Figure 2—figure supplement 1C–E ) .\",\n \"Beyond a 2 μm width , cells began to lose their rod shape as they became more spherical , and the predominantly circumferential orientation of MreB motion was lost ( Figure 2E , Figure 2—figure supplement 1E ) .\",\n \"This suggested that oriented MreB motion does not sense or rely on a specific cell radius; rather the orientation relies on differences between the two principal curvatures of the membrane .\",\n \"It appears that the motion of MreB filaments is oriented along the direction of greatest principal curvature: In rods , there is zero curvature along the rod length , and high curvature around the rod circumference , along which filaments orient .\",\n \"In contrast , in round cells where MreB motion is isotropic , the two principal curvatures are equal ( Figure 2F ) .\",\n \"To further verify that MreB filaments orient in response to overall cell shape , we externally imposed rod shape on cells with unoriented MreB motion .\",\n \"We loaded TagO-induced cells into long 1 . 5 ×1 . 5 μm microfluidic chambers , then reduced TagO expression to levels insufficient to produce rods in liquid culture ( Figure 3A , Figure 3—figure supplement 1A ) .\",\n \"After TagO depletion , cells expanded to fill the chamber indicating that WTA-depletion caused shape changes just as in bulk culture ( Figure 3A , Figure 3—figure supplement 1A ) .\",\n \"Within these chambers , cells grew as rods , but at a wider width ( 1 . 5 μm ) than wild-type cells , set by the chamber .\",\n \"When cells grew out of the chamber they swelled just as in bulk culture , showing confinement was required for rod shape at this induction level ( Figure 3B , Figure 3—figure supplement 1A ) .\",\n \"In the TagO-depleted cells confined into rod shapes , MreB moved circumferentially ( Figure 3C , Figure 3—video 1 ) , confirming that MreB orients in response to the cells having rod shape .\",\n \"This experiment demonstrates that the isotropic MreB motion observed in round cells arises from the lack of rod shape , and not from some other effect of our genetic perturbations .\",\n \"This experiment also showed another unexpected result: the doubling time of free ( unconstrained ) cells induced at similar TagO levels is long ( 53 ± 10 min ) , but confining them into rod shape restored their doubling time ( 44 ± 4 min ) toward wild-type times ( 39 ± 9 min ) ( Figure 3—figure supplement 1B ) .\",\n \"We next attempted to minimize any contribution to MreB filament orientation from ( A ) the directional motion of filaments , and ( B ) any pre-existing order within the sacculus .\",\n \"To accomplish this , we examined filament orientation in protoplasts ( cells that had their cell wall enzymatically removed ) confined into different shapes , using highly expressed GFP-MreB to assay long filaments , and GFP-Mbl to assay short filaments .\",\n \"We protoplasted cells in osmotically stabilized media ( Wyrick and Rogers , 1973 ) , then grew them under agar pads containing micro-patterned cross shapes .\",\n \"Cells in the center of these crosses ( ~5 μm diameter ) were forced to grow as spheres , whereas cells in the arms were constrained to grow into rods of various widths ranging from 2 to 5 μm ( Figure 3D ) .\",\n \"As reported previously ( Domínguez-Escobar et al . , 2011 ) , MreB filaments within protoplasts did not move directionally ( Figure 4—video 1 ) , likely because the cell wall provides the fixed surface along which the PG synthesis enzymes move .\",\n \"Within the protoplasts confined into the smallest rod shapes ( 2 μm ) , filaments oriented at a distribution of angles predominantly perpendicular to the cell length ( Figure 3E–F ) .\",\n \"The angular distributions of short GFP-Mbl filaments and longer GFP-MreB filaments were similar to each other , and also similar to the distribution of\",\n \"1 ) filament trajectories observed in intact , wild-type cells and\",\n \"2 ) filament trajectories of TagO depleted cells confined into the mother machine ( Figure 3Fi ) .\",\n \"As we increased the width of the imposed rod shape from 2 to 5 μm , filaments remained predominantly oriented in all cases ( Figure 3Fii ) , but their mean deviation from 90° increased as the rod width increased ( 34° at 2 μm , 35° at 4 μm , and 41° at 5 μm ) .\",\n \"In contrast to confinement in rods , both short and long filaments in spherically confined protoplasts remained unoriented ( Figure 3E ) .\",\n \"Together , these data demonstrate that MreB filaments orient to point around the rod width even in the absence of pre-existing cell wall or directional motion , as long as the cell has a rod shape .\",\n \"These experiments also demonstrate that MreB filaments will align even in wider rods , where the difference in principal curvatures is smaller than in wild-type cells , but that , as the difference in principal curvatures decreases , filament alignment becomes more disordered .\",\n \"To test if MreB filaments are themselves sufficient to align along the predominant direction of membrane curvature , we assembled purified T . maritima MreB within liposomes and visualized it using cryoelectron microscopy and tomography .\",\n \"While controlling the final concentration of protein encapsulated within liposomes ≤1 μm is difficult , we were able to assemble MreB inside liposomes at high concentrations .\",\n \"At these concentrations , MreB filaments tubulated liposomes , creating rod-like shapes ( Figure 4A , Figure 4—figure supplement 1A–B , Figure 4—video 1 ) .\",\n \"In tubulated regions , MreB filaments could be traced around the circumference of the liposome tube , while filaments in spherical regions were found in all possible orientations ( Figure 4A ) .\",\n \"At the highest concentrations , tubulated liposomes contained closely packed filament bundles , allowing us to observe a regular patterning of the canonical double filaments of MreB ( Figure 4B ) .\",\n \"Purified wild-type MreB did not bind to the outside surface of small liposomes contained within larger ones ( Figure 4A ) , indicating that MreB filaments preferentially polymerize on inward ( negative ) curvatures , akin to the inner leaflet of the bacterial membrane .\",\n \"In the absence of MreB , liposomes are spherical , with no deformations ( Figure 4—figure supplement 1C ) .\",\n \"Together , this data suggests that MreB filaments themselves are sufficient to align along the predominant direction of membrane curvature , as observed here with laterally associated filaments .\",\n \"We note that the experimental limitations of the liposomal system , combined with the tendency of MreB filaments to self-associate make it difficult for us to acquire and study the alignment of single filaments in vitro .\",\n \"Also , it remains to be determined if membrane-associated MreB filaments exist as bundles or isolated filaments in vivo .\",\n \"The above observations demonstrate that MreB filaments sense and align along the direction of greatest principal curvature , that is , the more curved inner surface of the rod circumference .\",\n \"The ultrastructure of MreB filaments provides a possible mechanism: MreB filaments are bent ( Salje et al . , 2011 ) , with the membrane-interacting surface on the outer face of the bend ( Figure 4C ) .\",\n \"This bent conformation could cause filaments to preferentially orient along the curved rod circumference , rather than the flat rod length , to maximize the burial of hydrophobic moieties into the membrane , a mechanism suggested by previous theory ( Wang and Wingreen , 2013 ) .\",\n \"As the curvature of MreB filaments bound to liposomes is much greater ( ~200 nm diameter [van den Ent et al . , 2014] ) than that of B . subtilis cells ( ~900 nm diameter ) , we performed analytical calculations to model how highly curved MreB filaments would align within a cell with a less curved surface ( Figure 4D–G , Appendix 1 ) .\",\n \"As many of the biochemical and physical parameters of MreB are still unknown , we first assumed a fixed set of parameters , and later verified that our results were robust over a large parameter range .\",\n \"We initially assumed a membrane interaction energy of 10 kT per monomer ( calculated from residues involved in membrane associations [Salje et al . , 2011] ) , and a similar Young’s modulus to actin ( 2 GPa ) .\",\n \"We modeled filaments as elastic beams made of two protofilaments .\",\n \"In addition , we used the Helfrich free energy to model the energetics of membrane deformation , and accounted for the work done against osmotic pressure due to changes in volume ( Appendix 1 ) .\",\n \"These calculations indicate that the total energy is minimized when filaments orient along the direction of maximal curvature ( Figure 4F ) and that , importantly , the energy penalty for incorrectly-oriented filaments is much greater than the energy of thermal fluctuations .\",\n \"Interestingly , this modeling indicates a decrease in energetic preference for the preferred filament orientation as the radius of the cell is increased ( Figure 4F ) , a prediction in qualitative agreement with our observations of alignment in protoplasts .\",\n \"Furthermore , our calculations indicate that orientation is robust over a large , biologically relevant range of parameters , including the membrane binding energy , filament length , and filament Young’s modulus ( Figure 4G ) .\",\n \"These calculations predict that filaments should orient circumferentially both if the membrane deforms to the filament ( at low osmotic pressures or if filaments are stiff ) ( Salje et al . , 2011 ) , or if filaments deform to the membrane ( at high osmotic pressures or if filaments are flexible ) ( Figure 4E ) .\",\n \"Our experimental data demonstrates MreB filament alignment across a range of pressures: high within cells , low to none within liposomes , and a pressure between the two within osmotically-stabilized protoplasts .\",\n \"In the absence of osmotic pressure , MreB filaments deform liposomes since it is energetically more favorable to bend the membranes than to bend the filaments , as observed in our in vitro data ( Figure 4A , Figure 4—figure supplement 1 ) .\",\n \"However , in live cells , our modeling predicts that MreB filaments cannot deform the inner membrane due to the large osmotic pressure , and instead deform to match the greatest principal membrane curvature .\",\n \"Hence filaments create curvature in liposomes and sense it in cells .\",\n \"Together , the above data demonstrate that MreB filaments are sufficient to preferentially orient along the direction of greatest principal membrane curvature .\",\n \"In rod-shaped cells , this direction is along the rod circumference .\",\n \"As filaments move along their length , their orientation constrains the spatial activity of the PG synthetic enzymes such that new cell wall is inserted in a mostly circumferential direction ( Hayhurst et al . , 2008 ) to reinforce rod shape ( Chang and Huang , 2014; Yao et al . , 1999 ) .\",\n \"While the ability of MreB filaments to orient in pre-existing rods can help explain how rod shape is maintained , we also wanted to understand how MreB filaments facilitate the de novo formation of rod shape .\",\n \"To explore this , we observed how cells interconvert between spheres and rods .\",\n \"We first examined how rod shape fails , by growing our TagO-inducible strain at induction levels that produced rods and then reducing the Mg2+ concentration to induce them to convert to spheres .\",\n \"This transition revealed that rods convert into round cells by continuous swelling: once a rod begins to widen , it continues to do so until reaching a fully spherical state with no reversion during the process ( Figure 5A ) .\",\n \"Similar rod to sphere transitions could be attained by holding Mg2+ constant while reducing TagO expression .\",\n \"Likewise , cells grown at intermediate TagO induction levels ( 8–12 mM ) grew as steady state populations of interconnected rods and spheres , indicating that cells underwent repeated cycles of rod shape formation followed by reversion to spheres ( Figure 1D , F ) .\",\n \"These results indicate that rod shape can be maintained only as long as the cell wall is sufficiently rigid to resist the internal osmotic pressure .\",\n \"We next examined how rod shape forms from round cells .\",\n \"As the recovery of protoplasted B . subtilis is so infrequent that it has never been directly visualized ( Mercier et al . , 2013 ) , we assayed how round cells with preexisting cell walls convert back into rods , using three systems: ( 1 ) re-inducing WTA expression within TagO-depleted , spherical cells , ( 2 ) holding TagO expression beneath the rod/sphere transition and increasing Mg2+ levels , and ( 3 ) re-inducing Pbp2a expression in spherical , Pbp2a-depleted cells .\",\n \"In all three cases , rods reformed in a discrete , local manner; spheres did not form into rods by progressively shrinking along one axis , but rather , rods abruptly emerged from one point on the cell , growing more rapidly than the parent sphere ( Figure 5B , Figure 5—video 1 and Figure 5—video 2 ) .\",\n \"This morphology is similar to the initial outgrowth of germinating B . subtilis spores ( Pandey et al . , 2013 ) .\",\n \"We occasionally observed another mode of recovery , occurring when round cells were constrained , or divided into , ovoid or near-rod shapes .\",\n \"Once these near-rod shaped cells formed , they immediately began rapid , rod-like elongation along their long axis ( Figure 5—figure supplement 1A ) .\",\n \"We focused on two salient features of the rod shape recoveries: ( 1 ) rod shape forms locally , most often at one point on the cell surface , and ( 2 ) once a rod-like region is formed , it appears self-reinforcing , both propagating rod shape and growing faster than adjacent or attached non-rod shaped cells .\",\n \"We first wanted to understand how rod shape initiates de novo from spherical cell surfaces .\",\n \"By examining the initial time points of recoveries , we found that rods begin as small outward bulges: local regions of outward ( positive Gaussian ) curvature flanked by regions of inward ( negative Gaussian ) curvature ( Figure 5C ) .\",\n \"These initial outward bulges showed a width distribution similar to that of the later emerging rods ( Figure 5D ) .\",\n \"Once these bulges formed , they immediately began rapid elongation into nascent rods , which would then thin down to wild type width over time .\",\n \"Bulge formation and rod recovery were independent of cell division , as cells depleted of FtsZ still recovered rod shape ( Figure 5—figure supplement 1B ) .\",\n \"Rather , these bulges appeared to arise randomly , evidenced by the fact that different cells produced rods at different times during WTA or Pbp2a repletion .\",\n \"We conclude that the appearance of a local outward bulge can act as the nucleating event of rod shape formation .\",\n \"As emerging rods appeared to grow faster than adjacent round cells , we tested if the doubling times of rod-shaped cells were faster than those of non-rods by measuring the doubling times in our inducible TagO strain at different induction levels using both OD600 measurements and single cell microscopy under steady state conditions ( Figure 3—figure supplement 1B ) .\",\n \"This revealed a sharp transition in doubling time that matched the conditions of the rod/sphere transition: growth is slow when cells are spheres , yet greatly increases when cells are rods ( Figure 3—figure supplement 1B , Figure 5—figure supplement 1C ) .\",\n \"Furthermore , the doubling times of recovering rods was similar to that of rods at steady state ( Figure 3—figure supplement 1B ) .\",\n \"We believe the lower doubling time of rods is likely due to cell shape and not another effect , such as the lack of WTAs , as ( 1 ) the doubling time of TagO-depleted cells confined in the microfluidic chambers matched that of wild type cells; and ( 2 ) both the doubling times and the boundary of the rod/sphere transition could be equivalently shifted by changing the Mg2+ concentration ( Figure 1F , Figure 1—figure supplement 1 , Figure 5—figure supplement 1C , Figure 5—video 2 ) .\",\n \"Combined , these results indicate that rod shape creates local , self-reinforcing regions that are poised for more rapid growth; once any small region of the cell approximates a rod shape , growth of the rod-like region is amplified , growing faster than other regions , and thereby outcompeting non-rod growth at the population level .\",\n \"We next sought to determine what features distinguished rods from round cells .\",\n \"As the elongation of rod-shaped cells requires a sufficiently rigid cell wall ( Figures 1D–E and 5A ) , the localized , self-reinforcing formation of rods in our system could arise from either of the two major cell wall components , PG and WTAs: ( 1 ) the PG strands could be arranged such that they better reinforce rod shape ( Amir and Nelson , 2012; Chang and Huang , 2014 ) , or ( 2 ) WTAs could be preferentially incorporated into the emerging rods to stabilize them .\",\n \"To assay the orientation of newly inserted cell wall , we imaged the motions of MreB as we induced TagO-depleted cells to recover into rods .\",\n \"This revealed that oriented MreB motion correlates with local shape: emerging rods displayed oriented MreB motion even at the initial points of their formation , while attached round parent cells displayed unaligned motion ( Figure 6A , Figure 6—video 1 ) .\",\n \"This demonstrates that oriented MreB motion correlates with local geometry and does not arise from a global , cell spanning change .\",\n \"We next examined the overall cellular distribution of MreB in recovering cells with confocal microscopy .\",\n \"This revealed that , immediately prior to rod emergence , MreB transiently accumulated in a bright ring oriented perpendicular to the direction of rod emergence , most often occurring at the interface of the bulge and the round cells ( Figure 6B , Figure 5—figure supplement 1D ) .\",\n \"To observe whether both spheres and rods inserted new PG during the process of rod shape recovery , we used fluorescent D-amino-acids ( FDAAs ) , which crosslink into newly inserted cell wall .\",\n \"We grew TagO-depleted cells in a microfluidic device in the presence of HADA , then switched the media to contain Cy3B-ADA as we re-induced TagO expression .\",\n \"During rod emergence , the old cell wall signal ( HADA ) remained in the sphere , while the emerging rod was almost entirely composed of new ( Cy3B-ADA ) material , confirming the discrete nature of rod shape recovery .\",\n \"However , the attached spheres also incorporated Cy3B-ADA , indicating PG synthesis occurs in both rods and spheres during recovery ( Figure 5—figure supplement 1E ) .\",\n \"The local reinforcement of rod shape in recovering cells could arise from preferential incorporation of the cell wall rigidifying WTAs .\",\n \"As the WTA ligases have been reported to interact with MreB ( Kawai et al . , 2011 ) , we tested if rod shape correlated with increased WTA accumulation in emerging rods .\",\n \"To test this , we labeled recovering cells with fluorescently labeled lectins that specifically bind to WTAs ( Figure 6—figure supplement 1A ) .\",\n \"Following TagO reinduction , WTAs in recovering cells were ( Figure 6—figure supplement 1B ) , equally present in the cell walls of both rods and spheres ( Figure 6C ) .\",\n \"To test if the WTA ligases move with MreB , we created GFP fusions to these proteins at their native locus and examined their dynamics with TIRFM .\",\n \"We were unable to observe any of the circumferential motions expected if the WTA ligases moved with MreB; instead they appeared to be rapidly diffusing on the membrane ( Figure 6—figure supplement 1C , Figure 6—video 2 , Appendix 2 ) .\",\n \"For further confirmation , we tracked the single molecule motions of these ligases , using JF549 labeled HaloTag fusions expressed at the native locus and promoter .\",\n \"We did not observe any directional motions of the ligases that would indicate they move or associate with MreB and Pbp2A ( Figure 6—figure supplement 2D–E , Figure 6—video 3 ) .\",\n \"In summary , these data gives new insights into what properties of the cell wall can be modulated to create and stabilize rod shape: rod shape is not formed by preferential localization of teichoic acids to rods , and both spheres and rods incorporate PG before and during rod shape recovery , in line with reports that PG synthesis is unchanged by the inhibition of WTA synthesis ( Pooley et al . , 1993 ) .\",\n \"Rather , the only differences we detected between rod shaped and round cells were increased growth rates and oriented MreB motion .\",\n \"We note that as WTAs have been shown to affect hydrolase activity ( Kasahara et al . , 2016 ) , their depletion may cause other rod-shape inhibiting PG abnormalities that we cannot observe .\"\n ],\n [\n \"To construct regular , micron-spanning shapes made of covalently crosslinked material , nature must devise strategies for coordinating the activities of disperse , nanometer-scale protein complexes .\",\n \"This work reveals that the role of MreB in creating rod shape is to locally sense and subsequently reinforce differences in principal curvatures .\",\n \"The local , short-range feedback between differences in curvature , MreB orientation , and shape-reinforcing cell wall synthesis could provide a robust , self-organizing mechanism for the stable maintenance and rapid reestablishment of rod shape , allowing the local activity of short MreB filaments to guide the emergence of a shape many times their size .\"\n ],\n [\n \"All B . subtilis strains were prepared for experimentation as follows: strains were streaked from −80°C freezer stocks onto lysogeny broth ( LB ) agar plates .\",\n \"Following >12 hr of growth at 37°C , single colonies were transferred to serially diluted overnight bulk liquid cultures in LB supplemented with 20 mM magnesium chloride , placed on a roller drum agitating at 60 rpm , and grown at 25°C .\",\n \"After >12 hr growth to OD600 <0 . 6 , these starter cultures were transferred to or inoculated into subsequent growth conditions .\",\n \"All strains with tagO under inducible control were grown overnight in the presence of 30 mM xylose unless otherwise noted .\",\n \"For the experiments in Figure 6C and Figure 3—figure supplement 1B , BEG300 cells were inoculated in the indicated medium ( LB with 20 mM MgCl2 unless otherwise stated ) from logarithmic phase overnights; ‘rods’ were grown from a low dilution with 30 mM xylose , and ‘spheres’ were grown with 0 mM xylose .\",\n \"For bulk culture doubling time measurements , doubling times were calculated from the slope of a graph of time vs . dilution for a succession of serial dilutions of a given strain .\",\n \"Time , the dependent variable , was taken as the time for a given dilution to pass the OD cutoff of OD600 = 0 . 20 .\",\n \"In place of technical replicates , a large number of replicates were performed on a continuous gradient of xylose induction , showing a consistent trend between the extreme values depicted in the figure .\",\n \"Single cell measurements were made in three ways .\",\n \"Phase contrast images were collected on a Nikon ( Tokyo , Japan ) Ti microscope equipped with a 6 . 5 μm-pixel Hamamatsu ( Hamamatsu City , Japan ) CMOS camera and a Nikon 100x NA 1 . 45 objective .\",\n \"Cells were collected by centrifugation at 6000 x g for 2 min and re-suspended in the original growth medium .\",\n \"Unless otherwise specified , cells were then placed on No . 1 . 5 cover glass , 24 × 60 mm , under a 1 mm thick agar pad ( 2–3% agar ) containing LB supplemented with 20 mM magnesium chloride .\",\n \"Unless otherwise noted , all cells were imaged at 37°C on a heated stage .\",\n \"Images were collected on a Nikon TI microscope with a 6 . 5 μm-pixel CMOS camera and a Nikon 100x NA 1 . 45 objective .\",\n \"Cells of strain BEG300 were grown overnight in LB supplemented with 30 mM xylose , 20 mM magnesium chloride , 1 μg/mL erythromycin , and 25 μg/mL lincomycin at 25°C at the specified xylose concentrations .\",\n \"11 μM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) was added to induce GFP-MreB and the cells were shifted to 37°C and allowed to grow for 2 hr before imaging .\",\n \"Cells of strain BEG202 ( ΔtagO ) with GFP-Mbl under a xylose-inducible promoter were grown overnight at 25°C in LB supplemented with 20 mM magnesium chloride and 0 . 125 mM xylose , and shifted to 37°C for 2 hr before imaging .\",\n \"Cells were placed on cleaned glass coverslips thickness No . 1 . 5 , as described in the next section .\",\n \"3–6% agar pads were prepared in LB supplemented with 20 mM magnesium chloride , 11 μM IPTG and the desired concentration of xylose .\",\n \"Images were collected for 3 min at 1 or 2 s intervals , as specified .\",\n \"30–50 cells were imaged in a day , and the experiments were repeated on at least one other day to test for technical variation .\",\n \"Coverslips were sonicated in 1 M KOH for 15 min , followed by five washes with water .\",\n \"Coverslips were washed twice with 100% ethanol , and then sonicated in 100% ethanol , followed by one more wash in 100% ethanol .\",\n \"They were stored in ethanol and dried for 10 min before use .\",\n \"Images were collected on a Nikon TI microscope with a Hamamatsu ImagEM ( EM-CCD ) camera ( effective pixel size 160 nm ) and Nikon 100x NA 1 . 45 TIRF objective .\",\n \"Z stacks were obtained at 0 . 2 μm slices .\",\n \"Total image depth was 3 μm .\",\n \"Only the top 3 slices of the cell were used in maximum intensity projections in Figure 3E .\",\n \"Cells were prepared as in ‘Imaging – MreB particle tracking’ above .\",\n \"Cells were placed under an agar pad in a MatTek ( Ashland , Massachusetts ) dish for imaging .\",\n \"Images were collected on an DeltaVision OMX SR Blaze system in SIM TIRF mode , using an Edge 5 . 5 sCMOS camera ( PCO ) and a 60x objective .\",\n \"75msec exposures from a 488 nm diode laser were used for each rotation .\",\n \"Spherical aberration was minimized using immersion oil matching .\",\n \"Raw images were reconstructed using Softworx ( Applied Precision ) .\",\n \"All image processing unless otherwise specified was performed in FIJI ( Schindelin et al . , 2012 ) .\",\n \"Images used for particle tracking were unaltered , except for trimming five pixels from the edges of some videos to remove edge artifacts detected by the tracking software .\",\n \"Phase contrast images and fluorescent images of protoplasts were adjusted for contrast .\",\n \"Phase contrast images presented in the manuscript collected from cells in the custom microfluidic device , which did not undergo quantitative processing , were gamma-adjusted ( γ = 1 . 5 ) to compensate for changes in brightness occurring at the device’s feature borders; such processing was not used for growth quantification .\",\n \"Images were background-subtracted for viewing purposes; unaltered images were used for quantitative processing in all cases .\",\n \"The custom microfluidic setup used to confine cells in Figure 3A–C , Figure 3—figure supplement 1A , and Figure 3—video 1 was previously described in Norman et al . ( 2013 ) .\",\n \"Briefly , a polydimethylsiloxane slab with surface features was bonded to a 22 × 60 mm glass coverslip by oxygen plasma treatment followed by heating to 65°C for >1 hr .\",\n \"The features in our setup differed from those described in Norman et al . ( 2013 ) , particularly in the omission of a second , wider layer in the cell chambers , which enhanced growth at timescales beyond that of our experiments .\",\n \"Syringes containing growth medium were connected to the microfluidic features using Tygon tubing stainless steel dispensing needles ( McMaster Carr Supply Company , Elmhurst , Illinois ) .\",\n \"Medium was supplied to cells at a constant rate of 2–5 μL/min using automatic syringe pumps .\",\n \"Imaging was carried out using phase contrast microscopy as described above .\",\n \"For the microfluidics experiments in Figures 5 and 6 and Figure 5—video 1 ( top ) , Figure 5—video 2 , and Figure 6—video 1 , the CellASIC platform from Merck Millipore ( Billerica , Massachusetts ) was used with B04A plates .\",\n \"The cell confinement experiment in Figure 3A–C was conducted by first loading cells into the chamber: BEG300 cells were grown to stationary phase ( OD6003 . 0–5 . 0 ) in LB supplemented with 20 mM magnesium chloride , passed through a 5 μm filter , and concentrated 100-fold before loading in the custom-made microfluidic device .\",\n \"Both phase contrast and fluorescent imaging were performed as described in the ‘Imaging’ section above .\",\n \"For observing MreB movement , MreB-GFP expression was induced with 50 μM IPTG upon loading into the microfluidic chamber , and cells were imaged every 2 s with a camera exposure time of 300 ms . The biological phenomena depicted in Figure 3A–C , Figure 3—figure supplement 1A , and Figure 3—video\",\n \"1 . are representative data drawn from three experiments for phase contrast data and two experiments for fluorescent data .\",\n \"Biological replicates , providing information on cell-to-cell clonal variation , for the purposes of this experiment should be considered individual channels , which are seeded by one or a few cells at the start of the experiment .\",\n \"Cells of strains bJS18 ( GFP-Mbl ) and bEG300 ( GFP-MreB ) were grown overnight at 25°C in the osmoprotective SMM media ( LB supplemented with 20 mM magnesium chloride , 17 mM maleic acid , 500 mM sucrose , brought to a pH of 7 . 0 ) with maximum xylose induction ( 30 mM ) ; cells were shifted to 37°C in the morning .\",\n \"For strain bEG300 , the SMM media was supplemented with 8 mM xylose ( for intermediate TagO induction ) .\",\n \"Following 2 hr of growth , 10 mg/mL of freshly suspended lysozyme was added to the cultures with OD600 >0 . 2 .\",\n \"After growing for 1–2 hr in lysozyme , the cells were spun and concentrated .\",\n \"6% agar pads made in LB-SMM were made using a polydimethylsiloxane ( PDMS ) mold with crosses ( 2 , 4 and 5 μm arms and 5 μm center ) .\",\n \"The cells were placed on the agar pad for 2 min , allowing the cells to settle in the crosses .\",\n \"The pad was then placed in a MatTek ( Ashland , Massachusetts ) dish for imaging .\",\n \"10–20 biological replicates were imaged per day and the experiment was replicated on 3 days .\",\n \"TagO depletions in Figure 2A were conducted using strain BEG300 in liquid culture .\",\n \"Cells were prepared as overnights , as described above , then grown at the specified xylose concentration at 37°C in LB with 20 mM magnesium chloride for 4 hr .\",\n \"The cells were then imaged as described above in the ‘Imaging – MreB particle tracking’ section .\",\n \"Pbp2A depletions shown in Figure 2 were conducted in liquid culture using strain BRB785 and BRB786 with an IPTG‐inducible Pbp2A fusion at the native locus with the redundant transpeptidase PbpH deleted .\",\n \"This strain was grown overnight in the presence of 2 mM IPTG , and then inoculated into CH media containing 2 mM IPTG , 0 . 015% xylose , and 20 mM magnesium chloride to stabilize the cells against lysis .\",\n \"At an OD600 of 0 . 6 , cells were spun down in a tabletop centrifuge and washed three times in CH media lacking IPTG .\",\n \"Cells were placed under agar pads containing 20 mM magnesium chloride , and spinning disk confocal images were taken every 5 s on a Nikon Ti microscope with a 100 × 1 . 49 TIRF objective and a Hamamatsu ImagEM C9100-13 EM-CCD camera ( effective pixel size of 160 nm ) .\",\n \"Depletions shown in Figure 5A were conducted using strain BEG300 .\",\n \"Cells were prepared as overnights in LB with 1 mM magnesium chloride and 12 mM xylose .\",\n \"In the morning , they were washed in LB with 12 mM xylose and no magnesium and placed under a 3% agar pad with the same medium .\",\n \"Phase contrast images were collected every 5 min using a Photometrics ( Tucson , Arizona ) CoolSNAP HQ2 CCD camera .\",\n \"The experiment was replicated on 2 days .\",\n \"Repletions of TagO or Pbp2a on pads , as shown in Figure 5B and Figure 5—video 1 ( bottom ) , were performed with strains BEG300 and BRB785 respectively .\",\n \"Cells were grown as overnights , as described above , then depleted at 37°C for >4 hr in LB with 20 mM magnesium chloride and collected by centrifugation at 6000 x g for 2 min .\",\n \"The cells were re-suspended in LB supplemented with 20 mM magnesium chloride and 1 mM IPTG ( BRB785 ) and 30 mM xylose ( BEG300 ) , placed under 5% agarose pads on coverslips with thickness No . 1 . 5 for imaging .\",\n \"Phase contrast images were collected every 5 min using a Photometrics CoolSNAP HQ2 CCD camera .\",\n \"For the repletions shown in Figures 5B–C and 6A , Figure 5—figure supplement 1 , and Figure 5—video 1 ( top ) and 2 , performed in the CellASIC microfluidic device in a B04A plate , BCW82 and BEG300 cells were grown to OD6001 . 2–1 . 5 in LB supplemented with 20 mM magnesium chloride , centrifuged to pellet large clumps for 3 min at <500 x g , and the supernatant loaded into the plate .\",\n \"Growth medium was supplied at 5–6 PSI .\",\n \"Cells were grown for at least an additional 30 min before the addition of inducer to the growth medium .\",\n \"Phase contrast images were collected every 10 min .\",\n \"Fluorescent images were collected on the imaging setup described in the ‘Imaging – MreB Particle Tracking’ section above: GFP-MreB was induced upon loading into the microfluidic chamber with 1 mM IPTG , and MreB dynamics were observed for 3 min after every 10 min , using 300 ms camera exposures taken every 2 s .\",\n \"The experiment was repeated six times , with the number of biological replicates per experiment limited by chance variation in the loading procedure , but on the order of one to four per experiment .\",\n \"For the repletions shown in Figure 6B and Figure 5—figure supplement 1D , the same procedure was used , but with imaging performed on the spinning disk confocal microscope described in ‘Imaging – Spinning Disk Confocal’ .\",\n \"Z-stacks were collected with a range of 3 μm around the focal plane and 0 . 2 μm steps .\",\n \"The MreB localization experiments were done using strain bEG300 with full induction of GFP-MreB ( 1 mM IPTG ) and recovering cells were imaged using the spinning disk microscope , collecting Z-stacks as described before .\",\n \"Where indicated , instead of visualizing MreB dynamics , fluorescent D-amino acids ( Kuru et al . , 2012 ) ( 7 μM ) were added to the growth medium in the CellASIC device: HADA during depletions of TagO ( 0 mM xylose ) and Cy3B-ADA during repletion of TagO ( 30 mM xylose ) .\",\n \"Cells were washed with LB supplemented with 20 mM magnesium chloride containing no D-amino acids for 1–2 min before imaging .\",\n \"To test if rod shape recovery occurs in the absence of cell division , three strains were tested ( BAB327 , BAB343 and BAB388 ) .\",\n \"Cells of BAB327 and BAB388 were grown in CH media with 25 mM magnesium chloride in the absence of xylose at 37°C until OD600 ~0 . 5 and diluted 10-fold in fresh media .\",\n \"After 2 hr of growth , IPTG was added to a final concentration of 1 mM ( MinCD and FtsA , respectively ) and cells were incubated for an extra 1 hr .\",\n \"Cells were imaged on a spinning disk confocal under pads with 1 mM IPTG and 60 mM xylose ( for TagO repletion ) .\",\n \"Phase-contrast and fluorescent images were acquired at 10 min intervals for a total of 8 hr .\",\n \"Cells of BAB343 were grown in LB supplemented with 20 mM , magnesium chloride in the absence of xylose at 25°C overnight .\",\n \"The next day , after 2 hr of growth in the same media at 37°C , IPTG was added to a final concentration of 1 mM ( MciZ ) and cells were incubated for an extra 1 hr .\",\n \"Cells were imaged on a spinning disk confocal under pads with 1 mM IPTG and 30 mM xylose ( for TagO repletion ) .\",\n \"Phase-contrast and fluorescent images were acquired at 10 min intervals for a total of 4 hr .\",\n \"For Figure 5—video 2 , cells of BCW51 were grown overnight at 25°C in LB supplemented with 8 mM xylose , 20 mM magnesium chloride , 1 μg/ml erythromycin and 25 μg/ml lincomycin ( MLS ) .\",\n \"Cells were shifted to 37°C for 2 hr and loaded into the CellASIC B04A plate at OD600 ~ 0 . 6 .\",\n \"At the start of imaging , magnesium was depleted by flowing in LB supplemented only with 8 mM xylose and MLS at 3 psi .\",\n \"Images were collected every 20 min over a 4 hr period .\",\n \"Magnesium was resupplied to the cells by changing to LB supplemented with 8 mM xylose , 20 mM magnesium chloride , and MLS .\",\n \"Imaging was continued every 20 min for an additional 4 hr . 5–10 cells were imaged and the experiment was done once .\",\n \"Cells were grown overnight at 25°C in LB supplemented with 30 mM xylose , 20 mM magnesium chloride , 1 μg/mL erythromycin and 25 μg/mL lincomycin .\",\n \"In the morning they were collected at OD600 ~ 0 . 2 , spun in a tabletop centrifuge at 9000 rpm for 3 min and washed in LB supplemented with various xylose ( 0–30 mM ) and magnesium chloride ( 0–20 mM ) levels .\",\n \"25-fold serial dilutions into LB supplemented with the same xylose and magnesium chloride concentrations were made and allowed to grow at 37°C for 4 hr .\",\n \"Cells at OD600 ~ 0 . 2 were concentrated by spinning in a tabletop centrifuge at 9000 rpm for 3 min .\",\n \"They were placed on a coverslip thickness No . 1 . 5 under 3% agarose pads made in LB supplemented with the same concentrations of xylose and magnesium chloride .\",\n \"Images were collected using the imaging setup described in the ‘Imaging – phase contrast microscopy’ section above , as well as with a Photometrics CoolSNAP HQ2 CCD camera .\",\n \"The magnification and pixel size were the same in both setups .\",\n \"The experiment was replicated on two days and the data pooled together .\",\n \"The MATLAB based software uTrack was used for particle tracking ( Jaqaman et al . , 2008 ) .\",\n \"We used the comet detection algorithm to detect filaments ( difference of Gaussian: one pixel low-pass to 4–6 pixels high pass , watershed segmentation parameters: minimum threshold 3–5 standard deviations with a step size of 1 pixel ) which , at our MreB induction levels gave better localization of the resultant asymmetric particles over algorithms that search for symmetric Gaussians .\",\n \"Visual inspection of detected particles confirmed that most of the particles and none of the noise were being detected .\",\n \"A minimum Brownian search radius of 0 . 1–0 . 2 pixels and a maximum of 1–2 pixels was applied to link particles with at least five successive frames .\",\n \"Directed motion propagation was applied , with no joins between gaps allowed .\",\n \"Tracks were visualized using the FIJI plug-in TrackMate ( Tinevez et al . , 2017 ) .\",\n \"For sphere to rod transitions and cells confined in microfluidic channels , movies were processed by subtracting every 8th frame from each frame to remove stationary spots using the FIJI plugin StackDifference before tracking .\",\n \"The tracking was done as described earlier in this section .\",\n \"Strains containing fluorescent fusions to TagT , TagU , and TagV were grown as described in the ‘Overnight culture growth’ section but in CH medium instead of LB .\",\n \"Cells were grown for 3 hr at 37°C before imaging , then collected by centrifugation at 6000 x g for 2 min and re-suspended in CH .\",\n \"Cells were then placed on a glass coverslip thickness No . 1 . 5 under an agar pad thickness 1 mm made from CH and 1 . 5% agarose .\",\n \"Timelapse images were collected with TIRF illumination , using continuous 100 ms 488 nm exposures .\",\n \"Epifluorescent illuminated images were collected from a single exposure , while maximal intensity projections were formed from a series of continuous 100 ms TIRF exposures .\",\n \"Technical replicates were not collected , but each strain was imaged under a variety of appropriate imaging conditions to establish that the phenomena observed were not an artifact of the experimental setup .\",\n \"For single-molecule experiments imaging HaloTag-15aa-TagU ( bAB196 ) , HaloTag-15aa-TagV ( bAB197 ) , HaloTag-15aa-TagT ( bAB198 ) , Pbp2A-30aa-HaloTag ( BSY201 ) and MreB-30aa-HaloTag ( BYS40 ) , cells were grown at 37°C in CH medium from fresh colonies until reaching mid-exponential phase ( OD600 = 0 . 5 ) , ten-fold diluted back in fresh CH medium and grown one more round until OD600 = 0 . 5 .\",\n \"Cultures were then incubated for 15 min with 25 nM ( HaloTag-15aa-TagU/V/T and Pbp2A-30aa-HaloTag ) and 25 pM ( MreB-30aa-HaloTag ) of HaloTag-JF549 ligand .\",\n \"Cells were spun for 1 min at 4000 xg , 10-fold concentrated and imaged under 2% agarose pads .\",\n \"Images were collected on a Nikon TI microscope equipped with an EMCCD camera , together with a Nikon 100x NA 1 . 45 objective .\",\n \"Exposure times were 0 . 25 s , and illumination was accomplished using a 561 nm laser .\",\n \"BEG300 cells were grown from overnights as described in ‘Overnight culture growth’ but in CH medium instead of LB .\",\n \"Cells were then grown at 37°C for 4 hr without xylose to deplete WTAs , then induced with 30 mM xylose for 1 . 5 hr to re-induce WTA expression .\",\n \"Cells were then moved to 25°C for at least 30 min and incubated with 25 μg/mL Concanavalin A conjugated to Alexa Fluor 647 .\",\n \"Cells were collected by centrifugation at 6000 x g for 2 min , washed with CH medium , then re-suspended in fresh CH medium .\",\n \"Cells were then placed on a glass coverslip thickness No . 1 . 5 under an agar pad thickness 1 mm made from CH medium and 1 . 5% agarose .\",\n \"For PY79 and BCW61 controls , lectin-Alexa Fluor conjugate concentration was 200 μg/mL .\",\n \"Separate technical replicates were collected with the microscopes described in ‘Imaging – MreB Particle Tracking’ and ‘Imaging – Spinning Disk Confocal’ .\",\n \"For quantitative analysis , the latter setup was used .\",\n \"Quantification was performed in FIJI .\",\n \"Five individual cells were selected arbitrarily and pooled for quantitative analysis .\",\n \"The contours of each cell were manually traced , and intensity along these contours measured , then corrected for the mean fluorescent background .\",\n \"The per cell average was calculated from the mean of each pixel in the contour less background ( calculated from an empty field ) .\",\n \"The per strain average was taken as the mean of the per cell average .\",\n \"The output of uTrack is the position coordinates of tracks over frames .\",\n \"We fit a line through these coordinates using orthogonal least squares regression to minimize the perpendicular distance of the points from the line of best fit .\",\n \"We used principal component analysis for orthogonal regression using custom written MATLAB code .\",\n \"The R2 values we obtain range from 0 . 5 to\",\n \"1 . We calculated mean track positions , angles and displacement using the line of best fit for all tracks .\",\n \"We also calculated the mean square displacement versus time of individual tracks and fit these curves to the quadratic equation MSD ( t ) = 4Dt+ ( Vt ) 2 , using nonlinear least squares fitting .\",\n \"As later times have fewer points and are noisier , we fit the first 80% of the data for each track .\",\n \"We determined α by fitting a straight line to the logMSDt vs . log ( t ) curve .\",\n \"The goodness of fit was evaluated by determining the R2 value .\",\n \"We selected tracks for linearity and directional motion , based on the following cutoffs: R2 >0 . 9 , displacement >0 . 2 μm , , velocity >1e−9 μm/s , and R2 of the linear fit of log ( MSD ( t ) ) vs . log ( t ) >0 . 6 .\",\n \"The MATLAB-based software Morphometrics ( Ursell et al . , 2017 ) was used to segment phase contrast images of cells .\",\n \"We used the phase contrast setting for rod-shaped and intermediate states and the peripheral fluorescence setting for spherical states , because in this latter condition , peripheral fluorescence empirically did a better job of fitting cell outlines .\",\n \"The cell contours obtained were visually inspected and any erroneous contours were removed by custom written MATLAB code .\",\n \"Track angles were calculated with respect to the cell midline as defined by the Morphometrics ‘Calculate Pill Mesh’ feature , which identifies the midline based on a unique discretization of the cell shape determined from its Voronoi diagram .\",\n \"The difference between the track angle and midline angle was then calculated .\",\n \"Since the track angles θt and midline angles θm both ranged from −90° to 90° , the range of angle differences ∆θ= θt-θm was −180° to 180° .\",\n \"We changed the range to 0 to 180° by the transformation: Δθ=180+ Δθ if Δθ<0 , and 0 to 90° by the transformation: Δθ=180− Δθ if Δθ>90 .\",\n \"The mean deviation from 90° ( σ90 ) for each distribution was calculated using the following formula , where xi is each angle in the distribution and N is the total number of angles:σ90= ∑i=1N ( xi-90 ) 2N Custom written MATLAB code was used to calculate the normalized dot product ( DP ) of track pairs along with the distance ( d ) between their mean positions x- and y- as follows: DPij= cos ( θi −θj ) , dij= ( x−i−x−j ) 2+ ( y−i−yj− ) 2 To eliminate out-of-cell tracks we only considered those that had three other tracks within a 5 μm radius of their mean position .\",\n \"The dot product of track pairs ( DP ) and distance ( d ) between them was stored in data files , along with all the previous information for each individual track ( R2 , velocities , angles , mean positions , displacement etc ) .\",\n \"The files were then parsed using the cutoffs described in the ‘Data analysis – selecting directional tracks’ section .\",\n \"The tracks were binned based on the distance and the mean dot product calculated for each distance range as follows: DP-= 1N∑i>jNcos ( θi -θj ) A cutoff of 3 μm was chosen as the maximum binning distance , which is the average length of a cell .\",\n \"A data file containing simulated tracks was created by a custom written MATLAB script , which generates random angles distributed randomly on a 100 × 100 μm area .\",\n \"Each track has R2 = 0 . 95 , velocity = 25 nm/s and displacement = 1 μm .\",\n \"The same analysis code was run on these simulated tracks to generate track pairs with dot product and distance stored in a new data file .\",\n \"The data file was parsed using the same cutoffs as the real data and the mean dot product for each distance range calculated .\",\n \"The total numbers of trajectories within the simulation were much higher than the actual data ( 2–10 times higher ) .\",\n \"Pill meshes were created using Morphometrics ( Ursell et al . , 2017 ) , which calculates the coordinates of line segments perpendicular to the cell long axis .\",\n \"For cell widths at various steady state TagO and Mg2+ levels , the distance of these line segments was calculated using a custom written MATLAB script ( Hussain , 2017; copy archived at https://github . com/elifesciences-publications/hussain-2017-elife ) and the maximum width along the length of the cell was taken as the cell width .\",\n \"When measuring cell width nearest to a track ( for calculating track angle as a function of cell width ) , the mean width of the 10 nearest contour points from the track was calculated using a custom written MATLAB script .\",\n \"Cell widths of emerging bulges and rods from round cells were measured manually in FIJI .\",\n \"Our ability to segment individual spherical cells was limited by their nonuniform contrast , perhaps arising from the nonuniform thickness of these cells in the Z dimension; consequently , Morphometrics-based width measurements in these cells was limited , especially in cells exceeding 2 μm in diameter .\",\n \"Sidewall curvature of cells was extracted from the pill mesh obtained from Morphometrics .\",\n \"The curvature values are calculated from three successive contour points and smoothed over two pixels .\",\n \"The mean curvature of 3 nearest points to each track were calculated from both sides of the cell contour and called the mean curvature .\",\n \"Principal curvature ratio was calculated by dividing the sidewall curvature with the curvature in the radial direction ( calculated from cell width assuming the cell is radially symmetric ) .\",\n \"For radial curvature we used the following expression , where rcell is half the cell width:κ2=1rcell A value close to one indicates the two principal curvatures are similar and the cells are round .\",\n \"Phase contrast images were used to show rod shape emergence from local bulges .\",\n \"Edges were enhanced in FIJI and contrast adjusted to give bright cell outlines in the images .\",\n \"The stack was then colored in time using temporal color code function in FIJI .\",\n \"To create the curvature plot , the phase contrast images were run through Morphometrics which calculates the curvature at each contour point along the cell outline .\",\n \"The contour points of interest were selected and plotted using a custom written MATLAB script , which colored each point according to its local curvature as calculated by Morphometrics .\",\n \"To provide a good resolution for positive curvatures , we rescaled the color map such that negative curvatures were colored blue and positive curvatures were scaled by their curvature value .\",\n \"Data from agar pads experiments was analyzed using custom written MATLAB code .\",\n \"Data from cellASIC experiments was analyzed in Morphometrics to get areas for each cell .\",\n \"For doubling times during sphere to rod transitions , the data was collected by manually measuring the areas of the sphere and rod regions of the same cell in FIJI .\",\n \"In all cases , the area of each cell per frame was calculated and the log plot of area vs time was fit to a line .\",\n \"The doubling time was calculated using the slope of this line .\",\n \"N = 15 cells ( transitioning from spheres to rods ) .\",\n \"Cell contours ( Figure 1—figure supplement 1 ) were used to calculate tangent angles using the equation: θi=tan-1yi+1-yixi+1-xi .\",\n \"The correlation between angles was calculated using the cosine of the angle difference binned as a function of number of points ( n ) between the angles: Gn= 1N∑i=1Ncos ( θi+n-θi ) .\",\n \"The number of points was converted to contour length using the pixel size of the camera to get the final correlation function: Gl= 1N∑i=1Ncos ( θi+l-θi ) .\",\n \"Tracks were generated using the TrackMate plugin in Fiji ( 2017 ) .\",\n \"Particles were detected with the Laplacian of Gaussians ( LoG ) detector , with a 0 . 4 μm spot diameter .\",\n \"Tracks were generated using the Simple LAP Tracker , with a 0 . 1 μm linking max distance and no frame gaps allowed .\",\n \"Tracks were exported into MATLAB for further processing .\",\n \"Mean squared displacement ( MSD ) was calculated for all tracks as a function of time delay ( t ) .\",\n \"For visualization of HaloTag tracks overlaid on phase images , we analyzed all tracks 10 frames or longer .\",\n \"For each track , the scaling exponent ( α ) was calculated by fitting MSD ( t ) =C ( t∧ α ) +4 ( σ∧2 ) using nonlinear least-squares fitting with constant C and where σ is the localization error ( Monnier et al . , 2012 ) .\",\n \"For analysis of alpha value and velocity frequencies tracks were first filtered using masks generated from phase images acquired after single-molecule imaging .\",\n \"We then included all tracks between 10 and 120 frames in length .\",\n \"α values were calculated by linear fitting log ( MSD ) versus log ( t ) .\",\n \"Velocity ( v ) was calculated by fitting MSD ( t ) =4D ( t ) + ( v*t ) ∧2 + 4 ( σ∧2 ) using nonlinear least-squares fitting , where D is the diffusion constant ( Monnier et al . , 2012 ) .\",\n \"For the graphs in Figure 6—figure supplement 2 , only plotted are molecules that moved in a consistent manner during their lifetime [>0 . 95 r2 fit to log ( MSD ) versus log ( t ) ] .\",\n \"Analysis of alpha value and velocity frequencies for MreB filaments was the same as for HaloTag , except we included all tracks between 10 and 60 frames in length and filtration by phase image masks was not necessary .\",\n \"Full length , un-tagged Thermotoga maritima MreB was purified as described previously ( Salje et al . , 2011 ) .\",\n \"The protein was encapsulated inside unilamellar liposomes following a previously published protocol ( Szwedziak et al . , 2014 ) .\",\n \"For this , 50 μL of E . coli total lipid extract , dissolved in chloroform at 10 mg/mL , was dried in a glass vial under a stream of nitrogen gas and left overnight under vacuum to remove traces of the solvent .\",\n \"The resulting thin lipid film was hydrated with 50 μL of TEN100 8 . 0 ( 50 mM Tris/HCl , 100 mM NaCl , 1 mM EDTA , 1 mM NaN3 , pH 8 . 0 ) , supplemented with 20 mM CHAPS ( Anatrace , Maumee , Ohio ) , and shaken vigorously at 800 rpm using a benchtop micro centrifuge tube shaker for 2 hr .\",\n \"The lipid-detergent solution was then sonicated for 1 min in a water bath sonicator .\",\n \"Subsequently , 50 μL of MreB protein solution at 30 μM , supplemented with 0 . 5 mM magnesium ATP ( Jena Bioscience , Germany ) was added and left for 30 min at room temperature .\",\n \"Next , the mixture was gradually diluted within 10–20 min to 600 μL with TEN100 8 . 0 plus 0 . 5 mM magnesium ATP ( without detergent ) to trigger spontaneous liposome formation .\",\n \"2 . 5 μL of the solution was mixed with 0 . 2 μL 10 nm IgG immunogold conjugate ( TAAB , UK ) and plunge-frozen onto Quantifoil R2/2 carbon grid , using a Vitrobot automated freeze plunger ( FEI Company , Hillsboro , Oregon ) into liquid ethane .\",\n \"2D electron cryomicroscopy images were taken on an FEI Polara TEM ( FEI Company ) operating at 300 kV with a 4k × 4 k Falcon II direct electron detector ( FEI Company ) at a pixel size of 1 . 8 Å .\",\n \"For electron cryotomography , samples were imaged using an FEI Titan Krios TEM ( FEI Company ) operating at 300 kV , equipped with a Gatan imaging filter set at zero-loss peak with a slit-width of 20 eV .\",\n \"A 4k × 4 k post-GIF K2 Summit direct electron detector ( Gatan , a subsidiary of Roper Technologies , Lakewood Ranch , Florida ) was used for data acquisition with SerialEM software ( 2005 ) at a pixel size of 3 . 8 Å at the specimen level .\",\n \"Specimens were tilted from −60˚ to +60˚ with uniform 1˚ increments .\",\n \"The defocus was set to between 8 and 10 μm , and the total dose for each tilt series was around 120–150 e/Å2 .\",\n \"Tomographic reconstructions from tilt series were calculated using RAPTOR ( 2008 ) and the IMOD tomography reconstruction package followed by SIRT reconstruction with the TOMO3D package ( 1996; 2011a ) .\",\n \"Movies showing liposomes were prepared with Chimera and PyMOL ( 2002; 2004 ) .\",\n \"Imaging of E . coli strain RM478 was conducted as in ‘Overnight culture growth’ , save the cells were grown in LB or M63-Glucose .\",\n \"When indicated , cells were grown and imaged under 2% agar pads made in the same media .\",\n \"For the media down shift ( LB at 37°C to M63-Glucose at 25°C ) , cells were first grown in LB at 37°C in a drum roller , then placed under agar pads made of M63-Glucose at 25°C and allowed to equilibrate 15 min prior to imaging .\",\n \"Imaging was conducted as in ‘Imaging – MreB particle tracking’ using a Nikon TI-E with TIRF illumination .\",\n \"All strains used in this study are available in Supplementary file\",\n \"1 . All primers used in this study are available in Supplementary file\",\n \"2 . BCW51 [ycgO::Pxyl-tagO , tagO::erm , amyE::sfGFP-mreB , sinR::phleo] was generated by transforming BEG300 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers Sinr_up_F and Sinr_up_R and template PY79 genomic DNA; ( 2 ) PCR with primers oJM028 and oJM029 and template plasmid pWX478a ( containing phleo ) ; ( 3 ) PCR with primers Sinr_DOWN_R and Sinr_DOWN_F and template genomic DNA .\",\n \"BCW61 [tagE::erm] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW054 and oCW055 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM028 and oCW057 and template plasmid pWX467a containing cat; ( 3 ) PCR with primers oCW058 and oCW059 and template PY79 genomic DNA .\",\n \"BCW72 [yvhJ::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW139 and oCW141 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW142 and oCW143 and template PY79 genomic DNA .\",\n \"BCW77 [ywtF::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW159 and oCW161 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW164 and oCW165 and template PY79 genomic DNA .\",\n \"BCW78 [ywtF::msfGFP-ywtF] was generated by transforming BCW77 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW160 and oCW161 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW163 and oCW165 and template PY79 genomic DNA .\",\n \"BCW79 [yvhJ::msfGFP-yvhJ] was generated by transforming BCW72 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW139 and oCW146 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW143 and oCW145 and template PY79 genomic DNA .\",\n \"BCW80 [lytR::PxylA-mazF ( cat ) ] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW101 and oCW109 and template PY79 genomic DNA; ( 2 ) PCR with primers oJM029 and oMK047 and template DNA consisting of a fusion of cat and the mazF counterselectable marker from pGDREF ( Yu et al . , 2010 ) ; ( 3 ) PCR with primers oCW100 and oCW125 and template PY79 genomic DNA .\",\n \"BCW81 [lytR::msfGFP-lytR] was generated by transforming BCW72 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oCW101 and oCW137 and template PY79 genomic DNA; ( 2 ) PCR with primers oCW072 and oCW073 and BMD61 genomic DNA; ( 3 ) PCR with primers oCW100 and oCW138 and template PY79 genomic DNA .\",\n \"BCW82 [tagO::erm , ycgO::PxylA-tagO , amyE::Pspac-gfp-mreB ( spec ) , dacA::kan] was generated by transforming BEG300 with genomic DNA from BGL19 .\",\n \"BEG202 [tagO::erm amyE::Pxyl-gfp-mbl ( spec ) ] was generated by transforming BEB1451 with genomic DNA from BJS18 .\",\n \"BEG281 [ycgO::PxylA-tagO] was generated by transforming with a plasmid created via ligating a Gibson assembly into pKM077 .\",\n \"pKM77 was digested with EcoRI and XhoI .\",\n \"The assembly was created with two fragments: ( 1 ) PCR with primers oEG85 and oEG86 and template py79 genomic DNA; ( 2 ) PCR with primers oEG87 and oEG88 .\",\n \"BEG291 [tagO::erm , ycgO::PxylA-tagO] was generated by transforming BEG281 with genomic DNA from BRB4282 .\",\n \"BEG300 [tagO::erm , ycgO::PxylA-tagO , amyE::Pspac-gfp-mreB ( spec ) ] was generated by transforming BEG291 with genomic DNA from BEG275 .\",\n \"BMD61 [mbl::mbl-msfGFP ( spec ) ] was generated by transforming py79 with a Gibson assembly consisting of four fragments: ( 1 ) PCR with primers oMD44 and oMD90 and template PY79 genomic DNA; ( 2 ) PCR with primers oMD47 and oMD56 and template synthetic , codon-optimized msfGFP; ( 3 ) PCR with primers oJM028 and oJM029 and template plasmid pWX466a ( containing spec ) ; ( 4 ) PCR with primers oMD48 and oMD50 and template genomic DNA .\",\n \"bSW99 [amyE::spc-Pspac-mciZ] was generated by transforming PY79 with a Gibson assembly consisting of five fragments: ( 1 ) PCR with primers oMD191 and oMD108 and template PY79 genomic DNA ( containing upstream region of amyE ) ; ( 2 ) PCR with primers oJM29 and oJM28 and template plasmid pWX466a ( containing spec ) ; ( 3 ) PCR with primers oMD234 and oSW76 and template plasmid pBOSE1400 ( a gift from Dr . Briana Burton , containing spec ) ; ( 4 ) PCR with primers oAB307 and oAB291 and template PY79 genomic DNA ( containing mciZ ) ; ( 5 ) PCR with primers oMD196 and oMD197 and template PY79 genomic DNA ( containing downstream region of amyE ) .\",\n \"bAB343 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Pspac-mciZ , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB185 ( Bisson-Filho et al . , 2017 ) with genomic DNA from bSW99 .\",\n \"The resultant strain was then transformed with the genomic DNA from BEG291 and selected for Cm resistance .\",\n \"Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 30 mM of xylose and 25 mM MgCl2 .\",\n \"bAB327 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Physpank-minCD , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB185 ( Bisson-Filho et al . , 2017 ) with genomic DNA from JB60 ( a gift from Dr . Frederico Gueiros-Filho ) .\",\n \"The resultant strain was then transformed with the genomic DNA from BEG291 and selected for Cm resistance .\",\n \"Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 60 mM xylose and 25 mM MgCl2 .\",\n \"bAB388 [tagO::erm , ycgO::cat-PxylA-tagO , amyE::spc-Physpank-ftsA , ftsAZ::ftsA-mNeonGreen-ftsZ] was generated by transforming bAB199 ( Bisson-Filho et al . , 2017 ) with genomic DNA from BEG291 and selected for Cm resistance .\",\n \"Subsequently , the resultant strain was transformed again with genomic DNA from BEG291 , but colonies were selected for MLS resistance in the presence of 60 mM xylose and 25 mM MgCl2 .\",\n \"bAB196 [tagU::erm-Pxyl-HaloTag-15aa-tagU] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagU with primers oCW155 and oCW109 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagU with primers oCW138 and oCW156 and PY79 template genomic DNA .\",\n \"bAB197 [tagV::erm-Pxyl-HaloTag-15aa-tagV] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagV with primers oCW140 and oCW141 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagV with primers oCW144 and oCW145 and PY79 template genomic DNA .\",\n \"bAB198 [tagT::erm-Pxyl-HaloTag-15aa-tagT] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR amplifying the upstream region from tagT with primers oCW162 and oCW159 and template PY79 genomic DNA; ( 2 ) PCR amplifying the erm-Pxyl-HaloTag-15aa fragment with primers oJM029 and oCW73 and template bGS62 genomic; ( 3 ) PCR amplifying the downstream region from tagT with primers oCW163 and oCW166 and PY79 template genomic DNA .\",\n \"bYS09 [mreB::mreB-mNeonGreen] was generated by transforming BMD88 ( Schirner et al . , 2015 ) with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD134 and oMD262 and bMD135 template genomic DNA ( containing the upstream of mreB and 40aa linker ) ; ( 2 ) PCR with primers oYS007 and oYS008 and gBlocks gene fragment containing mNeonGreen; ( 3 ) PCR with primers oMD92 and oMD116 and template PY79 genomic DNA .\",\n \"bYS40 [mreB::mreB-HaloTag] was generated by transforming BMD88 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD134 and oYS602 and bMD135 template genomic DNA; ( 2 ) PCR with primers oYS603 and oYS604 and template plasmid cdr1086 ( containing HALO ) ; ( 3 ) PCR with primers oMD92 and oMD116 and template PY79 genomic DNA .\",\n \"bYS201 [HaloTag-Pbp2A::cat] was generated by transforming PY79 with a Gibson assembly consisting of three fragments: ( 1 ) PCR with primers oMD083 and oYS136 and bMD98 sfGFP-Pbp2A::cat template genomic DNA ( 2 ) PCR with primers oYS599 and oYS598 and template plasmid cdr1086 ( containing HALO ) ; ( 3 ) PCR with primers oMD069 and oMD082 and template PY79 genomic DNA .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["MreB is essential for rod shape in many bacteria .","Membrane-associated MreB filaments move around the rod circumference , helping to insert cell wall in the radial direction to reinforce rod shape .","To understand how oriented MreB motion arises , we altered the shape of Bacillus subtilis .","MreB motion is isotropic in round cells , and orientation is restored when rod shape is externally imposed .","Stationary filaments orient within protoplasts , and purified MreB tubulates liposomes in vitro , orienting within tubes .","Together , this demonstrates MreB orients along the greatest principal membrane curvature , a conclusion supported with biophysical modeling .","We observed that spherical cells regenerate into rods in a local , self-reinforcing manner: rapidly propagating rods emerge from small bulges , exhibiting oriented MreB motion .","We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting , self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape ."],"string":"[\n \"MreB is essential for rod shape in many bacteria .\",\n \"Membrane-associated MreB filaments move around the rod circumference , helping to insert cell wall in the radial direction to reinforce rod shape .\",\n \"To understand how oriented MreB motion arises , we altered the shape of Bacillus subtilis .\",\n \"MreB motion is isotropic in round cells , and orientation is restored when rod shape is externally imposed .\",\n \"Stationary filaments orient within protoplasts , and purified MreB tubulates liposomes in vitro , orienting within tubes .\",\n \"Together , this demonstrates MreB orients along the greatest principal membrane curvature , a conclusion supported with biophysical modeling .\",\n \"We observed that spherical cells regenerate into rods in a local , self-reinforcing manner: rapidly propagating rods emerge from small bulges , exhibiting oriented MreB motion .\",\n \"We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting , self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Many bacteria are surrounded by both a cell membrane and a cell wall – a rigid outer covering made of sugars and short protein chains .","The cell wall often determines which of a variety of shapes – such as rods or spheres – the bacteria grow into .","One protein required to form the rod shape is called MreB .","This protein forms filaments that bind to the bacteria’s cell membrane and associate with the enzymes that build the cell wall .","Together , these filament-enzyme complexes rotate around the cell to build and reinforce the cell wall in a hoop-like manner .","But how do the MreB filaments know how to move around the circumference of the rod , instead of moving in any other direction ?","Using a technique called total internal reflection microscopy to study how MreB filaments move across bacteria cells , Hussain , Wivagg et al . show that the filaments sense the shape of a bacterium by orienting along the direction of greatest curvature .","As a result , the filaments in rod-shaped cells orient and move around the rod , while in spherical bacteria they move in all directions .","However , spherical bacteria can regenerate into rods from small surface ‘bulges’ .","The MreB filaments in the bulges move in an oriented way , helping them to generate the rod shape .","Hussain , Wivagg et al . also found that forcing cells that lack a cell wall into a rod shape caused the MreB filaments bound to the cell membrane to orient and circle around the rod .","This shows that the organization of the filaments is sufficient to shape the cell wall .","In the future , determining what factors control the activity of the MreB filaments and the enzymes they associate with might reveal new targets for antibiotics that disrupt the cell wall and so kill the bacteria .","This will require higher resolution microscopes to be used to examine the cell wall in more detail .","The activity of all the proteins involved in building cell walls will also need to be extensively characterized ."],"string":"[\n \"Many bacteria are surrounded by both a cell membrane and a cell wall – a rigid outer covering made of sugars and short protein chains .\",\n \"The cell wall often determines which of a variety of shapes – such as rods or spheres – the bacteria grow into .\",\n \"One protein required to form the rod shape is called MreB .\",\n \"This protein forms filaments that bind to the bacteria’s cell membrane and associate with the enzymes that build the cell wall .\",\n \"Together , these filament-enzyme complexes rotate around the cell to build and reinforce the cell wall in a hoop-like manner .\",\n \"But how do the MreB filaments know how to move around the circumference of the rod , instead of moving in any other direction ?\",\n \"Using a technique called total internal reflection microscopy to study how MreB filaments move across bacteria cells , Hussain , Wivagg et al . show that the filaments sense the shape of a bacterium by orienting along the direction of greatest curvature .\",\n \"As a result , the filaments in rod-shaped cells orient and move around the rod , while in spherical bacteria they move in all directions .\",\n \"However , spherical bacteria can regenerate into rods from small surface ‘bulges’ .\",\n \"The MreB filaments in the bulges move in an oriented way , helping them to generate the rod shape .\",\n \"Hussain , Wivagg et al . also found that forcing cells that lack a cell wall into a rod shape caused the MreB filaments bound to the cell membrane to orient and circle around the rod .\",\n \"This shows that the organization of the filaments is sufficient to shape the cell wall .\",\n \"In the future , determining what factors control the activity of the MreB filaments and the enzymes they associate with might reveal new targets for antibiotics that disrupt the cell wall and so kill the bacteria .\",\n \"This will require higher resolution microscopes to be used to examine the cell wall in more detail .\",\n \"The activity of all the proteins involved in building cell walls will also need to be extensively characterized .\"\n]","isConversational":false},"year":{"kind":"string","value":"2018"}}},{"rowIdx":291,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["short report","medicine"],"string":"[\n \"short report\",\n \"medicine\"\n]","isConversational":false},"title":{"kind":"string","value":"Performance of a deep learning based neural network in the selection of human blastocysts for implantation"},"id":{"kind":"string","value":"elife-55301-v2"},"sections":{"kind":"list like","value":[["Assisted reproductive technologies ( ART ) such as in vitro fertilization ( IVF ) , while a solution to many infertile couples have been inefficient with an average success rate of approximately 30% reported in 2015 in the US ( CDC , 2015 ) .","IVF is also an expensive solution costing patients well over $10 , 000 out-of-pocket for each ART cycle in the US with many patients requiring multiple cycles to achieve successful pregnancy ( CDC , 2015; Birenbaum-Carmeli , 2004; Toner , 2002 ) .","Although multiple factors such as maternal age , medical diagnosis , gamete and embryo quality , and endometrium receptivity determine the success of ART cycles , the challenge of non-invasive selection of the highest available quality from a patient’s cohort of embryos ( top-quality embryo ) for transfer remains as one of the most important factors in achieving successful ART outcomes ( Vaegter et al . , 2017; Barash et al . , 2017; Conaghan et al . , 2013; Wong et al . , 2013; Racowsky et al . , 2015; Filho et al . , 2010; Machtinger and Racowsky , 2013; Demko et al . , 2016; Einarsson et al . , 2017; Hill et al . , 1989; Erenus et al . , 1991; Paulson et al . , 1990; Osman et al . , 2015 ) .","Traditional methods of embryo selection rely on visual embryo morphological assessment and are highly practice-dependent and subjective ( Storr et al . , 2017; Baxter Bendus et al . , 2006; Paternot et al . , 2009 ) .","Fully automated assessments of embryos are challenging owing to the complexity of embryo morphologies .","Emulating the skill of highly trained embryologists in efficient embryo assessment in a fully automated system is a major challenge in all of the previous work done in computer-aided assessments of embryos due to focus on measuring specific expert-defined parameters such as zona pellucida thickness variation , number of blastomeres , degree of cell symmetry and cytoplasmic fragmentation , etc . ( Rocha et al . , 2017a; Rocha et al . , 2017b ) .","Machine learning is loosely defined as a computer program that learns a given task over time through experience and improves itself to achieve the best possible task performance .","In the past decade , advances in hardware compute performance and machine learning techniques have significantly improved their applicability in real-world medical and non-medical problems .","Recently , machine learning has been proposed as a solution for automated analysis of embryo morphologies ( Rocha et al . , 2017b; Bormann et al . , 2020; Dimitriadis et al . , 2019; Thirumalaraju et al . , 2019; Khosravi et al . , 2019; Kanakasabapathy et al . , 2019a ) .","This work makes use of a deep convolutional neural network ( CNN ) , a representation learning technique , that has been proven to be effective in image classification tasks .","Unlike most prior computer-aided algorithms , including some techniques of machine learning used for embryo assessment , the reported CNN architecture allows automated embryo feature selection and analysis at the pixel level without any interference by an embryologist ( Rocha et al . , 2017a; Rocha et al . , 2017b ) .","Such networks do not depend on human-specified features and can develop an ability to evaluate embryos categorically through iterative learning from thousands of examples .","The use of deep-learning in IVF has also been explored; however , these recent neural network-based approaches have focused on either classifying embryos based on morphological quality and were not evaluated for transfer outcomes , or were developed with the use of time-lapse series of images toward the evaluation of implantation ( Khosravi et al . , 2019; Tran et al . , 2019 ) .","It is important to emphasize here that most fertility centers do not possess time-lapse imaging hardware even in the United States of America ( Dolinko et al . , 2017 ) .","The lack of availability of such hardware limits an otherwise promising technology mostly to resource-rich settings and fail to improve quality of and access to care in resource-constrained settings where such advances are sorely needed ( Wahl et al . , 2018; Hosny and Aerts , 2019 ) .","Furthermore , in current clinical practice , embryos with the highest morphological grades ( top-quality ) are the first to be transferred , however , clinically these decisions are performed manually , even with time-lapse imaging systems .","The development of networks that can measure an embryo’s potential for implantation and help in rank ordering embryos in a patient embryo cohort for transfer have utility in virtually all fertility centers .","Conventionally , embryo transfers are performed at the cleavage or the blastocyst stage of development .","Embryos are at the cleavage stage 2–3 days after fertilization and develop further in suitable culture conditions to reach the blastocyst stage 5–7 days after fertilization .","Blastocyst embryo transfers , in particular , have been associated with better implantation rates and have helped lower the number of embryos transferred at a time ( De Croo et al . , 2019 ) .","Therefore , in this study , we have investigated the use of a CNN pre-trained with 1 . 4 million ImageNet images and transfer-learned using 2440 static human embryo images recorded at a single time-point of 113 hr post insemination ( hpi ) for the development of neural networks that can help identify embryos capable of implantation and for identifying the top quality embryos ( Figure 1 ) .","The top-quality embryos were identified by combining a previously developed network ( Xception architecture ) trained to classify embryos based on its blastocyst quality with a genetic algorithm scheme ( Figure 1; Thirumalaraju et al . , 2020 ) .","The original neural network was trained on a hierarchical system of categorization , derived from a clinical Gardener-based grading system , to minimize data sparsity and improve overall network learning ( Kanakasabapathy et al . , 2019a; Thirumalaraju et al . , 2020; Kanakasabapathy et al . , 2019b; Esteva et al . , 2017 ) .","The two major categories of non-blastocysts and blastocysts made up the inference classes , which included the training classes 1 , 2 , and 3 , 4 , 5 , respectively ( Figure 1 ) .","Pre-training with a large dataset of images from ImageNet honed the ability of the developed CNN to identify the shape , structure , and texture variations between morphologically complex embryos with minimal data requirements while the genetic algorithm helped in rank ordering embryos by generating unified scores ( Figure 1 ) .","The developed network was evaluated using an independent test set comprising of 97 patient-embryo cohorts .","Embryos of the highest quality that were selected from the patient cohorts were evaluated using known implantation outcomes .","Additionally , we also investigated if the neural network can be trained to directly differentiate between embryos based on their potential for implantation ( Figure 1 ) .","Our tests with patient cohorts using the algorithm does not account for the ploidy status of the embryos .","Since pre-implantation genetic screened ( PGS ) euploid embryos are associated with higher implantation chance , we also designed a neural network to evaluate the network performance in refining the screened embryos based on their implantation potential .","The evaluations using the patient cohorts tend to yield embryo selections with unknown outcomes or ploidy status , therefore , for this section of the study , we utilized a test set of 97 euploid embryos with known implantation outcomes .","The CNN was trained and evaluated in identifying euploid embryos capable of implantation and the performance was compared against those of 15 embryologists from five different fertility centers across the United States of America ."],["In our evaluations of the CNN in categorizing embryos imaged at 113 hpi based on their morphology , the network performed with an accuracy of 90 . 97% ( area under the curve: 0 . 96 ) in differentiating between blastocysts and non-blastocysts ( n = 742 ) ( Kanakasabapathy et al . , 2019a; Thirumalaraju et al . , 2020; Figure 2—figure supplement 1 ) .","The high accuracy indicated that the trained network was concordant with embryologists in categorizing embryos .","These categorization scores ( five values per embryo ) need to be used by taking into account the scores of other embryos in the cohort to establish a rank order .","In order to use the five probability values effectively for calculating the embryo score , we utilized a genetic algorithm , which is well-suited for optimization problems with multiple existing solutions .","Here , the genetic algorithm empowered the developed CNN to make selections of the top-quality embryos within a patient’s embryo cohort at 113 hpi .","Therefore , once we established that the network was capable of categorizing embryos based on their morphologies with high accuracy , we used a genetic algorithm and the network defined probability values of the embryos , belonging to each of the five training classes , to rank order the embryos for transfer .","The 5 × 1 vector weights generated by the genetic algorithm during its training phase were used in evaluating retrospectively collected embryo cohorts from 97 patients .","The final weights utilized in this study were −10 . 01226347 , –3 . 63697951 , −3 . 32090987 , 2 . 15367795 , and 2 . 8715555 for classes 1 through 5 , respectively .","Embryos were ranked by the algorithm from highest to the lowest .","According to the American Society for Reproductive Medicine guidelines on the limits to the number of embryos per transfer , one embryo is transferred for high prognosis patients with <37 years of age and two or more embryos are transferred for patients with >37 years of age as well as younger patients with low prognosis ( Practice Committee of the American Society for Reproductive Medicine . Electronic address: ASRM@asrm . org and Practice Committee of the Society for Assisted Reproductive Technology , 2017 ) .","Therefore , in this study , the selection accuracy was assessed for scenarios of single embryo transfers ( SET ) and double embryo transfers ( DET ) .","Using embryo cohort images ( n = 732 ) from the 97 patients , the accuracy of 5 well-trained embryologists’ selections were evaluated in comparison to selections made by the CNN + genetic algorithm ( CNNg ) .","The rank-ordering performed by the algorithm may not utilize the same features used by embryologists in identifying the top embryos for transfer .","Therefore , we initially evaluated the ability of both groups to effectively select","( i ) blastocyst ( s ) for transfer and","( ii ) the highest quality of blastocyst ( s ) ( HQB ) available for transfer .","High-quality blastocysts are defined as embryos that met the freezing criteria ( >3 CC blastocyst grade; see Materials and methods ) of the Massachusetts General Hospital ( MGH ) fertility clinic .","For blastocyst selections at 113 hpi , the CNNg algorithm performed with an accuracy of 98 . 96% for SET , which was similar ( p>0 . 05 ) to the average accuracy of the embryologists ( 96 . 91% , CI: 94 . 69% to 99 . 12% ) ( n = 5 ) ( Figure 2A ) .","However , when two embryo selections for DET were allowed based on blastocyst and non-blastocyst classification , the CNNg algorithm performed with an accuracy of 100 . 00% , which was better ( p<0 . 05 ) than embryologists ( n = 5 ) who performed with an average accuracy of 98 . 76% ( CI: 97 . 69% to 99 . 83% ) ( Figure 2B ) .","Toward the selection of HQB at 113 hpi , the accuracy of the CNNg algorithm for SET was 89 . 69% similar ( p>0 . 05 ) to the embryologists ( n = 5 ) who performed with an average accuracy of 90 . 31% ( CI: 87 . 50% to 93 . 11% ) ( Figure 2C ) .","When two embryo selections for DET at 113 hpi were allowed , the system performed with a better ( p<0 . 05 ) accuracy of 97 . 94% in comparison to the embryologists who performed with an average accuracy of 96 . 91% ( CI: 96 . 00% to 97 . 81% ) ( Figure 2D ) .","The evaluations indicated that the two groups made selections that were of similar quality or marginally different quality .","Since the network was trained on the MGH classification criteria , the comparable performance of the CNNg algorithm and embryologists indicated that the neural network has trained itself sufficiently and made selections that were of clinically acceptable quality .","In our evaluations , the selections made by each group , while were of similar quality , were observed to not necessarily be the same embryos from each cohort , and thus their transfer outcomes may be different .","It is critical to evaluate the system performance in selecting the patient embryos based on pregnancy ( implantation ) outcome .","Typically , in a clinical IVF cycle , the top-quality embryo is selected from the cohort of available embryos and is transferred to the patient .","Embryos , which are similarly of a high-quality , are often frozen based on the freezing criteria used by the fertility center , for transfers in subsequent procedures for the same patient if needed .","Frozen cycle transfers are not performed for all patients .","Hence , the CNNg algorithm was evaluated in embryo selection for SET at 113 hpi using patient embryo cohorts based on actual implantation outcomes of the selected embryos and associated cycle characteristics ( n = 97 ) are provided in Supplementary file 1-table 1 .","The test dataset was retrospectively collected based on pre-defined selection criteria and evaluations of transfer outcomes were performed using fresh embryo transfer cycles .","The system selected 97 embryos in 97 patient embryo cohorts ( 742 embryos in total ) , out of which 44 embryos had known implantation outcomes .","The accuracy of the system in SET through embryo selection at 113 hpi based on its implantation outcome was 59 . 1% while the implantation success rate for the 102 transferred embryos at the MGH fertility center was 44 . 1% for blastocyst transfers ( Supplementary file 1-table 2 ) .","Furthermore , prior reports suggest that in general practice , the average implantation rates for manual-based embryo selection and transfers at blastocyst stages can be as low as 34% ( Martins et al . , 2017 ) .","A limitation of a retrospective study is that not all embryos are transferred .","Implantation outcomes of all embryos selected by the CNNg algorithm cannot be evaluated .","Therefore , although the dataset was prepared not taking into consideration the availability of subsequent frozen cycle transfers , we investigated with the fertility center if the patients of the test set had any subsequent embryo transfers using the frozen embryos from the test set .","When we consider subsequent frozen embryo transfers , five embryos originally selected by the CNNg algorithm at 113 hpi had known implantation outcomes of which four led to successful implantations ( Supplementary file 1-table 2 ) .","The accuracy of the CNNg algorithm in SET , when both fresh and frozen embryo transfers were considered , was 61 . 2% .","In such a scenario , for this specific dataset , the implantation success rate at MGH fertility center was 48 . 5% for blastocyst transfers when including both frozen and fresh transfers .","The results suggest that the CNNg algorithm has the potential to improve clinical transfer outcomes .","It should , however , be emphasized that in this particular analysis the performance of the system was evaluated by only using the embryos selected by the network and the embryologists .","Furthermore , to evaluate if a CNN can potentially measure implantation potential through morphology alone , a pooled set of 29 embryo images with known transfer outcomes in a pilot study was used by the network to evaluate embryos based on their potential for implantation .","The network was trained as a binary classifier and the SoftMax probability values outputted by the network was used as the embryo’s implantation potential .","The CNN was retrained using 281 embryo images with known implantation outcomes that did not overlap with the test set and the final classification layer was replaced with the two classes- negative implantation and positive for implantation .","The ability to differentiate embryo was measured through a receiver operating characteristic curve ( ROC ) analysis , establishing area under the curve ( AUC ) of 0 . 771 ( CI: 0 . 579 to 0 . 906 ) ( p<0 . 05 ) and the CNN performed with an accuracy of 82 . 76% ( CI: 64 . 23% to 94 . 15% ) ( Figure 3A ) .","Ten out of 11 embryos had implanted with an implantation potential of over 0 . 47 and similarly , for embryos that scored less than 0 . 47 , 12 out of 18 embryos did not implant according to the patient cycle history .","After we observed high performance in the artificial intelligence ( AI ) -based implantation potential prediction when compared with historical clinical data , we further conducted a multi-center AI system evaluation by comparing the implantation potential prediction accuracies obtained from the AI system and the embryo selections of 15 embryologists from five different fertility clinics .","Here , we used 97 genetically screened euploid embryos transferred at 113 hpi to remove the effect of chromosomal abnormalities as a confounder , which existed in the pilot study ( 29 patient embryo ) .","The IVF cycle characteristics in which these embryos were used are provided in Supplementary file 1-table 3 .","The system performed with an accuracy of 75 . 25% while the embryologists performed with an average accuracy of 67 . 35% ( CI: 64 . 52% to 70 . 19% ) in differentiating euploid embryos based on their implantation outcome ( Figure 3B ) .","A one-sample t-test revealed that the CNN significantly outperformed ( p<0 . 05 ) the embryologists in predicting embryo implantation by measuring the implantation potential of euploid embryos using a static image obtained at a single time-point of 113 hpi .","The average implantation score of euploid embryos misclassified based on their implantation outcome using the CNN was 0 . 57 .","95% of the misclassified euploid embryos possessed scores ranging between 0 . 51 and 0 . 63 .","Implantation scores closer to 0 . 5 indicate lower confidence in system predictions while implantation scores closer to 0 or 1 indicate higher confidence in system predictions ( Figure 3—figure supplement 1 ) .","These results indicate that the majority of system errors in misclassifying the euploids occur among the embryos with the lowest confidence .","Approximately 91% of euploid embryos with implantation potential scores of 0 . 80 or higher , and nearly 81% of embryos with implantation potential scores above 0 . 66 successfully implanted when transferred ( Figure 3—figure supplement 1 ) .","Similarly , around 78% of euploid embryos with an implantation potential <0 . 33 , failed to successfully implant when transferred ( Figure 3—figure supplement 1 ) .","These results suggest that the network’s implantation scores agree well with transfer outcomes even in high-quality euploid embryos ."],["Deep neural networks hold value in aiding clinical decision making and have received significant attention from the IVF community .","The deep-neural network-based approach showcased here is an objective approach to one of the more subjective but important parts of a clinical IVF process-embryo selections for transfer ( Bormann et al . , 2020 ) .","Since over 80% of fertility clinics rely on non-time lapse imaging systems as part of their clinical processes , such neural network-based algorithms that rely purely on static single timepoint images can effectively assist in decision making ( Dolinko et al . , 2017 ) .","In our study , we have evaluated two neural network-based approaches for improving embryo selection .","Firstly , we have demonstrated that a deep-neural network in combination with a genetic algorithm ( CNNg ) can yield a continuous score that represents the quality of the embryo and that objective orders of transfer can be determined for a given set of embryos using such scores .","The ranking algorithm studied here was able to consistently select embryos of the highest available morphological quality .","Although the network was trained to classify embryos based on their quality , it performed well even in differentiating between embryos of the same class when combined with a genetic algorithm .","The benefit of such systems is particularly evident in cases where selections made by the clinic/embryologist , although of similar grade , resulted in lower overall transfer success rates .","Our networks only focused on the morphological features for embryo quality assessments due to data scarcity .","The network’s learning can be compounded with data from additional timepoints , morphokinetics , and patient and cycle-specific information for more personalized IVF predictions and outcomes .","Recently , Tran et al . studied the use of a deep-learning model ( IVY ) that can analyze whole time-lapse videos instead of specific time points for fetal heartbeat prediction ( Tran et al . , 2019 ) .","However , the study was flawed since embryos with unknown outcomes ( non-transferred embryos ) were considered as negative outcome cases , which made up most of their dataset ( ~90% ) .","The heavy class bias in their dataset and improper study design severely limits any conclusions that can be drawn from the work .","A major hurdle for the development of networks capable of analyzing multi-timepoint images and with additional patient-specific information is the limited availability of diversified data with known clinical outcomes .","During training , the lack of availability of such data prevents the networks from effectively learning relevant outcome-associated patterns in data .","The need for data scales with the complexity of the task and the number of variables introduced .","While this work focuses primarily on the utility of deep-learning algorithm for embryo evaluations at 113 hpi , it is also possible to develop similar networks for embryo evaluations at different timepoints , provided that sufficient data with matched outcomes/annotations are available .","We have evaluated a similar network for use with cleavage-stage embryos ( 70 hpi ) and showed that deep-learning approaches can outperform trained embryologists in certain tasks such as embryo selection ( Thirumalaraju et al . , 2019; Kanakasabapathy et al . , 2020 ) .","A major concern in any clinical practice , however , is the loss of viable embryos due to system errors .","Therefore , the AI-based embryo selection algorithm reported here does not make any suggestion on discarding embryos .","All embryos assessed by the CNNg in the selection process may be cryopreserved as per clinical practice .","Thereby , our approach will not negatively affect the cumulative pregnancy rate since viable embryos will not be lost .","However , it may improve the pregnancy rate as the system may be able to improve the chance of achieving a pregnancy faster with fewer embryos transferred .","Furthermore , it is important to note that in its current stage this system is intended to act only as an assistive tool for embryologists .","The embryologists can include the system’s prediction to make better judgments during embryo selection .","The scores provided by the algorithm are continuous , but it can also be easily modified to present its scoring results in both binary and a more categorical format .","Clinically , besides morphological features , various other important metrics and parameters are considered by embryologists at the time of decision making such as taking into account the ploidy status of the transferable embryos .","PGS verified euploid embryos have been shown to possess a higher probability of successful outcome but cost a hefty premium on top of the cycle costs at most fertility centers in the United States ( Drazba et al . , 2014 ) .","Furthermore , for patients with two or more euploid embryos , additional assessments of embryo morphology are required to select the best embryo based on their morphology for transfer , since euploids do not inherently guarantee implantation .","Thus far , to the best of our knowledge , no system , deep-learning-based or otherwise , has been shown to be capable of differentiating between euploid blastocysts based on their capacity for implantation .","Euploid embryos are usually of the highest available quality and differentiating between them objectively and reliably through manual analysis can be extremely challenging .","The CNN-based approach , through direct estimations of implantation potential from 113 hpi embryo morphology , outperformed trained embryologists in identifying implanting embryos from a set of PGS euploid embryos .","This accomplishment exhibits the potential of artificial intelligence-based approaches to improve success rates in the IVF lab .","Our observations indicated that the system performed with a significantly better agreement with the actual implantation outcome for embryos with implantation scores closer to 1 or 0 ( Higher confidence ) .","Furthermore , the comparison between the decisions made by 15 embryologists from different fertility centers in the US and the deep-neural network showcased that neural networks can outperform embryologists in identifying embryos capable of implantation .","Hence , by applying the suggestions of a CNN , a trained embryologist can improve their selection of the embryo with the highest implantation potential .","Advances in artificial intelligence have fostered numerous applications that have the potential to improve standard-of-care in the different fields of medicine .","While other groups have also evaluated different use cases for machine learning in assisted reproductive medicine , this approach is novel in how it used a CNN trained on a large dataset to make predictions based on static images .","The approach has shown the potential of CNNs to be used in aiding embryologists to select the embryo with the highest implantation potential , especially amongst high-quality euploid embryos .","Although the current retrospective study shows that these systems can perform better than highly-trained embryologists , randomized control trials are required before routine use in clinical practice is adopted ."],["Data were collected at the Massachusetts General Hospital ( MGH ) fertility center in Boston , Massachusetts .","We used 3469 recorded videos of embryos collected from 543 patients with informed consent for research and publication , under an institutional review board approval for secondary research use .","Videos were collected for research after institutional review board approval by the Massachusetts General Hospital Institutional Review Board ( IRB#2017P001339 and IRB#2019P002392 ) .","All the experiments were performed in compliance with the relevant laws and institutional guidelines of the Massachusetts General Hospital , Brigham and Women's Hospital , and Partners Healthcare .","The videos were collected using a commercial time-lapse imaging system ( Vitrolife Embryoscope ) .","The imaging system used a Leica 20x objective that collected images at 10-min intervals under illumination from a single 635 nm LED .","Each patient’s set of embryos were exported as videos ( . avi ) using the imaging system software .","The videos of individual embryos were broken down into their respective frames to extract images from all timepoints post insemination .","The images were identified by their timestamps and only images collected at 113 ± 0 . 05 hr post insemination were processed and used in this study .","The extracted images were 250 × 250 pixels and they were cropped to 210 × 210 pixels .","The cropping removed both the timestamps and identifiers present in the frame .","All embryos used in the study were annotated using images from the fixed time-points ( 113 hpi ) by senior-level embryologists with a minimum of 5 years of human IVF training .","Annotations for embryo implantation were assigned based on clinical outcomes .","Out-of-focus images were included in the datasets and used for both testing and training .","Only images of embryos that were completely non-discernable were removed from the study as part of the data cleaning procedure .","The two networks in this study used two categorization systems .","The network focused on the rank ordering of embryos used a hierarchical categorization system .","The embryo images at 113 hpi time point were categorized between training classes 1 through five as described in detail elsewhere ( Thirumalaraju et al . , 2020 ) .","Briefly , degenerated embryos , which did not begin compaction formed Class 1 while Class 2 embryos were those that reached the morula stage by 113 hpi .","Classes 1 and 2 together formed ‘non-blastocysts’ inference class .","Class three embryos exhibit features of an early blastocyst which is highlighted by the presence of blastocoel cavity and thick zona pellucida but lack expansion .","Class four embryos were blastocysts with blastocoel cavities occupying over half of the embryo volume but either their inner cell mass ( ICM ) or trophectoderm ( TE ) was of poor quality .","They are non-freezable quality embryos ( <3 CC ) , where three represents the degree of expansion ( range 1–6 ) and C represents the quality of ICM and TE ( range A-D ) , respectively .","Class 5 embryos , however , met cryopreservation criteria ( >3 CC ) and included full blastocysts to hatched blastocysts .","Classes 3 , 4 , and 5 together formed ‘blastocysts’ inference class .","The two inference classes are used since the differentiation of blastocysts and non-blastocysts is a universally accepted categorization that is relevant to embryologists , while the five class categorization is specific to the neural network training , performance and evaluation ( Thirumalaraju et al . , 2020 ) .","Networks that were focused on estimating an embryo’s implantation potential used a two-class training and inference system- positive for implantation and negative for implantation .","The 113 hpi evaluation dataset included images of 2440 embryos categorized across five classes post-cleaning based on their clinical annotations made at 113 hpi .","Our training set for this classification task used 1188 images with a validation dataset of 510 images obtained at 113 hpi .","With the availability of unskewed validation sets prior to augmentation , we used a data generator during training , which performed random rotations and flips across all classes on the fly .","The system performing with an accuracy of 90 . 97% was used in this study in combination with our genetic algorithm .","The genetic algorithm was trained and tested with the training data prior to testing it with our independent test data .","No human interaction was required/performed once the images were provided to the system during testing , as the entire process was fully automated .","The independent non-overlapping test set consisted of 742 images of embryos originating from 97 patients .","The selections were compared with embryologist selections .","The network was also trained to classify embryos with successful and unsuccessful implantation .","281 embryo images with known implantation outcomes were used for training .","Implantation signifies the attachment of a blastocyst into the endometrium .","The status of implantation was clinically verified by ultrasound ~6 weeks after embryo transfer .","Ninety-seven euploid embryos were evaluated by 15 embryologists , including director level embryologists from five different fertility centers .","A genetic algorithm was designed to perform selections in combination with the neural network .","The genetic algorithm component utilizes the probability scores of every embryo belonging to each of the five different classes to generate a transfer score that can be used to effectively identify the best embryo available in a cohort .","For system evaluations , we used an independent set of embryos ( 100 patients; 2–12 embryos per patient ) , with no overlap with the training data set used for any prior exercise .","The patient cohorts were chosen under the following criteria:","( i ) each patient embryo cohort had to possess at least two 2PN embryos , and","( ii ) at least one embryo of the patient embryo cohort developed to blastocyst stage by 113 hpi .","We trained a genetic algorithm to select the morphologically highest quality embryo from a given cohort .","There are four phases namely initialization , selection , crossover , and mutation .","The classified embryos for each patient were sorted according to their identifier numbers allotted by the deep neural network .","A population of weights was generated at random during initialization .","A population size of 100 was generated with a 5 × 1 matrix representing each weight .","Each weight defined a possible solution for the rank-ordering of embryos based on their quality using the five training classes .","The dot product of the weights with the output logits provided by the CNN was used in the calculation of the fitness .","The algorithm runs multiple cycles to select the optimal set of weight towards achieving the appropriately suitable rank order of embryos based on their qualities .","At each cycle , all the weight sets obtained using the given population were used rank-ordering embryos within the training set .","The best 20 wt sets were selected in each cycle .","These selected weights ( specimens ) were then bred with each other with a probability set to 20% .","It randomly selected two specimens from the selected top pool and created a random binary 5 × 1 matrix , where one represents that the given element should be switched in cohort and 0 represents that given element should not be switched within the cohort .","The fitness function checks if the selected embryo belongs to the highest class available within the tested cohort .","It checks if the selected solution ( specimen ) picked the embryo belonging to the top class in a given cohort of patient embryos .","If the selected embryo belonged to the top class , the score was increased and if it did not , the score was not modified .","After iterating for all patients’ cohorts , the total scores were used to select the best 20 weights of the given population and were taken for crossover and mutation to repeat the process .","The new specimens replaced their parents in the top selected group of embryos .","Otherwise , the matrix remained the same .","After breeding , each specimen from the top selected group was mutated to give five mutations by adding a random float 5 × 1 matrix with a probability of 20% .","These mutations were then added to the new population and the selection step was repeated with the new population of 100 .","The genetic algorithm ran until the entire population converged to the same score after which a random weight was selected from the population as the final weight .","Thus , final generated weights were used to further test the embryo cohorts within our test set ."]],"string":"[\n [\n \"Assisted reproductive technologies ( ART ) such as in vitro fertilization ( IVF ) , while a solution to many infertile couples have been inefficient with an average success rate of approximately 30% reported in 2015 in the US ( CDC , 2015 ) .\",\n \"IVF is also an expensive solution costing patients well over $10 , 000 out-of-pocket for each ART cycle in the US with many patients requiring multiple cycles to achieve successful pregnancy ( CDC , 2015; Birenbaum-Carmeli , 2004; Toner , 2002 ) .\",\n \"Although multiple factors such as maternal age , medical diagnosis , gamete and embryo quality , and endometrium receptivity determine the success of ART cycles , the challenge of non-invasive selection of the highest available quality from a patient’s cohort of embryos ( top-quality embryo ) for transfer remains as one of the most important factors in achieving successful ART outcomes ( Vaegter et al . , 2017; Barash et al . , 2017; Conaghan et al . , 2013; Wong et al . , 2013; Racowsky et al . , 2015; Filho et al . , 2010; Machtinger and Racowsky , 2013; Demko et al . , 2016; Einarsson et al . , 2017; Hill et al . , 1989; Erenus et al . , 1991; Paulson et al . , 1990; Osman et al . , 2015 ) .\",\n \"Traditional methods of embryo selection rely on visual embryo morphological assessment and are highly practice-dependent and subjective ( Storr et al . , 2017; Baxter Bendus et al . , 2006; Paternot et al . , 2009 ) .\",\n \"Fully automated assessments of embryos are challenging owing to the complexity of embryo morphologies .\",\n \"Emulating the skill of highly trained embryologists in efficient embryo assessment in a fully automated system is a major challenge in all of the previous work done in computer-aided assessments of embryos due to focus on measuring specific expert-defined parameters such as zona pellucida thickness variation , number of blastomeres , degree of cell symmetry and cytoplasmic fragmentation , etc . ( Rocha et al . , 2017a; Rocha et al . , 2017b ) .\",\n \"Machine learning is loosely defined as a computer program that learns a given task over time through experience and improves itself to achieve the best possible task performance .\",\n \"In the past decade , advances in hardware compute performance and machine learning techniques have significantly improved their applicability in real-world medical and non-medical problems .\",\n \"Recently , machine learning has been proposed as a solution for automated analysis of embryo morphologies ( Rocha et al . , 2017b; Bormann et al . , 2020; Dimitriadis et al . , 2019; Thirumalaraju et al . , 2019; Khosravi et al . , 2019; Kanakasabapathy et al . , 2019a ) .\",\n \"This work makes use of a deep convolutional neural network ( CNN ) , a representation learning technique , that has been proven to be effective in image classification tasks .\",\n \"Unlike most prior computer-aided algorithms , including some techniques of machine learning used for embryo assessment , the reported CNN architecture allows automated embryo feature selection and analysis at the pixel level without any interference by an embryologist ( Rocha et al . , 2017a; Rocha et al . , 2017b ) .\",\n \"Such networks do not depend on human-specified features and can develop an ability to evaluate embryos categorically through iterative learning from thousands of examples .\",\n \"The use of deep-learning in IVF has also been explored; however , these recent neural network-based approaches have focused on either classifying embryos based on morphological quality and were not evaluated for transfer outcomes , or were developed with the use of time-lapse series of images toward the evaluation of implantation ( Khosravi et al . , 2019; Tran et al . , 2019 ) .\",\n \"It is important to emphasize here that most fertility centers do not possess time-lapse imaging hardware even in the United States of America ( Dolinko et al . , 2017 ) .\",\n \"The lack of availability of such hardware limits an otherwise promising technology mostly to resource-rich settings and fail to improve quality of and access to care in resource-constrained settings where such advances are sorely needed ( Wahl et al . , 2018; Hosny and Aerts , 2019 ) .\",\n \"Furthermore , in current clinical practice , embryos with the highest morphological grades ( top-quality ) are the first to be transferred , however , clinically these decisions are performed manually , even with time-lapse imaging systems .\",\n \"The development of networks that can measure an embryo’s potential for implantation and help in rank ordering embryos in a patient embryo cohort for transfer have utility in virtually all fertility centers .\",\n \"Conventionally , embryo transfers are performed at the cleavage or the blastocyst stage of development .\",\n \"Embryos are at the cleavage stage 2–3 days after fertilization and develop further in suitable culture conditions to reach the blastocyst stage 5–7 days after fertilization .\",\n \"Blastocyst embryo transfers , in particular , have been associated with better implantation rates and have helped lower the number of embryos transferred at a time ( De Croo et al . , 2019 ) .\",\n \"Therefore , in this study , we have investigated the use of a CNN pre-trained with 1 . 4 million ImageNet images and transfer-learned using 2440 static human embryo images recorded at a single time-point of 113 hr post insemination ( hpi ) for the development of neural networks that can help identify embryos capable of implantation and for identifying the top quality embryos ( Figure 1 ) .\",\n \"The top-quality embryos were identified by combining a previously developed network ( Xception architecture ) trained to classify embryos based on its blastocyst quality with a genetic algorithm scheme ( Figure 1; Thirumalaraju et al . , 2020 ) .\",\n \"The original neural network was trained on a hierarchical system of categorization , derived from a clinical Gardener-based grading system , to minimize data sparsity and improve overall network learning ( Kanakasabapathy et al . , 2019a; Thirumalaraju et al . , 2020; Kanakasabapathy et al . , 2019b; Esteva et al . , 2017 ) .\",\n \"The two major categories of non-blastocysts and blastocysts made up the inference classes , which included the training classes 1 , 2 , and 3 , 4 , 5 , respectively ( Figure 1 ) .\",\n \"Pre-training with a large dataset of images from ImageNet honed the ability of the developed CNN to identify the shape , structure , and texture variations between morphologically complex embryos with minimal data requirements while the genetic algorithm helped in rank ordering embryos by generating unified scores ( Figure 1 ) .\",\n \"The developed network was evaluated using an independent test set comprising of 97 patient-embryo cohorts .\",\n \"Embryos of the highest quality that were selected from the patient cohorts were evaluated using known implantation outcomes .\",\n \"Additionally , we also investigated if the neural network can be trained to directly differentiate between embryos based on their potential for implantation ( Figure 1 ) .\",\n \"Our tests with patient cohorts using the algorithm does not account for the ploidy status of the embryos .\",\n \"Since pre-implantation genetic screened ( PGS ) euploid embryos are associated with higher implantation chance , we also designed a neural network to evaluate the network performance in refining the screened embryos based on their implantation potential .\",\n \"The evaluations using the patient cohorts tend to yield embryo selections with unknown outcomes or ploidy status , therefore , for this section of the study , we utilized a test set of 97 euploid embryos with known implantation outcomes .\",\n \"The CNN was trained and evaluated in identifying euploid embryos capable of implantation and the performance was compared against those of 15 embryologists from five different fertility centers across the United States of America .\"\n ],\n [\n \"In our evaluations of the CNN in categorizing embryos imaged at 113 hpi based on their morphology , the network performed with an accuracy of 90 . 97% ( area under the curve: 0 . 96 ) in differentiating between blastocysts and non-blastocysts ( n = 742 ) ( Kanakasabapathy et al . , 2019a; Thirumalaraju et al . , 2020; Figure 2—figure supplement 1 ) .\",\n \"The high accuracy indicated that the trained network was concordant with embryologists in categorizing embryos .\",\n \"These categorization scores ( five values per embryo ) need to be used by taking into account the scores of other embryos in the cohort to establish a rank order .\",\n \"In order to use the five probability values effectively for calculating the embryo score , we utilized a genetic algorithm , which is well-suited for optimization problems with multiple existing solutions .\",\n \"Here , the genetic algorithm empowered the developed CNN to make selections of the top-quality embryos within a patient’s embryo cohort at 113 hpi .\",\n \"Therefore , once we established that the network was capable of categorizing embryos based on their morphologies with high accuracy , we used a genetic algorithm and the network defined probability values of the embryos , belonging to each of the five training classes , to rank order the embryos for transfer .\",\n \"The 5 × 1 vector weights generated by the genetic algorithm during its training phase were used in evaluating retrospectively collected embryo cohorts from 97 patients .\",\n \"The final weights utilized in this study were −10 . 01226347 , –3 . 63697951 , −3 . 32090987 , 2 . 15367795 , and 2 . 8715555 for classes 1 through 5 , respectively .\",\n \"Embryos were ranked by the algorithm from highest to the lowest .\",\n \"According to the American Society for Reproductive Medicine guidelines on the limits to the number of embryos per transfer , one embryo is transferred for high prognosis patients with <37 years of age and two or more embryos are transferred for patients with >37 years of age as well as younger patients with low prognosis ( Practice Committee of the American Society for Reproductive Medicine . Electronic address: ASRM@asrm . org and Practice Committee of the Society for Assisted Reproductive Technology , 2017 ) .\",\n \"Therefore , in this study , the selection accuracy was assessed for scenarios of single embryo transfers ( SET ) and double embryo transfers ( DET ) .\",\n \"Using embryo cohort images ( n = 732 ) from the 97 patients , the accuracy of 5 well-trained embryologists’ selections were evaluated in comparison to selections made by the CNN + genetic algorithm ( CNNg ) .\",\n \"The rank-ordering performed by the algorithm may not utilize the same features used by embryologists in identifying the top embryos for transfer .\",\n \"Therefore , we initially evaluated the ability of both groups to effectively select\",\n \"( i ) blastocyst ( s ) for transfer and\",\n \"( ii ) the highest quality of blastocyst ( s ) ( HQB ) available for transfer .\",\n \"High-quality blastocysts are defined as embryos that met the freezing criteria ( >3 CC blastocyst grade; see Materials and methods ) of the Massachusetts General Hospital ( MGH ) fertility clinic .\",\n \"For blastocyst selections at 113 hpi , the CNNg algorithm performed with an accuracy of 98 . 96% for SET , which was similar ( p>0 . 05 ) to the average accuracy of the embryologists ( 96 . 91% , CI: 94 . 69% to 99 . 12% ) ( n = 5 ) ( Figure 2A ) .\",\n \"However , when two embryo selections for DET were allowed based on blastocyst and non-blastocyst classification , the CNNg algorithm performed with an accuracy of 100 . 00% , which was better ( p<0 . 05 ) than embryologists ( n = 5 ) who performed with an average accuracy of 98 . 76% ( CI: 97 . 69% to 99 . 83% ) ( Figure 2B ) .\",\n \"Toward the selection of HQB at 113 hpi , the accuracy of the CNNg algorithm for SET was 89 . 69% similar ( p>0 . 05 ) to the embryologists ( n = 5 ) who performed with an average accuracy of 90 . 31% ( CI: 87 . 50% to 93 . 11% ) ( Figure 2C ) .\",\n \"When two embryo selections for DET at 113 hpi were allowed , the system performed with a better ( p<0 . 05 ) accuracy of 97 . 94% in comparison to the embryologists who performed with an average accuracy of 96 . 91% ( CI: 96 . 00% to 97 . 81% ) ( Figure 2D ) .\",\n \"The evaluations indicated that the two groups made selections that were of similar quality or marginally different quality .\",\n \"Since the network was trained on the MGH classification criteria , the comparable performance of the CNNg algorithm and embryologists indicated that the neural network has trained itself sufficiently and made selections that were of clinically acceptable quality .\",\n \"In our evaluations , the selections made by each group , while were of similar quality , were observed to not necessarily be the same embryos from each cohort , and thus their transfer outcomes may be different .\",\n \"It is critical to evaluate the system performance in selecting the patient embryos based on pregnancy ( implantation ) outcome .\",\n \"Typically , in a clinical IVF cycle , the top-quality embryo is selected from the cohort of available embryos and is transferred to the patient .\",\n \"Embryos , which are similarly of a high-quality , are often frozen based on the freezing criteria used by the fertility center , for transfers in subsequent procedures for the same patient if needed .\",\n \"Frozen cycle transfers are not performed for all patients .\",\n \"Hence , the CNNg algorithm was evaluated in embryo selection for SET at 113 hpi using patient embryo cohorts based on actual implantation outcomes of the selected embryos and associated cycle characteristics ( n = 97 ) are provided in Supplementary file 1-table 1 .\",\n \"The test dataset was retrospectively collected based on pre-defined selection criteria and evaluations of transfer outcomes were performed using fresh embryo transfer cycles .\",\n \"The system selected 97 embryos in 97 patient embryo cohorts ( 742 embryos in total ) , out of which 44 embryos had known implantation outcomes .\",\n \"The accuracy of the system in SET through embryo selection at 113 hpi based on its implantation outcome was 59 . 1% while the implantation success rate for the 102 transferred embryos at the MGH fertility center was 44 . 1% for blastocyst transfers ( Supplementary file 1-table 2 ) .\",\n \"Furthermore , prior reports suggest that in general practice , the average implantation rates for manual-based embryo selection and transfers at blastocyst stages can be as low as 34% ( Martins et al . , 2017 ) .\",\n \"A limitation of a retrospective study is that not all embryos are transferred .\",\n \"Implantation outcomes of all embryos selected by the CNNg algorithm cannot be evaluated .\",\n \"Therefore , although the dataset was prepared not taking into consideration the availability of subsequent frozen cycle transfers , we investigated with the fertility center if the patients of the test set had any subsequent embryo transfers using the frozen embryos from the test set .\",\n \"When we consider subsequent frozen embryo transfers , five embryos originally selected by the CNNg algorithm at 113 hpi had known implantation outcomes of which four led to successful implantations ( Supplementary file 1-table 2 ) .\",\n \"The accuracy of the CNNg algorithm in SET , when both fresh and frozen embryo transfers were considered , was 61 . 2% .\",\n \"In such a scenario , for this specific dataset , the implantation success rate at MGH fertility center was 48 . 5% for blastocyst transfers when including both frozen and fresh transfers .\",\n \"The results suggest that the CNNg algorithm has the potential to improve clinical transfer outcomes .\",\n \"It should , however , be emphasized that in this particular analysis the performance of the system was evaluated by only using the embryos selected by the network and the embryologists .\",\n \"Furthermore , to evaluate if a CNN can potentially measure implantation potential through morphology alone , a pooled set of 29 embryo images with known transfer outcomes in a pilot study was used by the network to evaluate embryos based on their potential for implantation .\",\n \"The network was trained as a binary classifier and the SoftMax probability values outputted by the network was used as the embryo’s implantation potential .\",\n \"The CNN was retrained using 281 embryo images with known implantation outcomes that did not overlap with the test set and the final classification layer was replaced with the two classes- negative implantation and positive for implantation .\",\n \"The ability to differentiate embryo was measured through a receiver operating characteristic curve ( ROC ) analysis , establishing area under the curve ( AUC ) of 0 . 771 ( CI: 0 . 579 to 0 . 906 ) ( p<0 . 05 ) and the CNN performed with an accuracy of 82 . 76% ( CI: 64 . 23% to 94 . 15% ) ( Figure 3A ) .\",\n \"Ten out of 11 embryos had implanted with an implantation potential of over 0 . 47 and similarly , for embryos that scored less than 0 . 47 , 12 out of 18 embryos did not implant according to the patient cycle history .\",\n \"After we observed high performance in the artificial intelligence ( AI ) -based implantation potential prediction when compared with historical clinical data , we further conducted a multi-center AI system evaluation by comparing the implantation potential prediction accuracies obtained from the AI system and the embryo selections of 15 embryologists from five different fertility clinics .\",\n \"Here , we used 97 genetically screened euploid embryos transferred at 113 hpi to remove the effect of chromosomal abnormalities as a confounder , which existed in the pilot study ( 29 patient embryo ) .\",\n \"The IVF cycle characteristics in which these embryos were used are provided in Supplementary file 1-table 3 .\",\n \"The system performed with an accuracy of 75 . 25% while the embryologists performed with an average accuracy of 67 . 35% ( CI: 64 . 52% to 70 . 19% ) in differentiating euploid embryos based on their implantation outcome ( Figure 3B ) .\",\n \"A one-sample t-test revealed that the CNN significantly outperformed ( p<0 . 05 ) the embryologists in predicting embryo implantation by measuring the implantation potential of euploid embryos using a static image obtained at a single time-point of 113 hpi .\",\n \"The average implantation score of euploid embryos misclassified based on their implantation outcome using the CNN was 0 . 57 .\",\n \"95% of the misclassified euploid embryos possessed scores ranging between 0 . 51 and 0 . 63 .\",\n \"Implantation scores closer to 0 . 5 indicate lower confidence in system predictions while implantation scores closer to 0 or 1 indicate higher confidence in system predictions ( Figure 3—figure supplement 1 ) .\",\n \"These results indicate that the majority of system errors in misclassifying the euploids occur among the embryos with the lowest confidence .\",\n \"Approximately 91% of euploid embryos with implantation potential scores of 0 . 80 or higher , and nearly 81% of embryos with implantation potential scores above 0 . 66 successfully implanted when transferred ( Figure 3—figure supplement 1 ) .\",\n \"Similarly , around 78% of euploid embryos with an implantation potential <0 . 33 , failed to successfully implant when transferred ( Figure 3—figure supplement 1 ) .\",\n \"These results suggest that the network’s implantation scores agree well with transfer outcomes even in high-quality euploid embryos .\"\n ],\n [\n \"Deep neural networks hold value in aiding clinical decision making and have received significant attention from the IVF community .\",\n \"The deep-neural network-based approach showcased here is an objective approach to one of the more subjective but important parts of a clinical IVF process-embryo selections for transfer ( Bormann et al . , 2020 ) .\",\n \"Since over 80% of fertility clinics rely on non-time lapse imaging systems as part of their clinical processes , such neural network-based algorithms that rely purely on static single timepoint images can effectively assist in decision making ( Dolinko et al . , 2017 ) .\",\n \"In our study , we have evaluated two neural network-based approaches for improving embryo selection .\",\n \"Firstly , we have demonstrated that a deep-neural network in combination with a genetic algorithm ( CNNg ) can yield a continuous score that represents the quality of the embryo and that objective orders of transfer can be determined for a given set of embryos using such scores .\",\n \"The ranking algorithm studied here was able to consistently select embryos of the highest available morphological quality .\",\n \"Although the network was trained to classify embryos based on their quality , it performed well even in differentiating between embryos of the same class when combined with a genetic algorithm .\",\n \"The benefit of such systems is particularly evident in cases where selections made by the clinic/embryologist , although of similar grade , resulted in lower overall transfer success rates .\",\n \"Our networks only focused on the morphological features for embryo quality assessments due to data scarcity .\",\n \"The network’s learning can be compounded with data from additional timepoints , morphokinetics , and patient and cycle-specific information for more personalized IVF predictions and outcomes .\",\n \"Recently , Tran et al . studied the use of a deep-learning model ( IVY ) that can analyze whole time-lapse videos instead of specific time points for fetal heartbeat prediction ( Tran et al . , 2019 ) .\",\n \"However , the study was flawed since embryos with unknown outcomes ( non-transferred embryos ) were considered as negative outcome cases , which made up most of their dataset ( ~90% ) .\",\n \"The heavy class bias in their dataset and improper study design severely limits any conclusions that can be drawn from the work .\",\n \"A major hurdle for the development of networks capable of analyzing multi-timepoint images and with additional patient-specific information is the limited availability of diversified data with known clinical outcomes .\",\n \"During training , the lack of availability of such data prevents the networks from effectively learning relevant outcome-associated patterns in data .\",\n \"The need for data scales with the complexity of the task and the number of variables introduced .\",\n \"While this work focuses primarily on the utility of deep-learning algorithm for embryo evaluations at 113 hpi , it is also possible to develop similar networks for embryo evaluations at different timepoints , provided that sufficient data with matched outcomes/annotations are available .\",\n \"We have evaluated a similar network for use with cleavage-stage embryos ( 70 hpi ) and showed that deep-learning approaches can outperform trained embryologists in certain tasks such as embryo selection ( Thirumalaraju et al . , 2019; Kanakasabapathy et al . , 2020 ) .\",\n \"A major concern in any clinical practice , however , is the loss of viable embryos due to system errors .\",\n \"Therefore , the AI-based embryo selection algorithm reported here does not make any suggestion on discarding embryos .\",\n \"All embryos assessed by the CNNg in the selection process may be cryopreserved as per clinical practice .\",\n \"Thereby , our approach will not negatively affect the cumulative pregnancy rate since viable embryos will not be lost .\",\n \"However , it may improve the pregnancy rate as the system may be able to improve the chance of achieving a pregnancy faster with fewer embryos transferred .\",\n \"Furthermore , it is important to note that in its current stage this system is intended to act only as an assistive tool for embryologists .\",\n \"The embryologists can include the system’s prediction to make better judgments during embryo selection .\",\n \"The scores provided by the algorithm are continuous , but it can also be easily modified to present its scoring results in both binary and a more categorical format .\",\n \"Clinically , besides morphological features , various other important metrics and parameters are considered by embryologists at the time of decision making such as taking into account the ploidy status of the transferable embryos .\",\n \"PGS verified euploid embryos have been shown to possess a higher probability of successful outcome but cost a hefty premium on top of the cycle costs at most fertility centers in the United States ( Drazba et al . , 2014 ) .\",\n \"Furthermore , for patients with two or more euploid embryos , additional assessments of embryo morphology are required to select the best embryo based on their morphology for transfer , since euploids do not inherently guarantee implantation .\",\n \"Thus far , to the best of our knowledge , no system , deep-learning-based or otherwise , has been shown to be capable of differentiating between euploid blastocysts based on their capacity for implantation .\",\n \"Euploid embryos are usually of the highest available quality and differentiating between them objectively and reliably through manual analysis can be extremely challenging .\",\n \"The CNN-based approach , through direct estimations of implantation potential from 113 hpi embryo morphology , outperformed trained embryologists in identifying implanting embryos from a set of PGS euploid embryos .\",\n \"This accomplishment exhibits the potential of artificial intelligence-based approaches to improve success rates in the IVF lab .\",\n \"Our observations indicated that the system performed with a significantly better agreement with the actual implantation outcome for embryos with implantation scores closer to 1 or 0 ( Higher confidence ) .\",\n \"Furthermore , the comparison between the decisions made by 15 embryologists from different fertility centers in the US and the deep-neural network showcased that neural networks can outperform embryologists in identifying embryos capable of implantation .\",\n \"Hence , by applying the suggestions of a CNN , a trained embryologist can improve their selection of the embryo with the highest implantation potential .\",\n \"Advances in artificial intelligence have fostered numerous applications that have the potential to improve standard-of-care in the different fields of medicine .\",\n \"While other groups have also evaluated different use cases for machine learning in assisted reproductive medicine , this approach is novel in how it used a CNN trained on a large dataset to make predictions based on static images .\",\n \"The approach has shown the potential of CNNs to be used in aiding embryologists to select the embryo with the highest implantation potential , especially amongst high-quality euploid embryos .\",\n \"Although the current retrospective study shows that these systems can perform better than highly-trained embryologists , randomized control trials are required before routine use in clinical practice is adopted .\"\n ],\n [\n \"Data were collected at the Massachusetts General Hospital ( MGH ) fertility center in Boston , Massachusetts .\",\n \"We used 3469 recorded videos of embryos collected from 543 patients with informed consent for research and publication , under an institutional review board approval for secondary research use .\",\n \"Videos were collected for research after institutional review board approval by the Massachusetts General Hospital Institutional Review Board ( IRB#2017P001339 and IRB#2019P002392 ) .\",\n \"All the experiments were performed in compliance with the relevant laws and institutional guidelines of the Massachusetts General Hospital , Brigham and Women's Hospital , and Partners Healthcare .\",\n \"The videos were collected using a commercial time-lapse imaging system ( Vitrolife Embryoscope ) .\",\n \"The imaging system used a Leica 20x objective that collected images at 10-min intervals under illumination from a single 635 nm LED .\",\n \"Each patient’s set of embryos were exported as videos ( . avi ) using the imaging system software .\",\n \"The videos of individual embryos were broken down into their respective frames to extract images from all timepoints post insemination .\",\n \"The images were identified by their timestamps and only images collected at 113 ± 0 . 05 hr post insemination were processed and used in this study .\",\n \"The extracted images were 250 × 250 pixels and they were cropped to 210 × 210 pixels .\",\n \"The cropping removed both the timestamps and identifiers present in the frame .\",\n \"All embryos used in the study were annotated using images from the fixed time-points ( 113 hpi ) by senior-level embryologists with a minimum of 5 years of human IVF training .\",\n \"Annotations for embryo implantation were assigned based on clinical outcomes .\",\n \"Out-of-focus images were included in the datasets and used for both testing and training .\",\n \"Only images of embryos that were completely non-discernable were removed from the study as part of the data cleaning procedure .\",\n \"The two networks in this study used two categorization systems .\",\n \"The network focused on the rank ordering of embryos used a hierarchical categorization system .\",\n \"The embryo images at 113 hpi time point were categorized between training classes 1 through five as described in detail elsewhere ( Thirumalaraju et al . , 2020 ) .\",\n \"Briefly , degenerated embryos , which did not begin compaction formed Class 1 while Class 2 embryos were those that reached the morula stage by 113 hpi .\",\n \"Classes 1 and 2 together formed ‘non-blastocysts’ inference class .\",\n \"Class three embryos exhibit features of an early blastocyst which is highlighted by the presence of blastocoel cavity and thick zona pellucida but lack expansion .\",\n \"Class four embryos were blastocysts with blastocoel cavities occupying over half of the embryo volume but either their inner cell mass ( ICM ) or trophectoderm ( TE ) was of poor quality .\",\n \"They are non-freezable quality embryos ( <3 CC ) , where three represents the degree of expansion ( range 1–6 ) and C represents the quality of ICM and TE ( range A-D ) , respectively .\",\n \"Class 5 embryos , however , met cryopreservation criteria ( >3 CC ) and included full blastocysts to hatched blastocysts .\",\n \"Classes 3 , 4 , and 5 together formed ‘blastocysts’ inference class .\",\n \"The two inference classes are used since the differentiation of blastocysts and non-blastocysts is a universally accepted categorization that is relevant to embryologists , while the five class categorization is specific to the neural network training , performance and evaluation ( Thirumalaraju et al . , 2020 ) .\",\n \"Networks that were focused on estimating an embryo’s implantation potential used a two-class training and inference system- positive for implantation and negative for implantation .\",\n \"The 113 hpi evaluation dataset included images of 2440 embryos categorized across five classes post-cleaning based on their clinical annotations made at 113 hpi .\",\n \"Our training set for this classification task used 1188 images with a validation dataset of 510 images obtained at 113 hpi .\",\n \"With the availability of unskewed validation sets prior to augmentation , we used a data generator during training , which performed random rotations and flips across all classes on the fly .\",\n \"The system performing with an accuracy of 90 . 97% was used in this study in combination with our genetic algorithm .\",\n \"The genetic algorithm was trained and tested with the training data prior to testing it with our independent test data .\",\n \"No human interaction was required/performed once the images were provided to the system during testing , as the entire process was fully automated .\",\n \"The independent non-overlapping test set consisted of 742 images of embryos originating from 97 patients .\",\n \"The selections were compared with embryologist selections .\",\n \"The network was also trained to classify embryos with successful and unsuccessful implantation .\",\n \"281 embryo images with known implantation outcomes were used for training .\",\n \"Implantation signifies the attachment of a blastocyst into the endometrium .\",\n \"The status of implantation was clinically verified by ultrasound ~6 weeks after embryo transfer .\",\n \"Ninety-seven euploid embryos were evaluated by 15 embryologists , including director level embryologists from five different fertility centers .\",\n \"A genetic algorithm was designed to perform selections in combination with the neural network .\",\n \"The genetic algorithm component utilizes the probability scores of every embryo belonging to each of the five different classes to generate a transfer score that can be used to effectively identify the best embryo available in a cohort .\",\n \"For system evaluations , we used an independent set of embryos ( 100 patients; 2–12 embryos per patient ) , with no overlap with the training data set used for any prior exercise .\",\n \"The patient cohorts were chosen under the following criteria:\",\n \"( i ) each patient embryo cohort had to possess at least two 2PN embryos , and\",\n \"( ii ) at least one embryo of the patient embryo cohort developed to blastocyst stage by 113 hpi .\",\n \"We trained a genetic algorithm to select the morphologically highest quality embryo from a given cohort .\",\n \"There are four phases namely initialization , selection , crossover , and mutation .\",\n \"The classified embryos for each patient were sorted according to their identifier numbers allotted by the deep neural network .\",\n \"A population of weights was generated at random during initialization .\",\n \"A population size of 100 was generated with a 5 × 1 matrix representing each weight .\",\n \"Each weight defined a possible solution for the rank-ordering of embryos based on their quality using the five training classes .\",\n \"The dot product of the weights with the output logits provided by the CNN was used in the calculation of the fitness .\",\n \"The algorithm runs multiple cycles to select the optimal set of weight towards achieving the appropriately suitable rank order of embryos based on their qualities .\",\n \"At each cycle , all the weight sets obtained using the given population were used rank-ordering embryos within the training set .\",\n \"The best 20 wt sets were selected in each cycle .\",\n \"These selected weights ( specimens ) were then bred with each other with a probability set to 20% .\",\n \"It randomly selected two specimens from the selected top pool and created a random binary 5 × 1 matrix , where one represents that the given element should be switched in cohort and 0 represents that given element should not be switched within the cohort .\",\n \"The fitness function checks if the selected embryo belongs to the highest class available within the tested cohort .\",\n \"It checks if the selected solution ( specimen ) picked the embryo belonging to the top class in a given cohort of patient embryos .\",\n \"If the selected embryo belonged to the top class , the score was increased and if it did not , the score was not modified .\",\n \"After iterating for all patients’ cohorts , the total scores were used to select the best 20 weights of the given population and were taken for crossover and mutation to repeat the process .\",\n \"The new specimens replaced their parents in the top selected group of embryos .\",\n \"Otherwise , the matrix remained the same .\",\n \"After breeding , each specimen from the top selected group was mutated to give five mutations by adding a random float 5 × 1 matrix with a probability of 20% .\",\n \"These mutations were then added to the new population and the selection step was repeated with the new population of 100 .\",\n \"The genetic algorithm ran until the entire population converged to the same score after which a random weight was selected from the population as the final weight .\",\n \"Thus , final generated weights were used to further test the embryo cohorts within our test set .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Deep learning in in vitro fertilization is currently being evaluated in the development of assistive tools for the determination of transfer order and implantation potential using time-lapse data collected through expensive imaging hardware .","Assistive tools and algorithms that can work with static images , however , can help in improving the access to care by enabling their use with images acquired from traditional microscopes that are available to virtually all fertility centers .","Here , we evaluated the use of a deep convolutional neural network ( CNN ) , trained using single timepoint images of embryos collected at 113 hr post-insemination , in embryo selection amongst 97 clinical patient cohorts ( 742 embryos ) and observed an accuracy of 90% in choosing the highest quality embryo available .","Furthermore , a CNN trained to assess an embryo’s implantation potential directly using a set of 97 euploid embryos capable of implantation outperformed 15 trained embryologists ( 75 . 26% vs . 67 . 35% , p<0 . 0001 ) from five different fertility centers ."],"string":"[\n \"Deep learning in in vitro fertilization is currently being evaluated in the development of assistive tools for the determination of transfer order and implantation potential using time-lapse data collected through expensive imaging hardware .\",\n \"Assistive tools and algorithms that can work with static images , however , can help in improving the access to care by enabling their use with images acquired from traditional microscopes that are available to virtually all fertility centers .\",\n \"Here , we evaluated the use of a deep convolutional neural network ( CNN ) , trained using single timepoint images of embryos collected at 113 hr post-insemination , in embryo selection amongst 97 clinical patient cohorts ( 742 embryos ) and observed an accuracy of 90% in choosing the highest quality embryo available .\",\n \"Furthermore , a CNN trained to assess an embryo’s implantation potential directly using a set of 97 euploid embryos capable of implantation outperformed 15 trained embryologists ( 75 . 26% vs . 67 . 35% , p<0 . 0001 ) from five different fertility centers .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Around one in seven couples have trouble conceiving , which means there is a high demand for solutions such as in vitro fertilization , also known as IVF .","This process involves fertilizing and developing embryos in the laboratory and then selecting a few to implant into the womb of the patient .","IVF , however , only has a 30% success rate , is expensive and can be both mentally and physically taxing for patients .","Selecting the right embryos to implant is therefore extremely important , as this increases the chance of success , minimizes complications and ensures the baby will be healthy .","Currently the tools available for making this decision are limited , highly subjective , time-consuming , and often extremely expensive .","As a result , embryologists often rely on their experience and observational skills when choosing which embryos to implant , which can lead to a lot of variability .","An automated system based on artificial intelligence ( AI ) could therefore improve IVF success rates by assisting embryologists with this decision and ensuring more consistent results .","The AI system could learn how embryos develop over time and then uses this information to select the best embryos to implant from just a single image .","This would offer a cheaper alternative to current analysis tools that are only available at the most expensive IVF clinics .","Now , Bormann , Kanakasabapathy , Thirumalaraj et al . have developed an AI system for IVF based on thousands of images of embryos .","Using individual images , the system selected embryos of a comparable quality to those selected by a human specialist .","It also showed a greater ability to identify embryos that will lead to successful implantation .","Indeed , the software outperformed 15 embryologists from five different centers across the United States in detecting which embryos were most likely to implant out of a group of high-quality embryos with few visible differences .","Artificial intelligence has many potential applications to support expert clinical decision-making .","Systems like these could improve success , reduce errors and lead to faster , cheaper and more accessible results .","Beyond immediate IVF applications , this system could also be used in research and industry to help understand differences in embryo quality ."],"string":"[\n \"Around one in seven couples have trouble conceiving , which means there is a high demand for solutions such as in vitro fertilization , also known as IVF .\",\n \"This process involves fertilizing and developing embryos in the laboratory and then selecting a few to implant into the womb of the patient .\",\n \"IVF , however , only has a 30% success rate , is expensive and can be both mentally and physically taxing for patients .\",\n \"Selecting the right embryos to implant is therefore extremely important , as this increases the chance of success , minimizes complications and ensures the baby will be healthy .\",\n \"Currently the tools available for making this decision are limited , highly subjective , time-consuming , and often extremely expensive .\",\n \"As a result , embryologists often rely on their experience and observational skills when choosing which embryos to implant , which can lead to a lot of variability .\",\n \"An automated system based on artificial intelligence ( AI ) could therefore improve IVF success rates by assisting embryologists with this decision and ensuring more consistent results .\",\n \"The AI system could learn how embryos develop over time and then uses this information to select the best embryos to implant from just a single image .\",\n \"This would offer a cheaper alternative to current analysis tools that are only available at the most expensive IVF clinics .\",\n \"Now , Bormann , Kanakasabapathy , Thirumalaraj et al . have developed an AI system for IVF based on thousands of images of embryos .\",\n \"Using individual images , the system selected embryos of a comparable quality to those selected by a human specialist .\",\n \"It also showed a greater ability to identify embryos that will lead to successful implantation .\",\n \"Indeed , the software outperformed 15 embryologists from five different centers across the United States in detecting which embryos were most likely to implant out of a group of high-quality embryos with few visible differences .\",\n \"Artificial intelligence has many potential applications to support expert clinical decision-making .\",\n \"Systems like these could improve success , reduce errors and lead to faster , cheaper and more accessible results .\",\n \"Beyond immediate IVF applications , this system could also be used in research and industry to help understand differences in embryo quality .\"\n]","isConversational":false},"year":{"kind":"string","value":"2020"}}},{"rowIdx":292,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["chromosomes and gene expression","biochemistry and chemical biology"],"string":"[\n \"chromosomes and gene expression\",\n \"biochemistry and chemical biology\"\n]","isConversational":false},"title":{"kind":"string","value":"SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis"},"id":{"kind":"string","value":"elife-06659-v3"},"sections":{"kind":"list like","value":[["Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division .","Multi-subunit Structural Maintenance of Chromosomes ( SMC ) protein complexes—such as cohesin and condensin—are major determinants of chromosome structure and dynamics during the cell cycle in eukaryotes as well as in prokaryotes ( Hirano , 2006; Thadani et al . , 2012; Gruber , 2014 ) .","Condensin subunits were initially identified as abundant , non-histone components of mitotic chromosomes in metazoans ( Hirano and Mitchison , 1994 ) .","In mitosis , condensin localizes together with topoisomerase II in punctate structures to the longitudinal core of chromatids , called the chromosome axis ( Coelho et al . , 2003; Maeshima and Laemmli , 2003; Ono et al . , 2004 ) .","Inactivation of condensin subunits by mutation or depletion results in severe morphological aberrations and mechanical sensitivity of metaphase chromosomes , and subsequently to defects in their segregation during anaphase ( Hirano and Mitchison , 1994; Ono et al . , 2003; Gerlich et al . , 2006 ) .","In bacteria , Smc–ScpAB is the prevalent version of SMC protein complexes .","Its distant relatives MksBEF and MukBEF can be found scarcely scattered over most of the bacterial phylogenetic tree and in isolated branches of proteobacteria , respectively ( Gruber , 2011 ) .","In Bacillus subtilis and Streptococcus pneumoniae , Smc–ScpAB is recruited to a region around the replication origin by ParB/Spo0J protein bound to parS sites , thereby forming a discrete focus—also called condensation center—on each nascent copy of the chromosome ( Gruber and Errington , 2009; Sullivan et al . , 2009; Minnen et al . , 2011 ) .","Inactivation of Smc–ScpAB in B . subtilis under nutrient rich growth conditions blocks separation of sister replication origins and consequentially leads to lethal defects in chromosome partitioning ( Gruber et al . , 2014; Wang et al . , 2014 ) .","Smc–ScpAB thus promotes the initial stages of chromosome segregation in B . subtilis , likely by condensing and individualizing the emerging copies of the chromosome in preparation for their segregation to opposite halves of the cell .","The canonical SMC complex in bacteria comprises five subunits: ( 1 ) two Smc proteins , which each form a 45 nm long antiparallel coiled coil that connects an ABC-type ATPase ‘head’ domain at one end of the coiled coil with a ‘hinge’ homodimerization domain at the other end ( Hirano et al . , 2001 ) , ( 2 ) a single ScpA subunit , which belongs to the kleisin family of proteins and associates via its C-terminal winged-helix domain ( WHD ) with the bottom ‘cap’ surface of one Smc head and via its N-terminal helical domain with the ‘neck’ coiled coil region of the other Smc protein ( Bürmann et al . , 2013 ) , and ( 3 ) a dimer of ScpB protein , which binds to the central region of ScpA ( Bürmann et al . , 2013; Kamada et al . , 2013 ) .","Overall , the pentameric Smc–ScpAB complex displays a highly extended conformation harboring a central channel , which is surrounded by a closed tripartite ring formed by the Smc dimer and the ScpAB2 sub-complex .","The B . subtilis Smc coiled coils associate with one another to form rod-shaped Smc dimers ( Soh et al . , 2015 ) .","Furthermore , the Smc head domains can interact directly with one another—via a composite interface that includes two molecules of ATP .","Binding to ATP , head engagement and ATP hydrolysis likely control and drive the biochemical action of Smc–ScpAB .","Models for SMC condensation activity have been proposed based on observations made with isolated SMC dimers , SMC fragments or holo-complexes .","Such protein preparations support the bridging of given DNA molecules in vitro as indicated by the re-annealing of single stranded DNA , intermolecular DNA ligation , DNA catenation and the co-purification of labeled and unlabeled DNA molecules ( Sutani and Yanagida , 1997; Losada and Hirano , 2001; Cui et al . , 2008 ) .","Many SMC complexes bound to different segments of DNA might thus come together and anchor DNA in condensation centers or at the chromosome axis .","Oligomeric assemblies of bacterial Smc proteins have indeed been observed by Atomic Force Microscopy and Electron Microscopy ( Mascarenhas et al . , 2005; Fuentes-Perez et al . , 2012 ) .","This model provides a straightforward explanation for the compaction activity of SMC .","However , it is unclear how such apparently indiscriminate DNA aggregation would promote rather than block the individualization of sister chromosomes ( Gruber , 2014 ) .","Local wrapping of DNA around the SMC complex could result in well-defined lengthwise condensation of DNA .","However , too little SMC protein appears to be present in chromosomes to yield decent levels of compaction by simple wrapping .","A different hypothesis is based on the finding that the structurally related cohesin complex holds sister chromatids in eukaryotes together by entrapping sister DNA fibers within its ring ( Gruber et al . , 2003; Gligoris et al . , 2014 ) .","Accordingly , individual SMC complexes might entrap and expand loops of DNA , thereby driving lengthwise condensation of chromosomes with little limitations in the attainable levels of compaction ( Nasmyth , 2001; Alipour and Marko , 2012 ) .","Here , we investigate how the prokaryotic SMC–kleisin complex binds to chromosomes in vivo using a novel whole-chromosome assay ."],["We initially attempted to detect topological interactions between B . subtilis Smc–ScpAB and plasmid DNA using pull-down assays as previously described ( Ivanov and Nasmyth , 2005; Ghosh et al . , 2009; Cuylen et al . , 2011 ) .","However , several attempts failed to provide clear evidence for entrapment of small circular DNA by prokaryotic condensin .","Conceivably , Smc–ScpAB does not interact with these artificial substrates in a physiological manner .","To circumvent this possibility , we established an inverse assay by immobilizing whole chromosomes of B . subtilis in agarose plugs and monitoring their association with covalently closed rings of Smc–ScpA under harsh protein denaturing conditions ( Figure 1A ) .","To develop the chromosome entrapment assay we first performed experiments with the replicative sliding clamp , DnaN , in B . subtilis , which is known to entrap DNA in a topological manner .","Furthermore , most of cellular DnaN protein is maintained in the vicinity of active replication forks in B . subtilis , presumably by its topological association with leading and lagging strand DNA ( Su'etsugu and Errington , 2011 ) . 10 . 7554/eLife . 06659 . 003Figure 1 . Development of the chromosome entrapment assay using DnaN .","( A ) Scheme for the chromosome entrapment assay .","Cells are incubated with the cysteine cross-linker BMOE , lysed in agarose plugs and subjected to an electric field in the presence of SDS buffer .","Proteins stably bound to chromosomal DNA are re-isolated from nuclease treated agarose plugs , concentrated and analyzed by SDS-PAGE .","( B ) Crystal structure of S . pneumoniae DnaN ( PDB: 3D1F ) in surface representation .","The monomers of DnaN are shown in dark and light blue colours , respectively .","The positions of an engineered pair of cysteine residues ( N114C and V313C ) at the monomer–monomer interface of B . subtilis DnaN are indicated by arrows .","( C ) Chromosome entrapment by DnaN .","Cells of strains BSG1449 ( dnaN-HT ) and BSG1459 ( dnaN ( N114C , V313C ) -HT ) were cross-linked with BMOE and subjected to the chromosome entrapment assay .","Input and eluate fractions were analysed by in-gel detection of fluorescently labeled HT fused to DnaN ( top panel ) .","Eluate fractions of samples treated with or without nuclease during cell lysis are indicated as nuclease ‘+’ or ‘−’ , respectively .","Eluate fractions were further analyzed by silver staining revealing that another protein was consistently co-isolated during the chromosome entrapment assay ( bottom panel ) .","This protein—identified as flagellin by mass spectrometry—was retained independently of the integrity of the chromosome .","The following figure supplement is available: Figure 1—figure supplement 1: DNA entrapment by DnaN . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00310 . 7554/eLife . 06659 . 004Figure 1—figure supplement 1 . DNA entrapment by DnaN .","( A ) Cross-linking time course of DnaN-HT .","Cells of strain BSG1459 were incubated with the cross-linker BMOE and quenched at the indicated times .","Extracts were analysed by in-gel fluorescence detection of DnaN-HT .","At later time points the double cross-linked DnaN species ( ‘XX-DnaN-HT’ ) is enriched over non-cross-linked and single cross-linked DnaN-HT .","( B ) Cartoon represenation of the DnaN-HT construct based on the crystal structure of S . pneumoniae DnaN ( PDB: 3D1F ) and Rhodococcus Haloalkane dehalogenase ( PDB: 1BN6 ) .","The structure of the DnaN dimer ( monomers in green and blue colours , respectively ) was juxtaposed to the structure of the Haloalkane dehalogenase linked by a flexible peptide .","The binding pockets for HT ligand binding are indicated by orange arrows .","The positions of the engineered pair of cysteine residues ( N114C and V313C ) at the DnaN–DnaN interface are indicated by blue and green arrows .","( C ) Chromosome entrapment assay of strain BSG1459 ( dnaN ( N114C , V313C ) -HT ) in presence or absence of the cross-linker BMOE .","( D ) Effect of benzonase ( ‘nuclease’ ) on protein extracts of strains BSG1001 , BSG1449 and BSG1459 .","Cell extracts were analysed by in-gel fluorescence detection ( upper panel ) of DnaN-HT after long-time ( ‘80 min’ ) or short-time ( ‘10 min’ ) incubation with benzonase .","Lower panel shows Coomassie staining of the same SDS-PAGE gel to control for equal protein extraction efficiency . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 004 Based on the crystal structure of S . pneumoniae DnaN we engineered a pair of cysteine residues ( N114C , V313C ) into the B . subtilis protein so that DnaN can be cross-linked into covalent rings in the presence of a cysteine-specific cross-linker such as BMOE ( Figure 1B ) .","For detection a cys-less variant of the HaloTag ( ‘HT’ ) was fused to the C-terminus of DnaN ( Figure 1—figure supplement 1B ) and the construct was integrated into the genome of B . subtilis via allelic replacement at the endogenous locus .","The dnaN-ht genes with and without cysteine mutations supported normal growth of B . subtilis , implying that they encoded functional DnaN proteins ( data not shown ) .","In vivo cross-linking of DnaN-HT resulted in two additional , slow migrating bands in SDS-PAGE gels ( Figure 1C ) , corresponding to single and double cross-linked species of DnaN dimers , designated as X-DnaN-HT and XX-DnaN-HT , respectively ( Figure 1—figure supplement 1A ) .","We next embedded cells in agarose plugs and disrupted their cell walls by lysozyme digestion .","Agarose plugs were then subjected to an electric field in the presence of SDS to denature and remove any unattached proteins from chromosomes .","Plugs were finally treated with benzonase to digest genomic DNA and to release any stably entrapped protein .","DnaN-HT protein was then analysed by in-gel fluorescence .","Non-crosslinkable DnaN-HT was efficiently depleted from agarose plugs during the entrapment assay ( Figure 1C ) .","In contrast , the double cross-linked , circular form of DnaN ( N114C , V313C ) -HT ( XX-DnaN-HT ) was retained in the agarose plug during electrophoresis with high efficiency ( ∼50% of input ) .","A minor fraction of single cross-linked DnaN dimer ( X-DnaN-HT ) was also observed .","This is likely generated from XX-DnaN-HT by spontaneous hydrolysis of thiol-malemide adducts during protein isolation ( Kalia and Raines , 2007; Baldwin and Kiick , 2013 ) .","Importantly , the presence of benzonase during cell lysis eliminated all DnaN from the plug , indicating that circular DnaN is retained in plugs via its interaction with cellular DNA .","Furthermore , in the absence of the cross-linker BMOE , no DnaN-HT was detected in the eluate fraction ( Figure 1—figure supplement 1C ) .","The chromosome entrapment assay thus specifically detects a topological association of intact chromosomes with DNA sliding clamps and confirms that a major fraction ( at least 50% ) of DnaN is loaded onto DNA in rapidly growing cells .","Next , we used the newly developed chromosome entrapment assay to test for an association between native chromosomes and Smc–ScpAB complexes .","Cysteine pairs were introduced at the Smc–Smc and at both Smc–ScpA interfaces and a HT was fused at the C-terminus of Smc to allow in-gel fluorescence detection ( Figure 2A ) ( Bürmann et al . , 2013 ) .","Strains bearing the cysteine mutations and the Smc-HaloTag fusion supported normal growth on nutrient rich medium demonstrating the functionality of the modified Smc complex ( Figure 2—figure supplement 1A ) .","Cells were treated with BMOE and extracts were analysed by SDS-PAGE .","As internal control for the chromosome entrapment assay we employed the DnaN ( N114C , V313C ) protein , whose double cross-linked form was detected in input and eluate samples by immunoblotting ( Figure 2—figure supplement 1B ) .","Various species of Smc–ScpAB were identified in extracts of BMOE cross-linked cells by in-gel fluorescence .","These correspond to fully cross-linked Smc–ScpA–Smc rings and several intermediate cross-linking species as reported previously ( Figure 2B , C ) ( Bürmann et al . , 2013 ) .","To reveal the identity of all species , strains lacking one of six engineered cysteines were used as controls that collectively form several intermediate cross-linked species but no fully cross-linked rings of Smc–ScpAB ( Figure 2—figure supplement 1C ) .","In these control samples little or no Smc-HT protein was retained in agarose plugs under denaturing conditions as expected for any non-circular protein ( Figure 2B ) .","In the presence of all pairs of cysteine , however , a set of two closely migrating species was consistently detected at significant levels after the chromosome entrapment assay ( ∼10–20% of input material ) ( Figure 2B ) .","We argued that the two closely migrating species might correspond to Smc–ScpAB with a single or a double cross-link at the Smc hinge .","Consistent with this notion we find that only a single species of Smc2–ScpA accumulated during the chromosome entrapment assay when a single cysteine residue ( R643C ) was used to cross-link the Smc hinge domains ( Figure 2—figure supplement 2A ) .","These findings strongly suggest that Smc–ScpAB is bound to chromosomes via entrapment of chromosomal DNA .","If this were indeed the case , then its retention in agarose plugs should depend on the integrity of Smc–ScpAB rings and of chromosomal DNA .","Incubation of agarose plugs with the nuclease benzonase during cell lysis eliminated the Smc-HT and DnaN signal in the sample ( Figure 2B , Figure 2—figure supplement 1B ) .","To disrupt covalent Smc–ScpAB rings , we inserted cleavage sites for TEV protease into the linker region preceding the C-terminal WHD of ScpA and incubated cells during lysozyme treatment with recombinant TEV protease to open any circular Smc2–ScpA species .","As expected , little or no Smc-HT signal was detected in agarose plugs after TEV cleavage of ScpA ( Figure 2D ) .","To exclude any artefacts due to the presence of the HT on Smc we have repeated the chromosome entrapment assay with an untagged allele of Smc using immunoblotting with anti-Smc antibodies for the detection of cross-linked species , which yielded very similar results ( Figure 2—figure supplement 2C ) .","Furthermore , we found that Smc2-ScpA rings are stably trapped in agarose plugs over extended periods of time in constant or alternating electric fields ( data not shown ) .","Thus , our chromosome entrapment assay specifically detects the association between intact chromosomal DNA and rings of Smc–ScpAB in B . subtilis , demonstrating that DNA fibers pass through the Smc ring . 10 . 7554/eLife . 06659 . 005Figure 2 . Prokaryotic condensin entraps the chromosome .","( A ) Scheme for the cross-linking of Smc-HaloTag ( ‘HT’ ) and ScpA into a covalent Smc–ScpA–Smc ring .","( B ) Chromosome entrapment of covalent Smc2–ScpA rings .","Cells of strains BSG1782 , BSG1809-1813 and BSG1831 were cross-linked and subjected to the chromosome entrapment assay .","Cross-linked Smc-HT species were visualized by in-gel fluorescence detection .","The presence or absence of cysteine pairs at each of the three ring interfaces are indicated by ‘+’ and ‘−’ , respectively .","An aliquot of cells of strains BSG1782 was incubated with benzonase during cell lysis ( nuclease ‘+’ ) .","The positions of uncross-linked Smc-HT and fully cross-linked , circular Smc–ScpA–Smc species are indicated by ‘Smc-HT’ and ‘Smc-HT circ . ’; all species are labelled by colour-coded arrowheads ( see panel C for legend ) .","Circular species ( ‘h’ ) are labeled by a double pointed arrowhead .","( C ) Schematic depiction of the structure of cross-linked Smc–ScpA species ( ‘a’–‘i’ ) .","( D ) TEV cleavage of ScpA prevents entrapment of Smc–ScpAB in agarose plugs .","In-gel fluorescence detection of Smc-HT derived from strains BSG1807 and BSG1832 .","The presence or absence of TEV sites in ScpA and of TEV protease during cell lysis is indicated by ‘+’ and ‘−’ , respectively .","Cleavage of ScpA ( TEVs ) by TEV protease creates new species of cross-linked Smc-HT ( see ‘input’ samples ) and prevents entrapment of Smc-HT in agarose plugs ( see ‘eluate fraction’ ) ( top panel ) .","‘XX-DnaN’ serves as internal assay control visualized by immunoblotting of cross-linked species of DnaN protein ( bottom panel ) .","The following figure supplement is available: Figure 2—figure supplement 1: DNA entrapment by wild-type Smc–ScpAB ( I ) and Figure 2—figure supplement 2: DNA entrapment by wild-type Smc–ScpAB ( II ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00510 . 7554/eLife . 06659 . 006Figure 2—figure supplement 1 . DNA entrapment by wild-type Smc–ScpAB ( I ) .","( A ) Colony formation assays of strains BSG1002 , BSG1007 , BSG1782 , 1809–1813 and BSG1831 were performed on minimal medium agar ( SMG ) or nutrient agar ( NA ) .","Cultures were diluted 9^2-fold ( top row ) and 9^5-fold ( bottom row ) and grown at 37°C for 24 hr ( SMG ) and 14 hr ( NA ) , respectively .","The presence and absence of cysteine pairs for cross-linking of the hinge , cap and neck interface are indicated by ‘+’ and ‘−’ , respectively .","( B ) Immunoblot of SDS-PAGE gels shown in Figure 2B using anti-DnaN antibody .","All strains carry cysteine mutations for DnaN cross-linking .","Doubly cross-linked DnaN ( ‘XX-DnaN’ ) serves as internal assay control as visualized by immunoblotting .","( C ) Identification of cross-linked species of Smc-HT and ScpA .","The gel image is identical to Figure 2A with higher contrast settings to visualize low-intensity species .","All cross-linked species are labelled ( with a colour-coded letter ) ( ‘a’–‘i’ ) .","Circular species ( ‘h’ ) are labeled by a double pointed arrowhead .","( D ) Same as Figure 2 , panel C . Schematic depiction of the structure of cross-linked Smc–ScpA species ( ‘a’–‘i’ ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00610 . 7554/eLife . 06659 . 007Figure 2—figure supplement 2 . DNA entrapment by wild-type Smc–ScpAB ( II ) .","( A ) Cross-linking of the Smc hinge interface with different cysteine residues .","Cross-linking of the Smc hinge using cysteine residue ( ‘R643C’ ) instead of the ‘R558C’ and ‘N643C’ cysteine pair results in different migration pattern of cross-linked Smc-ScpA species ( see input fractions ) .","In this case species ‘g’ and ‘i’ have the same migration behavior as the circular form ‘h’ .","Only a single species of Smc-ScpA is retained in the agarose plug after the entrapment assay ( see eluate fractions ) .","( B ) Colony formation assay of strains BSG1002 , BSG1007 , BSG1807 and BSG1832 were performed as described before .","The smc and scpA loci lacking mutations ( ‘WT’ ) , harbouring cysteine mutations for cross-linking alone ( ‘Cys’ ) or in combination with a TEV cleavage site ( ‘Cys-TEV site’ ) are denoted .","( C ) Chromosome entrapment of Smc–ScpAB lacking fusion tags ( see scheme on the left ) .","The right panel shows immunoblotting of input and eluate fractions using an anti-Smc antibody .","All strains harbour cysteine residues for crosslinking of Smc-ScpA in an otherwise wild-type background ( ‘WT’ ) in BSG680 , combined with a deletion of the parB gene ( ‘ΔparB’ ) in BSG1991 or with the E1118Q mutation at the smc locus in BSG1995 .","The position of fully cross-linked , circular Smc2-ScpA species is indicated by double pointed arrowhead labelled ‘h’ ( see Figure 2—figure supplement 1C ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 007 Next , we established the requirements for the formation of interconnections between Smc–ScpAB rings and chromosomes .","The intrinsic ATPase activity of cohesin has previously been implicated in stable association with chromosomes ( Arumugam et al . , 2003; Weitzer et al . , 2003 ) .","More specifically , ATP hydrolysis has been hypothesized to transiently open an entry gate for DNA in the cohesin ring during its loading onto chromosomes ( Gruber et al . , 2006; Hu et al . , 2011 ) .","To test what steps of the ATP hydrolysis cycle in Smc–ScpAB are involved in the entrapment of chromosomal DNA , we made use of smc alleles harboring mutations that specifically prevent ATP binding ( K37I ) , engagement of Smc head domains ( S1090R ) or ATP hydrolysis ( E1118Q ) ( Figure 3A ) ( Hirano and Hirano , 2004 ) .","The three mutant proteins are expressed at normal levels in B . subtilis being indicative of proper protein folding ( Figure 3—figure supplement 1A ) .","However , they do not support growth on nutrient rich medium similar to smc null mutants , implying that all steps of the ATPase cycle are essential for Smc functionality ( Figure 3—figure supplement 1B ) ( Gruber et al . , 2014 ) .","For the chromosome entrapment assay , these Smc ATPase mutations were combined with cysteine mutations for BMOE cross-linking .","To support their viability , the resulting strains as well as the wild-type controls were grown in minimal medium .","The three mutant Smc proteins assembled into normal Smc–ScpAB complexes as judged by Smc–ScpA cross-linking , albeit there is a slight decrease in the fraction of ScpA proteins bridging Smc dimers and a concomitant minor increase in ScpA subunits bound to single Smc proteins ( Figure 3—figure supplement 1C , species ‘e’ and ‘d’ , respectively ) ( Bürmann et al . , 2013 ) .","Intriguingly , the ATP binding and engagement mutants abolished the fraction of covalent ring species retained in the agarose plug during the chromosome entrapment assay ( Figure 3B ) .","In case of the ATP hydrolysis mutant Smc ( E1118Q ) only minute amounts of cross-linked rings were recovered from SDS treated plugs .","This small fraction of stably bound condensin conceivably arises as a consequence of residual levels of ATP hydrolysis activity in Smc ( E1118Q ) ( Hirano and Hirano , 2004 ) .","Thus , ATP binding and ATP dependent Smc head engagement—and most probably also ATP hydrolysis—are essential for entrapment of chromosomal DNA by condensin in bacteria , as has been supposed for cohesin in yeast .","Furthermore , the strict requirement of several steps of the ATPase cycle strongly suggests that entrapment of DNA corresponds to the physiological form of association with the bacterial chromosome . 10 . 7554/eLife . 06659 . 008Figure 3 . The Smc ATPase is required for loading of DNA into Smc–ScpAB .","( A ) A scheme for the ATP hydrolysis cycle of Smc .","Schematic positions for Walker A , Walker B and ABC-signature motifs on the Smc head domain are shown ( top row ) .","ATP binding to the Walker A domain is blocked in Smc ( K37I ) ‘ ( 1 ) ’ .","ATP-dependent engagement of two Smc heads is abolished in the Smc ( S1090R ) mutant ‘ ( 2 ) ’ .","The E1118Q mutation strongly reduces ATP hydrolysis ‘ ( 3 ) ’ .","( B ) Smc ATPase mutations abolish chromosomal loading of Smc–ScpAB .","In-gel fluorescence detection of Smc-HT of input and eluate fractions from a representative chromosome entrapment assay performed with strains BSG1782 and BSG1784-6 .","Protein extracts ( 10% of input ) were loaded next to samples subjected to the entrapment assay .","Selected cross-linked species of Smc-HT are labeled ( top panel ) .","Detection of cross-linked species of DnaN by immunoblotting was used as internal assay control ( bottom panel ) .","The following figure supplement is available: Figure 3—figure supplement 1: ATPase mutants of Smc–ScpAB . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00810 . 7554/eLife . 06659 . 009Figure 3—figure supplement 1 . ATPase mutants of Smc-ScpAB .","( A ) Upper panel shows Smc expression levels of strains with mutant smc alleles ( BSG1002 , 1007 , 1008 , 1045 , 1046 , 1047 and 1074 ) determined by immunoblotting using anti-Smc antibodies .","Lower panel shows Coomassie staining of a SDS-PAGE gel loaded with the same whole cell extract samples to control for equal protein extraction efficiency .","( B ) Overnight cultures of strains ( BSG1002 , 1007 , 1008 , 1045 , 1046 , 1047 ) were spotted on SMG and NA as described before ( see Figure 2—figure supplement 1 ) .","Smc locus was without mutation ( ‘WT’ ) , a smc deletion ( ‘Δsmc’ ) or mutations in the Smc ATPase domain .","( C ) Smc ATPase mutations show slightly decreased levels of cross-linking species ‘e’ and increased levels of ‘d’ .","Image is identical to Figure 3B , with higher contrast . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 009 What other factors might be required for the loading of condensin onto DNA ?","The ScpB subunit forms homodimers that bind in an asymmetric manner to the central region of a single ScpA monomer .","It thus is in close proximity of the Smc ATPase domains .","Together with ScpA it putatively plays a role in the regulation of the Smc ATPase activity ( Kamada et al . , 2013 ) .","Its precise molecular function , however , is not clear yet .","To test whether ScpB is involved in the association of Smc–ScpA rings with chromosomes we combined the cysteine mutations in Smc and ScpA with an scpB in-frame deletion ( Figure 4—figure supplement 1 ) .","Ring formation was only mildly affected by the absence of ScpB as judged by BMOE cross-linking and in-gel fluorescence detection ( Figure 4A ) ( Bürmann et al . , 2013 ) .","However , Smc complexes lacking ScpB subunits failed to entrap chromosomes altogether demonstrating that ScpB is absolutely required for loading of prokaryotic condensin onto chromosomal DNA . 10 . 7554/eLife . 06659 . 010Figure 4 . ScpB and ParB are essential for efficient DNA entrapment by Smc complexes .","( A ) Deletion of scpB eliminates loading of chromosomal DNA into Smc complexes .","In-gel fluorescence detection of Smc-HT in input and eluate fractions is shown from chromosome entrapment assays performed with strains BSG1782 ( ‘WT’ ) and BSG1850 ( ‘ΔscpB’ ) ( top panel ) .","DnaN was used as internal control ( bottom panel ) .","( B ) Several parB mutations interfere with efficient chromosomal loading of Smc–ScpAB .","Input and eluate fractions from chromosome entrapment assays with strains BSG1782 , BSG1783 and BSG1960-3 were analysed by in-gel fluorescence detection of Smc-HT ( top panel ) .","DnaN was used as internal control ( middle panel ) .","Immunoblotting using polyclonal rabbit anti-ParB antiserum confirms near-normal expression of mutant ParB proteins ( bottom panel ) .","The following figure supplement is available: Figure 4—figure supplement 1: Growth of smc , parB double mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01010 . 7554/eLife . 06659 . 011Figure 4—figure supplement 1 . Growth of smc ( Cys ) mutants . Overnight cultures of strains ( BSG1002 , BSG1007 , BSG1782 , BSG1850 and BSG1783 ) were serially diluted and spotted as described before ( Figure 2—figure supplement 1 ) .","Strains were wild-type ( ‘WT’ ) , harboured the smc deletion ( ‘Δ’ ) or cysteine mutations for cross-linking the Smc-ScpA ring ( ‘Cys’ ) .","In addition , scpB ( ‘ΔscpB’ ) or parB ( ‘ΔparB’ ) were deleted where indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 011 ParB proteins—bound to parS sites—are crucial for efficient targeting of Smc–ScpAB to a large region of the chromosome near the replication origin ( Gruber and Errington , 2009; Sullivan et al . , 2009; Minnen et al . , 2011 ) .","ParB might act by simply increasing the local concentration of Smc–ScpAB around oriC either before or after its loading onto the chromosome .","Alternatively , ParB bound to parS sites might be more directly involved in the loading reaction itself , for example , as catalytic factor , and its absence might thus affect levels of chromosomal condensin .","To test this , we performed the chromosome entrapment assay with cells lacking the parB gene .","Intriguingly , the levels of Smc–ScpAB entrapping chromosomal DNA were strongly reduced in the parB null mutant as judged by the limited retention of Smc–ScpA species in agarose plugs ( Figure 4B ) .","Thus , ParB protein likely promotes the entrapment of chromosomal DNA by Smc–ScpAB .","This strongly suggests that most condensin is loaded onto the chromosome at parS sites , where ParB protein is bound .","In all other parts of the chromosome entrapment of DNA fibers by Smc–ScpAB might be very inefficient .","The cysteine bearing smc allele causes growth defects when combined with ΔparB ( Figure 4—figure supplement 1 ) .","Therefore , we cannot formally exclude the possibility that the decreased loading of Smc observed in ΔparB are due to the cysteine modifications in Smc and that chromosomal loading of wild-type Smc is not or much less affected by parB deletion .","Previously , two parB point mutations ( N112S and R149G ) , which prevent the formation of Smc-GFP foci , have been isolated in B . subtilis ( Gruber and Errington , 2009 ) .","We found that these mutations strongly impair loading of Smc onto the chromosome in the entrapment assay similar to ΔparB ( Figure 4B ) .","The R149G mutation is positioned on the helix-turn-helix motif of ParB and might thus directly affect binding to parS sites ( Leonard et al . , 2004 ) .","The N112S mutation , however , is located in another highly conserved region , which has been implicated in the ‘spreading’ of ParB protein from parS sequences into adjacent DNA ( Leonard et al . , 2004; Graham et al . , 2014 ) .","The spreading of ParB along several kb of DNA is a feature conserved in plasmid and chromosome derived ParB proteins , however , the underlying mechanism is only poorly understood ( Rodionov et al . , 1999 ) .","It might possibly involve the formation of a large nucleoprotein complex ( Broedersz et al . , 2014 ) .","Several other mutants of ParB ( including B . subtilis ParB G77S and R80A ) have been reported to be defective in spreading from parS sites ( Breier and Grossman , 2007; Graham et al . , 2014 ) .","Intriguingly , also these mutations resulted in largely reduced levels of Smc on the chromosome in our entrapment assay , being comparable to the levels found in a parB deletion mutant ( Figure 4B ) .","This implies that ParB spreading from parS sites or formation of large nucleoprotein complexes might be essential for loading of DNA into the Smc ring by ParB .","These findings are consistent with the observation that formation of Smc-GFP foci near the origin of replication are affected by the G77S mutation ( Sullivan et al . , 2009 ) .","In summary , these results demonstrate that several factors—including ScpB protein , a ParB/parS nucleoprotein complex and the Smc ATPase cycle—are required to promote efficient loading of condensin rings onto the chromosome .","Smc proteins and fragments thereof exhibit affinity for single- and double-stranded DNA in vitro ( Chiu et al . , 2004; Hirano and Hirano , 2006; Soh et al . , 2015 ) .","The physical contacts with DNA might occur once condensin has been successfully loaded onto chromosomes and thus be a permanent feature of chromosomal Smc–ScpAB .","Alternatively , the direct association with DNA might be restricted to certain intermediates in the chromosomal loading reaction .","To test for interactions between Smc–ScpAB and specific chromosomal DNA fragments , we have affinity-purified endogenous Smc–ScpAB from B . subtilis cell lysates using a short Avitag peptide fused to the C-terminus of the Smc protein , which gets biotinylated when the biotin ligase gene birA is co-expressed ( ‘Smc-Avitag’ ) .","We then examined fractions for the co-purification of fragments of chromosomal DNA—generated by restriction digest with XbaI—using quantitative PCR with primer pairs specific for different parts of the chromosome .","Since we worried that Smc–ScpAB might not be sufficiently stable in diluted cell extracts , we cross-linked the three ring interfaces in Smc–ScpAB using BMOE cross-linking of engineered pairs of cysteines .","A small fraction of chromosomal DNA was reproducibly co-purified with wild-type Smc-Avitag , whereas the yield of co-purified DNA was significantly improved by the presence of cross-linkable cysteine residues in Smc–ScpAB ( Figure 5 ) .","In both cases origin-proximal regions ( yyaD , parS-359 , dnaA and dnaN ) of the chromosome were more efficiently enriched than distal regions ( amyE , trnS and ter ) by the co-purification with Smc implying that the observed Smc-DNA contacts are dependent on chromosomal loading of Smc–ScpAB by ParB protein at parS sites and are thus physiologically relevant ( Figure 5 , Figure 5—figure supplement 1 ) ( Gruber and Errington , 2009 ) .","The association of DNA with wild-type and BMOE cross-linked Smc–ScpAB was highly sensitive to washes with a salt solution ( 2M NaCl ) , suggesting that it was dependent on electrostatic contacts between DNA and protein .","These DNA contacts are presumably formed by the Smc–ScpAB complex itself .","Alternatively , albeit less likely , other chromosomal proteins physically bound to DNA could prevent the release of condensin from DNA by blocking its sliding towards DNA ends . 10 . 7554/eLife . 06659 . 012Figure 5 . A physical interaction of Smc–ScpAB rings and chromosomal DNA . Co-purification of chromosomal DNA fragments with native Smc–ScpAB .","Cells of strains BSG1104-5 and BSG1107-8 were treated with the cross-linker BMOE prior to cell lysis .","Strains carrying ( ‘+’ ) or lacking ( ‘−’ ) cysteine mutations ( ‘6xCys’ ) in the Smc-AviTag construct were expressed in presence ( ‘+’ ) or absence ( ‘−’ ) of the biotin ligase ( ‘BirA’ ) .","Beads were washed in the presence of either a 150 mM ammonium acetate buffer ( ‘low salt’ ) or a 2 M sodium chloride buffer ( ‘high salt’ ) .","The co-purification of DNA fragments with Smc-biotin on streptavidin beads was measured by quantitative PCR using primer pairs specific for genomic positions indicated on a representation of the circular B . subtilis genome .","Mean values and standard deviations were calculated from two independent biological replicates .","The following figure supplement is available: Figure 5—figure supplement 1: Chromatin immuno-precipitation of Smc . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01210 . 7554/eLife . 06659 . 013Figure 5—figure supplement 1 . Chromatin immuno-precipitation of Smc . All Smc variants used in Figure 5 behave similarly in standard ChIP experiments using anti-Smc antibodies .","Cells of strains BSG1104-5 and 1107-8 were grown to mid-exponential phase and subjected to chromatin immunoprecipitation ( ChIP ) using polyclonal rabbit anti-Smc serum .","Input and eluate DNA samples were analysed by quantitative PCR using specific primer pairs for the genomic positions indicated on a circular representation of the B . subtilis genome .","Pull-down efficiency ( ChIP-DNA/input-DNA *100% ) was plotted for each primer pair .","The lack and presence of the BirA biotin ligase or cysteines for cross-linking of Smc and ScpA proteins are denoted as ‘BirA−’ and ‘BirA+’ or ‘6xCys−’ and ‘6xCys+’ , respectively .","It is noted that the highly transcribed tRNA locus , trnS , is highly enriched by Smc-Avitag using standard ChIP ( but not native ChIP , see Figure 5 ) , being consistent with the notion that enrichment of highly transcribed genes by ChIP is prone to artefacts due to non-uniform formaldehyde cross-linking . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 013"],["In many cases , it is challenging to measure the activity and outcome of biochemical processes in the living cell .","Here , we report the establishment of a straight-forward method to determine the physical association of ring-shaped protein complexes with whole bacterial chromosomes .","Two examples , the SMC condensin complex and the sliding clamp DnaN , document the significant potential of our simple entrapment approach .","In principle , similar assays should also be possible with eukaryotic cells and for many other chromosomal proteins such as for example hexameric helicases and certain transcription factors .","Furthermore , analogous procedures might be useful to address biological questions related to other denaturation-resistant cellular structures such as cell wall polymers ( e . g . , made up of peptidoglycan , chitin or cellulose ) .","SMC–kleisin complexes are major governors of chromosome superstructure in most branches of the phylogenetic tree .","The eukaryotic variants cohesin and condensin have been suggested to work as concatenases , which hold selected stretches of DNA together by simple embracement in their ring ( Haering et al . , 2008; Cuylen et al . , 2011 ) .","Whether DNA entrapment is an ancestral and thus fundamentally conserved function of SMC–kleisin complexes , however , remained elusive so far .","Furthermore , interaction studies developed for cohesin and condensin are based on small , artificial DNA substrates and might thus not necessarily reflect the mode of binding to native chromosomes .","These assays also fall short of providing an estimate for the fraction of SMC complexes involved in interlocked associations with DNA and thus leave open the possibility that DNA entrapment might be an insignificant side reaction .","Finally , it has not been tested under physiological conditions , whether the ATPase cycle is required for proper loading of DNA into any SMC–kleisin complex .","To provide answers to these questions , we have established the chromosome entrapment assay to determine the association of prokaryotic condensin with native chromosomes .","Our results clearly demonstrate that chromosomal DNA is loaded into condensin complexes in B . subtilis—in a manner that depends on the non-SMC subunit ScpB and at least one full cycle of Smc ATPase activity .","The chromosome entrapment assay recovers about 10–20% of the fully cross-linked input material .","This number probably understates the real proportion of chromosomally entrapped Smc complexes due to the loss of material during protein re-isolation from agarose plugs , due to possible adverse effects of cysteine mutations on Smc–ScpAB loading and because cysteine-maleimide linkages are vulnerable to hydrolytic reversal during and after the entrapment assay ( Kalia and Raines , 2007; Baldwin and Kiick , 2013 ) .","Interestingly , a recent single-molecule tracking study in B . subtilis revealed two major populations of Smc: 80% of Smc-YFP proteins are displaying highly dynamic behavior on the nucleoid , whereas the other 20% ( and most ScpA-YFP protein ) are immobile and constrained within a small volume of the cell ( Kleine Borgmann et al . , 2013 ) .","This immobile fraction possibly represents Smc–ScpAB complexes embracing origin proximal DNA after loading at parS sites as observed in the entrapment assay .","Our results show that embracement of chromosomal DNA is a predominant feature of Smc–ScpAB , which has been evolutionarily retained in cohesin and likely all other SMC–kleisin complexes as well .","Since the chromosome entrapment assay is based on the immobilization of intact replicating chromosomes , which possibly represent internally knotted and branched DNA structures , it is conceivable that Smc–ScpAB rings are linked to chromosomal DNA by non-topological capture of DNA loops , which themselves might be interlinked ( i . e . , knotted ) with other parts of the chromosome .","Therefore , it remains to be determined whether DNA entrapment by Smc–ScpAB is of topological ( Figure 6A ) and/or non-topological ( Figure 6B ) nature . 10 . 7554/eLife . 06659 . 014Figure 6 . Models for entrapment of chromosomal DNA by Smc–ScpAB .","( A ) Loop capture model .","DNA loops might be pre-formed within ParB/parS nucleoprotein assemblies .","Driven by ATP dependent engagement of Smc head domains Smc–ScpAB adopts a ring-like configuration .","Occasional opening of the Smc hinge then allows capture of ParB-DNA loops within Smc–ScpAB .","Subsequent ATP hydrolysis by Smc locks the hinge in a closed state and stabilizes the structure .","( B ) Loop formation model .","ParB/parS might serve as a landing platform for Smc–ScpAB allowing Smc–ScpAB in its ring-like conformation to guide DNA into its central cavity .","Continuous extrusion of DNA through Smc–ScpAB then drives lengthwise condensation of chromosomes .","Ring opening is not required in this model and DNA entrapment by Smc–ScpAB is thus strictly non-topological here .","The following figure supplement is available: Figure 6—figure supplement 1: Quantitative Blotting of Smc protein and parB DNA . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01410 . 7554/eLife . 06659 . 015Figure 6—figure supplement 1 . Quantitative Blotting of Smc protein and parB DNA . To estimate Smc protein and parS-359 DNA abundance in B . subtilis cells , levels of Smc and parS-359 in extracts of wild-type cells were compared to defined amounts of purified Smc protein and parS-359 DNA spiked into equivalent extracts from cell harboring a tagged version of Smc and a modified parS-359 locus ( see panel C ) .","The ratio of Smc protein ( see panel B ) to parS-359 DNA ( see panel A ) was calculated as average from three independent experiments ( biological replicates ) to be 61 ( ±12 SEM , standard error of mean ) Smc monomers per parS-359 DNA .","Due to potential loss of chromosomal DNA during genomic DNA preparation this might be a slight over-estimation of the real number .","( A ) Quantitative Southern blotting to estimate the number of molecules ( in fmol ) of the parS359 locus in a given number of cells .","Genomic DNA from 52× cell culture equivalents ( 1 equivalent equals 0 . 025 ml*OD600 ) of wild type ( ‘WT’ , BSG1001 ) has been digested with the PstI restriction enzyme ( see panel C ) and loaded into the left lane .","DNA loaded into the second and third lane is derived from mixtures of wild-type and tagged ( ‘parB-GFP’ , BSG2058 ) cells .","Defined amounts of a purified PCR product covering the wild-type parS-359 locus have been spiked into cell extracts of the tagged strain .","Digested DNA was detected using a Southern probe specific for the parS-359 locus .","( B ) Quantitative Western blot to estimate the number of molecules of the Smc protein ( in fmol ) in a given number of cells .","Cultures were identical to the ones used in ( A ) .","Whole cell protein extract corresponding to 4 culture equivalents of wild-type cells ( ‘WT’ , BSG1001 ) was loaded into the left lane .","Other lanes were loaded with samples derived from wild-type and tagged cells or mixtures thereof with or without spiked-in purified Smc protein as indicated in the figure panel .","Smc protein was detected by immunoblotting using a polyclonal rabbit anti-Smc antibody .","( C ) Protein and DNA spike-in was performed in extracts derived from cells ( BSG2058 ) with modifications at the parS-359 locus ( blue filled circle ) and the smc gene ( purple filled circle ) .","PstI restriction sites ( in red ) in the parB gene ( light blue box ) and the neighbouring yyaC gene ( grey box ) generate a fragment of 1270 bp in size in case of the wild-type locus .","From parB-gfp cells ( BSG2058 ) , however , a bigger DNA fragment of 3147 bp in size is generated .","Strain BSG2058 also harboured a modified smc gene , which is fused to a halotag cassette ( pink box ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 015 Smc–ScpAB plays a crucial role in the segregation of replication origins in B . subtilis cells ( Gruber , 2014; Wang et al . , 2014 ) , presumably by organizing nascent sister chromosomes so that their spatial overlap and entanglement is minimized .","It is tempting to speculate that ParB/parS not only enriches Smc in the vicinity of the replication origin but also sets up lengthwise compaction of chromosomes by presenting DNA of a certain topology to the Smc–ScpAB complex .","Consistent with this notion , we found that several parB mutants , which are defective in the ability to form large nucleoprotein complexes and to spread from parS sites , fail to promote loading of Smc–ScpAB onto the chromosome .","Thus , Smc–ScpAB might capture , stabilize and expand structures–such as DNA loops or coils–that are pre-formed within larger ParB/parS nucleoprotein assemblies ( Figure 6A ) .","Alternatively , ParB/parS might serve as an elaborate landing platform on the chromosome , where Smc–ScpAB initiates with the help of ParB the lengthwise compaction of chromosomes by forming and extruding loops of DNA ( Figure 6B ) .","Extrusion of DNA might involve the translocation of Smc–ScpAB along DNA fibers made up of naked or ParB coated DNA .","These models are not mutually exclusive .","A reasonable first step towards understanding the architecture of Smc/ParB/parS assemblies might be the investigation of physical interactions between ParB/parS and Smc–ScpAB and their functional interconnection with the Smc ATPase cycle .","In the future , Smc/ParB/parS structures could serve as a relatively simple paradigm for chromosome organization by the more intricately regulated cohesin and condensin complexes in eukaryotes .","ParB serves a supplemental—albeit important—role in the Smc loading process .","In contrast , Smc functionality and its loading onto DNA in vivo is critically dependent on the ScpB subunit and the ATPase cycle .","It remains unclear though what the exact role of ScpB and ATP hydrolysis in the entrapment of DNA might be .","If loading indeed depends on transient opening of a DNA entry gate , the open state would likely represent an energetically unfavorable reaction intermediate ( Figure 6A ) .","Timely opening would require energy input as well as tight regulation .","We have recently demonstrated that the ATP dependent engagement of Smc head domains—together with DNA binding to the Smc hinge domain—can transform the configuration of the Smc coiled coil from a rod to a more open ring-like conformation ( Soh et al . , 2015 ) .","Hydrolysis of ATP and/or ScpB binding could drive a subsequent conformational change that might open the SMC–kleisin ring ( Figure 6A ) .","Alternatively , ScpB and/or ATP hydrolysis might stabilize Smc–ScpAB once loaded onto DNA or promote Smc's sliding along DNA to allow efficient extrusion of DNA loops by the Smc–ScpAB ring ( Figure 6B ) .","Deletion or mutation of the parB gene results in a clear drop in the levels of chromosomally bound condensin ( ∼5–10 fold less ) in our chromosome entrapment assay ( Figure 4B , Figure 2—figure supplement 2C ) .","In addition to this loading defect , also the specific recruitment of Smc–ScpAB towards the replication origin is lost in the absence of ParB ( Gruber and Errington , 2009; Sullivan et al . , 2009 ) .","Thus , in parB mutants only a very small proportion of cellular Smc–ScpAB is bound to chromosomes within the replication origin region , where it presumably performs its essential function by promoting the separation of nascent sister chromosomes ( Gruber et al . , 2014; Wang et al . , 2014 ) .","Nevertheless , defects in chromosome segregation are rather mild in parB mutants when compared to mutants of smc .","Using quantitative blotting of Smc protein and replication origin DNA from cell extracts , we have estimated the average number of Smc protein to be around 30 dimers per replication origin in a fast growing population of cells ( Figure 6—figure supplement 1 ) .","Assuming that all Smc complexes entrap chromosomal DNA in wild-type cells , only three to six Smc dimers ( 10–20% of total ) are loaded onto the chromosome in parB mutants according to our measurements .","Thus , a handful of Smc–ScpAB complexes , which are presumably randomly distributed over the chromosome , appears to be capable of supporting near-normal chromosome segregation under these conditions—when chromosome segregation is already compromised by the loss of the parABS system .","Few Smc–ScpAB therefore seem to be able to provide enough organization to the replication origin region and the remainder of the chromosome to prevent lethal accumulation of inter-linked sister chromosomes .","It is conceivable that individual Smc–ScpAB complexes are able to organize large chunks of a bacterial chromosome , possible by forming giant loops of DNA .","Alternatively , Smc activity might be needed only at a limited number of defined locations on the B . subtilis chromosome and/or for very short periods of time .","However , when levels of functional Smc dimers are in addition reduced for example by hypomorphic mutations in the smc gene itself , the loss of ParB protein becomes lethal ( Gruber and Errington , 2009 ) .","This work reveals the mode of association of Smc–ScpAB with bacterial chromosomes , highlights its striking evolutionary conservation and demonstrates the involvement of the SMC ATPase cycle in chromosomal loading .","Future work must address the underlying biochemical mechanisms to get basic insight into the architectural role of SMC in chromosome biology ."],["Genetic modifications at smc , scpAB , parB and dnaN loci were generated via double cross-over recombination in strains derived from B . subtilis 1A700 or B . subtilis 168ED .","Genotypes of strains used in this study are listed in Supplementary file 1 .","Cells were transformed with plasmids or B . subtilis genomic DNA using a 2-step starvation protocol as previously described ( Hamoen et al . , 2002; Bürmann et al . , 2013 ) .","Transformants were selected by growth on nutrient agar ( NA ) plates ( Oxoid , UK ) supplemented with antibiotics as required: 5 µg ml−1 kanamycin , 80 µg ml−1 spectinomycin , 10 µg ml−1 tetracycline , 5 µg ml−1 chloramphenicol , 1 µg ml−1 erythromycin and 25 µg ml−1 lincomycin .","Strains displaying a condensin null phenotype were selected on SMG medium instead: SMM salt solution ( 2 g l−1 ammonium sulphate , 14 g l−1 dipotassium hydrogen phosphate , 6 g l−1 potassium dihydrogen phosphate , 1 g l−1 trisodium citrate , 0 . 2 g l−1 magnesium sulphate , 6 g l−1 potassium hydrogen phosphate ) supplemented with 5 g l−1 glucose , 20 mg l−1 tryptophan and 1 g l−1 glutamate with the respective antibiotics .","Strains were single-colony purified and grown in the absence of antibiotics for experiments .","Cells were pre-grown in a 96-well plate in SMG medium for 24 hr at 37°C .","Overnight cultures were diluted 9^2-fold ( high density spots ) or 9^5-fold ( low density spots ) and spotted onto NA or SMG agar plates .","Plates were incubated at 37°C for 12 hr on NA or 24 hr on SMG agar .","Cells were grown in either LB Miller medium ( 10 g l−1 tryptone , 5 g l−1 yeast extract , 10 g l−1 sodium chloride ) or SMG medium to mid-exponential phase at 37°C ( in LB Miller medium , OD600 of 0 . 4; in SMG medium , OD600 of 0 . 03 ) .","Cells were harvested by centrifugation or vacuum filtration and washed in ice-cold PBS supplemented with 0 . 1% glycerol ( ‘PBSG’ ) .","Cell aliquots ( corresponding to 1 ml at an OD600 of 1 . 25 ) were re-suspended in PBSG and incubated with the cross-linker BMOE ( bis-maleimidoethane , Applichem , Germany ) at a concentration of 1 mM ( diluted from a 20 mM stock solution in DMSO ) .","After a 10 min incubation on ice the reaction was quenched by addition of 2-mercaptoethanol ( ‘2-ME’ ) to a final concentration of 28 mM .","For the preparation of protein extracts ( ‘input’ ) a mixture of following components was added to an aliquot of cells: 400 units ready-lyse lysozyme ( Epicentre , Madison , WI ) , 12 . 5 units benzonase ( Sigma-Aldrich , St . Louis , MO ) and a protease-inhibitor cocktail ( Sigma ) .","In addition , 1 µM HT Oregon Green substrate ( Promega , Madison , WI ) was added to cell suspensions with HaloTag bearing alleles .","Samples were then incubated for 20 min at 37°C protected from light .","Finally , the samples were heated to 70°C for 5 min in LDS Sample Buffer ( NuPage , Thermo Scientific , Waltham , MA ) containing 200 mM DTT and loaded onto a SDS-PAGE gel ( see below ) .","Gels with Oregon Green labeled samples were scanned on a Typhoon scanner ( GE Healthcare , UK ) with Cy3-DIGE filter setup .","Cells were grown , cross-linked and quenched as described above .","Lysozyme stock solution , protease inhibitor and HT substrate were added to an aliquot of cells at concentrations given above .","The cell suspension was mixed immediately in a 1:1 ratio with a 2% solution of Megabase agarose ( BioRad , Hercules , CA ) or low-melt agarose ( BioRad ) and casted into 100 µl agarose plugs using plug molds ( BioRad ) .","Agarose plugs were incubated for 20 min at 37°C , protected from light , and then loaded into the wells of a 6% SDS-PAGE Tris-glycine gel .","The polyacrylamide mini-gel was run for 60 min at 25 mA protected from light .","Agarose plugs were then re-extracted from the gel and transferred into 1 . 5 ml Eppendorf tubes .","1 ml of Wash Buffer ( ‘WB’: 0 . 01 mM EDTA , 0 . 5 mM Tris , 0 . 5 mM MgCl2 , 0 . 01% SDS ) was added per agarose plug .","Plugs were incubated for 10 min with gentle agitation protected from light .","This step was repeated once .","Wash buffer was then discarded and replaced by 100 µl fresh WB supplemented with 50 units of benzonase ( Sigma ) .","Plugs were incubated at 37°C for 30 min .","Plugs were melted at 85°C for 2 min under vigorous agitation .","The samples were frozen at −80°C and stored overnight .","Samples were then thawed , centrifuged for 10 min at 4°C and 14 , 000×g and transferred to a 0 . 45 µm Cellulose acetate spin column ( Costar , Tewksbury , MA ) and spun for 1 min at 10 , 000×g .","The flow-through was concentrated in a Speed Vac ( Thermo Scientific , no heating , 2 . 5 hr running time ) .","The concentrated sample was resuspended in LDS Sample Buffer ( NuPage ) containing 200 mM DTT and heated for 3 min at 70°C .","Samples were loaded onto Tris-acetate gels ( 3–8% Novex , Thermo Scientific ) and run for 2 . 5 hr at 35 mA per gel at 4°C .","For DnaN detection Bis-Tris gels ( 8–12% Novex ) were run for 1 hr at 200 V at room temperature .","Gels were either scanned on a Typhoon scanner ( FLA 9000 , GE Healthcare ) with Cy3-DIGE filter setup or immuno-blotted using antibodies against DnaN or Smc ( see below ) .","For cleavage of ScpA ( TEVs ) or degradation of chromosomal DNA 15 units of His-TEV protease or 12 . 5 units of benzonase , respectively , was added before casting agarose plugs .","B . subtilis strains containing smc-tev-avitag alleles were grown to OD600 of 0 . 4 in 100 ml LB Miller at 37°C .","Part of the culture was fixed with formaldehyde and subjected to chromatin immunoprecipitation ( ChIP ) as described by ( Gruber and Errington , 2009 ) using a rabbit anti-Smc antiserum .","In parallel , 10 ml of the culture were mixed with ice , harvested by centrifugation and washed in cold PBSG .","Cells were resuspended in 200 µl PBSG and treated with 0 . 5 mM BMOE for 10 min on ice .","The reaction was quenched with 14 mM 2-ME and cells were washed once in CutSmart buffer ( New England Biolabs , Ipswich , MA ) .","Cells were resuspended in 200 µl CutSmart containing 10 kU Ready-Lyse lysozyme ( Epicentre ) , 40 U XbaI ( New England Biolabs ) and a protease inhibitor cocktail ( Sigma ) .","The suspension was incubated for 15 min at 37°C before addition of 1800 µl buffer LS ( 10 mM Tris/HCl , 150 mM NH4OAc , 1 mM EDTA , 6 mM 2-ME , 0 . 05% Tween-20 , 0 . 01% NaN3 , final pH 7 . 9 at 23°C ) .","Lysates were centrifuged for 5 min at 20 , 000×g .","Subsequently , 1400 µl of the extract were incubated with 100 µl Dynabeads Streptavidin C1 for 30 min at room temperature .","Beads were washed once in buffer LS , then split , resuspended either in buffer LS or in buffer HS ( 10 mM Tris/HCl , 2 M NaCl , 1 mM EDTA , 6 mM 2-ME , 0 . 05% Tween-20 , 0 . 01% NaN3 , final pH 7 . 9 at 23°C ) and incubated for 15 min at room temperature .","Beads were washed twice with buffer LS , and protein/DNA complexes were eluted for 1 hr at 22°C by incubation with 350 µl LS containing TEV protease and 1 mM DTT .","DNA from input and eluate fractions was purified by treatment with 0 . 5 mg/ml Proteinase K for 1 hr at 55°C followed by phenol/chloroform extraction .","Samples were analysed by quantitative PCR using the second derivative maximum of a four parameter logistic model similar to the method described by ( Zhao and Fernald , 2005 ) .","After gels were scanned for in-gel fluorescence detection , they were immediately transferred onto a PVDF membrane ( Immobilon-P , Merck Millipore , Germany ) using semi-dry transfer .","Membranes were blocked with 3 . 5% ( wt/vol ) milk powder in PBS with 0 . 1% Tween 20 .","Rabbit polyclonal sera against B . subtilis DnaN ( Lenhart et al . , 2013 ) , B . subtilis Smc ( this paper ) and B . subtilis ParB ( this paper ) were used as primary antibodies for immunoblotting at dilutions of 1:5000 each .","The membrane was developed with HRP-coupled secondary antibodies and chemiluminescence ( SuperSignal West Femto , Thermo Scientific ) and visualized on a LAS-3000 scanner ( FujiFilm , Germany ) .","To estimate DnaN cross-linking kinetics ( Figure 1—figure supplement 1A ) samples were grown as described above .","An aliquot of cells was incubated with the cross-linker BMOE ( 1 mM ) for the indicated length of time before the reaction was quenched with 2-ME ( 28 mM ) ."]],"string":"[\n [\n \"Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division .\",\n \"Multi-subunit Structural Maintenance of Chromosomes ( SMC ) protein complexes—such as cohesin and condensin—are major determinants of chromosome structure and dynamics during the cell cycle in eukaryotes as well as in prokaryotes ( Hirano , 2006; Thadani et al . , 2012; Gruber , 2014 ) .\",\n \"Condensin subunits were initially identified as abundant , non-histone components of mitotic chromosomes in metazoans ( Hirano and Mitchison , 1994 ) .\",\n \"In mitosis , condensin localizes together with topoisomerase II in punctate structures to the longitudinal core of chromatids , called the chromosome axis ( Coelho et al . , 2003; Maeshima and Laemmli , 2003; Ono et al . , 2004 ) .\",\n \"Inactivation of condensin subunits by mutation or depletion results in severe morphological aberrations and mechanical sensitivity of metaphase chromosomes , and subsequently to defects in their segregation during anaphase ( Hirano and Mitchison , 1994; Ono et al . , 2003; Gerlich et al . , 2006 ) .\",\n \"In bacteria , Smc–ScpAB is the prevalent version of SMC protein complexes .\",\n \"Its distant relatives MksBEF and MukBEF can be found scarcely scattered over most of the bacterial phylogenetic tree and in isolated branches of proteobacteria , respectively ( Gruber , 2011 ) .\",\n \"In Bacillus subtilis and Streptococcus pneumoniae , Smc–ScpAB is recruited to a region around the replication origin by ParB/Spo0J protein bound to parS sites , thereby forming a discrete focus—also called condensation center—on each nascent copy of the chromosome ( Gruber and Errington , 2009; Sullivan et al . , 2009; Minnen et al . , 2011 ) .\",\n \"Inactivation of Smc–ScpAB in B . subtilis under nutrient rich growth conditions blocks separation of sister replication origins and consequentially leads to lethal defects in chromosome partitioning ( Gruber et al . , 2014; Wang et al . , 2014 ) .\",\n \"Smc–ScpAB thus promotes the initial stages of chromosome segregation in B . subtilis , likely by condensing and individualizing the emerging copies of the chromosome in preparation for their segregation to opposite halves of the cell .\",\n \"The canonical SMC complex in bacteria comprises five subunits: ( 1 ) two Smc proteins , which each form a 45 nm long antiparallel coiled coil that connects an ABC-type ATPase ‘head’ domain at one end of the coiled coil with a ‘hinge’ homodimerization domain at the other end ( Hirano et al . , 2001 ) , ( 2 ) a single ScpA subunit , which belongs to the kleisin family of proteins and associates via its C-terminal winged-helix domain ( WHD ) with the bottom ‘cap’ surface of one Smc head and via its N-terminal helical domain with the ‘neck’ coiled coil region of the other Smc protein ( Bürmann et al . , 2013 ) , and ( 3 ) a dimer of ScpB protein , which binds to the central region of ScpA ( Bürmann et al . , 2013; Kamada et al . , 2013 ) .\",\n \"Overall , the pentameric Smc–ScpAB complex displays a highly extended conformation harboring a central channel , which is surrounded by a closed tripartite ring formed by the Smc dimer and the ScpAB2 sub-complex .\",\n \"The B . subtilis Smc coiled coils associate with one another to form rod-shaped Smc dimers ( Soh et al . , 2015 ) .\",\n \"Furthermore , the Smc head domains can interact directly with one another—via a composite interface that includes two molecules of ATP .\",\n \"Binding to ATP , head engagement and ATP hydrolysis likely control and drive the biochemical action of Smc–ScpAB .\",\n \"Models for SMC condensation activity have been proposed based on observations made with isolated SMC dimers , SMC fragments or holo-complexes .\",\n \"Such protein preparations support the bridging of given DNA molecules in vitro as indicated by the re-annealing of single stranded DNA , intermolecular DNA ligation , DNA catenation and the co-purification of labeled and unlabeled DNA molecules ( Sutani and Yanagida , 1997; Losada and Hirano , 2001; Cui et al . , 2008 ) .\",\n \"Many SMC complexes bound to different segments of DNA might thus come together and anchor DNA in condensation centers or at the chromosome axis .\",\n \"Oligomeric assemblies of bacterial Smc proteins have indeed been observed by Atomic Force Microscopy and Electron Microscopy ( Mascarenhas et al . , 2005; Fuentes-Perez et al . , 2012 ) .\",\n \"This model provides a straightforward explanation for the compaction activity of SMC .\",\n \"However , it is unclear how such apparently indiscriminate DNA aggregation would promote rather than block the individualization of sister chromosomes ( Gruber , 2014 ) .\",\n \"Local wrapping of DNA around the SMC complex could result in well-defined lengthwise condensation of DNA .\",\n \"However , too little SMC protein appears to be present in chromosomes to yield decent levels of compaction by simple wrapping .\",\n \"A different hypothesis is based on the finding that the structurally related cohesin complex holds sister chromatids in eukaryotes together by entrapping sister DNA fibers within its ring ( Gruber et al . , 2003; Gligoris et al . , 2014 ) .\",\n \"Accordingly , individual SMC complexes might entrap and expand loops of DNA , thereby driving lengthwise condensation of chromosomes with little limitations in the attainable levels of compaction ( Nasmyth , 2001; Alipour and Marko , 2012 ) .\",\n \"Here , we investigate how the prokaryotic SMC–kleisin complex binds to chromosomes in vivo using a novel whole-chromosome assay .\"\n ],\n [\n \"We initially attempted to detect topological interactions between B . subtilis Smc–ScpAB and plasmid DNA using pull-down assays as previously described ( Ivanov and Nasmyth , 2005; Ghosh et al . , 2009; Cuylen et al . , 2011 ) .\",\n \"However , several attempts failed to provide clear evidence for entrapment of small circular DNA by prokaryotic condensin .\",\n \"Conceivably , Smc–ScpAB does not interact with these artificial substrates in a physiological manner .\",\n \"To circumvent this possibility , we established an inverse assay by immobilizing whole chromosomes of B . subtilis in agarose plugs and monitoring their association with covalently closed rings of Smc–ScpA under harsh protein denaturing conditions ( Figure 1A ) .\",\n \"To develop the chromosome entrapment assay we first performed experiments with the replicative sliding clamp , DnaN , in B . subtilis , which is known to entrap DNA in a topological manner .\",\n \"Furthermore , most of cellular DnaN protein is maintained in the vicinity of active replication forks in B . subtilis , presumably by its topological association with leading and lagging strand DNA ( Su'etsugu and Errington , 2011 ) . 10 . 7554/eLife . 06659 . 003Figure 1 . Development of the chromosome entrapment assay using DnaN .\",\n \"( A ) Scheme for the chromosome entrapment assay .\",\n \"Cells are incubated with the cysteine cross-linker BMOE , lysed in agarose plugs and subjected to an electric field in the presence of SDS buffer .\",\n \"Proteins stably bound to chromosomal DNA are re-isolated from nuclease treated agarose plugs , concentrated and analyzed by SDS-PAGE .\",\n \"( B ) Crystal structure of S . pneumoniae DnaN ( PDB: 3D1F ) in surface representation .\",\n \"The monomers of DnaN are shown in dark and light blue colours , respectively .\",\n \"The positions of an engineered pair of cysteine residues ( N114C and V313C ) at the monomer–monomer interface of B . subtilis DnaN are indicated by arrows .\",\n \"( C ) Chromosome entrapment by DnaN .\",\n \"Cells of strains BSG1449 ( dnaN-HT ) and BSG1459 ( dnaN ( N114C , V313C ) -HT ) were cross-linked with BMOE and subjected to the chromosome entrapment assay .\",\n \"Input and eluate fractions were analysed by in-gel detection of fluorescently labeled HT fused to DnaN ( top panel ) .\",\n \"Eluate fractions of samples treated with or without nuclease during cell lysis are indicated as nuclease ‘+’ or ‘−’ , respectively .\",\n \"Eluate fractions were further analyzed by silver staining revealing that another protein was consistently co-isolated during the chromosome entrapment assay ( bottom panel ) .\",\n \"This protein—identified as flagellin by mass spectrometry—was retained independently of the integrity of the chromosome .\",\n \"The following figure supplement is available: Figure 1—figure supplement 1: DNA entrapment by DnaN . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00310 . 7554/eLife . 06659 . 004Figure 1—figure supplement 1 . DNA entrapment by DnaN .\",\n \"( A ) Cross-linking time course of DnaN-HT .\",\n \"Cells of strain BSG1459 were incubated with the cross-linker BMOE and quenched at the indicated times .\",\n \"Extracts were analysed by in-gel fluorescence detection of DnaN-HT .\",\n \"At later time points the double cross-linked DnaN species ( ‘XX-DnaN-HT’ ) is enriched over non-cross-linked and single cross-linked DnaN-HT .\",\n \"( B ) Cartoon represenation of the DnaN-HT construct based on the crystal structure of S . pneumoniae DnaN ( PDB: 3D1F ) and Rhodococcus Haloalkane dehalogenase ( PDB: 1BN6 ) .\",\n \"The structure of the DnaN dimer ( monomers in green and blue colours , respectively ) was juxtaposed to the structure of the Haloalkane dehalogenase linked by a flexible peptide .\",\n \"The binding pockets for HT ligand binding are indicated by orange arrows .\",\n \"The positions of the engineered pair of cysteine residues ( N114C and V313C ) at the DnaN–DnaN interface are indicated by blue and green arrows .\",\n \"( C ) Chromosome entrapment assay of strain BSG1459 ( dnaN ( N114C , V313C ) -HT ) in presence or absence of the cross-linker BMOE .\",\n \"( D ) Effect of benzonase ( ‘nuclease’ ) on protein extracts of strains BSG1001 , BSG1449 and BSG1459 .\",\n \"Cell extracts were analysed by in-gel fluorescence detection ( upper panel ) of DnaN-HT after long-time ( ‘80 min’ ) or short-time ( ‘10 min’ ) incubation with benzonase .\",\n \"Lower panel shows Coomassie staining of the same SDS-PAGE gel to control for equal protein extraction efficiency . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 004 Based on the crystal structure of S . pneumoniae DnaN we engineered a pair of cysteine residues ( N114C , V313C ) into the B . subtilis protein so that DnaN can be cross-linked into covalent rings in the presence of a cysteine-specific cross-linker such as BMOE ( Figure 1B ) .\",\n \"For detection a cys-less variant of the HaloTag ( ‘HT’ ) was fused to the C-terminus of DnaN ( Figure 1—figure supplement 1B ) and the construct was integrated into the genome of B . subtilis via allelic replacement at the endogenous locus .\",\n \"The dnaN-ht genes with and without cysteine mutations supported normal growth of B . subtilis , implying that they encoded functional DnaN proteins ( data not shown ) .\",\n \"In vivo cross-linking of DnaN-HT resulted in two additional , slow migrating bands in SDS-PAGE gels ( Figure 1C ) , corresponding to single and double cross-linked species of DnaN dimers , designated as X-DnaN-HT and XX-DnaN-HT , respectively ( Figure 1—figure supplement 1A ) .\",\n \"We next embedded cells in agarose plugs and disrupted their cell walls by lysozyme digestion .\",\n \"Agarose plugs were then subjected to an electric field in the presence of SDS to denature and remove any unattached proteins from chromosomes .\",\n \"Plugs were finally treated with benzonase to digest genomic DNA and to release any stably entrapped protein .\",\n \"DnaN-HT protein was then analysed by in-gel fluorescence .\",\n \"Non-crosslinkable DnaN-HT was efficiently depleted from agarose plugs during the entrapment assay ( Figure 1C ) .\",\n \"In contrast , the double cross-linked , circular form of DnaN ( N114C , V313C ) -HT ( XX-DnaN-HT ) was retained in the agarose plug during electrophoresis with high efficiency ( ∼50% of input ) .\",\n \"A minor fraction of single cross-linked DnaN dimer ( X-DnaN-HT ) was also observed .\",\n \"This is likely generated from XX-DnaN-HT by spontaneous hydrolysis of thiol-malemide adducts during protein isolation ( Kalia and Raines , 2007; Baldwin and Kiick , 2013 ) .\",\n \"Importantly , the presence of benzonase during cell lysis eliminated all DnaN from the plug , indicating that circular DnaN is retained in plugs via its interaction with cellular DNA .\",\n \"Furthermore , in the absence of the cross-linker BMOE , no DnaN-HT was detected in the eluate fraction ( Figure 1—figure supplement 1C ) .\",\n \"The chromosome entrapment assay thus specifically detects a topological association of intact chromosomes with DNA sliding clamps and confirms that a major fraction ( at least 50% ) of DnaN is loaded onto DNA in rapidly growing cells .\",\n \"Next , we used the newly developed chromosome entrapment assay to test for an association between native chromosomes and Smc–ScpAB complexes .\",\n \"Cysteine pairs were introduced at the Smc–Smc and at both Smc–ScpA interfaces and a HT was fused at the C-terminus of Smc to allow in-gel fluorescence detection ( Figure 2A ) ( Bürmann et al . , 2013 ) .\",\n \"Strains bearing the cysteine mutations and the Smc-HaloTag fusion supported normal growth on nutrient rich medium demonstrating the functionality of the modified Smc complex ( Figure 2—figure supplement 1A ) .\",\n \"Cells were treated with BMOE and extracts were analysed by SDS-PAGE .\",\n \"As internal control for the chromosome entrapment assay we employed the DnaN ( N114C , V313C ) protein , whose double cross-linked form was detected in input and eluate samples by immunoblotting ( Figure 2—figure supplement 1B ) .\",\n \"Various species of Smc–ScpAB were identified in extracts of BMOE cross-linked cells by in-gel fluorescence .\",\n \"These correspond to fully cross-linked Smc–ScpA–Smc rings and several intermediate cross-linking species as reported previously ( Figure 2B , C ) ( Bürmann et al . , 2013 ) .\",\n \"To reveal the identity of all species , strains lacking one of six engineered cysteines were used as controls that collectively form several intermediate cross-linked species but no fully cross-linked rings of Smc–ScpAB ( Figure 2—figure supplement 1C ) .\",\n \"In these control samples little or no Smc-HT protein was retained in agarose plugs under denaturing conditions as expected for any non-circular protein ( Figure 2B ) .\",\n \"In the presence of all pairs of cysteine , however , a set of two closely migrating species was consistently detected at significant levels after the chromosome entrapment assay ( ∼10–20% of input material ) ( Figure 2B ) .\",\n \"We argued that the two closely migrating species might correspond to Smc–ScpAB with a single or a double cross-link at the Smc hinge .\",\n \"Consistent with this notion we find that only a single species of Smc2–ScpA accumulated during the chromosome entrapment assay when a single cysteine residue ( R643C ) was used to cross-link the Smc hinge domains ( Figure 2—figure supplement 2A ) .\",\n \"These findings strongly suggest that Smc–ScpAB is bound to chromosomes via entrapment of chromosomal DNA .\",\n \"If this were indeed the case , then its retention in agarose plugs should depend on the integrity of Smc–ScpAB rings and of chromosomal DNA .\",\n \"Incubation of agarose plugs with the nuclease benzonase during cell lysis eliminated the Smc-HT and DnaN signal in the sample ( Figure 2B , Figure 2—figure supplement 1B ) .\",\n \"To disrupt covalent Smc–ScpAB rings , we inserted cleavage sites for TEV protease into the linker region preceding the C-terminal WHD of ScpA and incubated cells during lysozyme treatment with recombinant TEV protease to open any circular Smc2–ScpA species .\",\n \"As expected , little or no Smc-HT signal was detected in agarose plugs after TEV cleavage of ScpA ( Figure 2D ) .\",\n \"To exclude any artefacts due to the presence of the HT on Smc we have repeated the chromosome entrapment assay with an untagged allele of Smc using immunoblotting with anti-Smc antibodies for the detection of cross-linked species , which yielded very similar results ( Figure 2—figure supplement 2C ) .\",\n \"Furthermore , we found that Smc2-ScpA rings are stably trapped in agarose plugs over extended periods of time in constant or alternating electric fields ( data not shown ) .\",\n \"Thus , our chromosome entrapment assay specifically detects the association between intact chromosomal DNA and rings of Smc–ScpAB in B . subtilis , demonstrating that DNA fibers pass through the Smc ring . 10 . 7554/eLife . 06659 . 005Figure 2 . Prokaryotic condensin entraps the chromosome .\",\n \"( A ) Scheme for the cross-linking of Smc-HaloTag ( ‘HT’ ) and ScpA into a covalent Smc–ScpA–Smc ring .\",\n \"( B ) Chromosome entrapment of covalent Smc2–ScpA rings .\",\n \"Cells of strains BSG1782 , BSG1809-1813 and BSG1831 were cross-linked and subjected to the chromosome entrapment assay .\",\n \"Cross-linked Smc-HT species were visualized by in-gel fluorescence detection .\",\n \"The presence or absence of cysteine pairs at each of the three ring interfaces are indicated by ‘+’ and ‘−’ , respectively .\",\n \"An aliquot of cells of strains BSG1782 was incubated with benzonase during cell lysis ( nuclease ‘+’ ) .\",\n \"The positions of uncross-linked Smc-HT and fully cross-linked , circular Smc–ScpA–Smc species are indicated by ‘Smc-HT’ and ‘Smc-HT circ . ’; all species are labelled by colour-coded arrowheads ( see panel C for legend ) .\",\n \"Circular species ( ‘h’ ) are labeled by a double pointed arrowhead .\",\n \"( C ) Schematic depiction of the structure of cross-linked Smc–ScpA species ( ‘a’–‘i’ ) .\",\n \"( D ) TEV cleavage of ScpA prevents entrapment of Smc–ScpAB in agarose plugs .\",\n \"In-gel fluorescence detection of Smc-HT derived from strains BSG1807 and BSG1832 .\",\n \"The presence or absence of TEV sites in ScpA and of TEV protease during cell lysis is indicated by ‘+’ and ‘−’ , respectively .\",\n \"Cleavage of ScpA ( TEVs ) by TEV protease creates new species of cross-linked Smc-HT ( see ‘input’ samples ) and prevents entrapment of Smc-HT in agarose plugs ( see ‘eluate fraction’ ) ( top panel ) .\",\n \"‘XX-DnaN’ serves as internal assay control visualized by immunoblotting of cross-linked species of DnaN protein ( bottom panel ) .\",\n \"The following figure supplement is available: Figure 2—figure supplement 1: DNA entrapment by wild-type Smc–ScpAB ( I ) and Figure 2—figure supplement 2: DNA entrapment by wild-type Smc–ScpAB ( II ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00510 . 7554/eLife . 06659 . 006Figure 2—figure supplement 1 . DNA entrapment by wild-type Smc–ScpAB ( I ) .\",\n \"( A ) Colony formation assays of strains BSG1002 , BSG1007 , BSG1782 , 1809–1813 and BSG1831 were performed on minimal medium agar ( SMG ) or nutrient agar ( NA ) .\",\n \"Cultures were diluted 9^2-fold ( top row ) and 9^5-fold ( bottom row ) and grown at 37°C for 24 hr ( SMG ) and 14 hr ( NA ) , respectively .\",\n \"The presence and absence of cysteine pairs for cross-linking of the hinge , cap and neck interface are indicated by ‘+’ and ‘−’ , respectively .\",\n \"( B ) Immunoblot of SDS-PAGE gels shown in Figure 2B using anti-DnaN antibody .\",\n \"All strains carry cysteine mutations for DnaN cross-linking .\",\n \"Doubly cross-linked DnaN ( ‘XX-DnaN’ ) serves as internal assay control as visualized by immunoblotting .\",\n \"( C ) Identification of cross-linked species of Smc-HT and ScpA .\",\n \"The gel image is identical to Figure 2A with higher contrast settings to visualize low-intensity species .\",\n \"All cross-linked species are labelled ( with a colour-coded letter ) ( ‘a’–‘i’ ) .\",\n \"Circular species ( ‘h’ ) are labeled by a double pointed arrowhead .\",\n \"( D ) Same as Figure 2 , panel C . Schematic depiction of the structure of cross-linked Smc–ScpA species ( ‘a’–‘i’ ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00610 . 7554/eLife . 06659 . 007Figure 2—figure supplement 2 . DNA entrapment by wild-type Smc–ScpAB ( II ) .\",\n \"( A ) Cross-linking of the Smc hinge interface with different cysteine residues .\",\n \"Cross-linking of the Smc hinge using cysteine residue ( ‘R643C’ ) instead of the ‘R558C’ and ‘N643C’ cysteine pair results in different migration pattern of cross-linked Smc-ScpA species ( see input fractions ) .\",\n \"In this case species ‘g’ and ‘i’ have the same migration behavior as the circular form ‘h’ .\",\n \"Only a single species of Smc-ScpA is retained in the agarose plug after the entrapment assay ( see eluate fractions ) .\",\n \"( B ) Colony formation assay of strains BSG1002 , BSG1007 , BSG1807 and BSG1832 were performed as described before .\",\n \"The smc and scpA loci lacking mutations ( ‘WT’ ) , harbouring cysteine mutations for cross-linking alone ( ‘Cys’ ) or in combination with a TEV cleavage site ( ‘Cys-TEV site’ ) are denoted .\",\n \"( C ) Chromosome entrapment of Smc–ScpAB lacking fusion tags ( see scheme on the left ) .\",\n \"The right panel shows immunoblotting of input and eluate fractions using an anti-Smc antibody .\",\n \"All strains harbour cysteine residues for crosslinking of Smc-ScpA in an otherwise wild-type background ( ‘WT’ ) in BSG680 , combined with a deletion of the parB gene ( ‘ΔparB’ ) in BSG1991 or with the E1118Q mutation at the smc locus in BSG1995 .\",\n \"The position of fully cross-linked , circular Smc2-ScpA species is indicated by double pointed arrowhead labelled ‘h’ ( see Figure 2—figure supplement 1C ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 007 Next , we established the requirements for the formation of interconnections between Smc–ScpAB rings and chromosomes .\",\n \"The intrinsic ATPase activity of cohesin has previously been implicated in stable association with chromosomes ( Arumugam et al . , 2003; Weitzer et al . , 2003 ) .\",\n \"More specifically , ATP hydrolysis has been hypothesized to transiently open an entry gate for DNA in the cohesin ring during its loading onto chromosomes ( Gruber et al . , 2006; Hu et al . , 2011 ) .\",\n \"To test what steps of the ATP hydrolysis cycle in Smc–ScpAB are involved in the entrapment of chromosomal DNA , we made use of smc alleles harboring mutations that specifically prevent ATP binding ( K37I ) , engagement of Smc head domains ( S1090R ) or ATP hydrolysis ( E1118Q ) ( Figure 3A ) ( Hirano and Hirano , 2004 ) .\",\n \"The three mutant proteins are expressed at normal levels in B . subtilis being indicative of proper protein folding ( Figure 3—figure supplement 1A ) .\",\n \"However , they do not support growth on nutrient rich medium similar to smc null mutants , implying that all steps of the ATPase cycle are essential for Smc functionality ( Figure 3—figure supplement 1B ) ( Gruber et al . , 2014 ) .\",\n \"For the chromosome entrapment assay , these Smc ATPase mutations were combined with cysteine mutations for BMOE cross-linking .\",\n \"To support their viability , the resulting strains as well as the wild-type controls were grown in minimal medium .\",\n \"The three mutant Smc proteins assembled into normal Smc–ScpAB complexes as judged by Smc–ScpA cross-linking , albeit there is a slight decrease in the fraction of ScpA proteins bridging Smc dimers and a concomitant minor increase in ScpA subunits bound to single Smc proteins ( Figure 3—figure supplement 1C , species ‘e’ and ‘d’ , respectively ) ( Bürmann et al . , 2013 ) .\",\n \"Intriguingly , the ATP binding and engagement mutants abolished the fraction of covalent ring species retained in the agarose plug during the chromosome entrapment assay ( Figure 3B ) .\",\n \"In case of the ATP hydrolysis mutant Smc ( E1118Q ) only minute amounts of cross-linked rings were recovered from SDS treated plugs .\",\n \"This small fraction of stably bound condensin conceivably arises as a consequence of residual levels of ATP hydrolysis activity in Smc ( E1118Q ) ( Hirano and Hirano , 2004 ) .\",\n \"Thus , ATP binding and ATP dependent Smc head engagement—and most probably also ATP hydrolysis—are essential for entrapment of chromosomal DNA by condensin in bacteria , as has been supposed for cohesin in yeast .\",\n \"Furthermore , the strict requirement of several steps of the ATPase cycle strongly suggests that entrapment of DNA corresponds to the physiological form of association with the bacterial chromosome . 10 . 7554/eLife . 06659 . 008Figure 3 . The Smc ATPase is required for loading of DNA into Smc–ScpAB .\",\n \"( A ) A scheme for the ATP hydrolysis cycle of Smc .\",\n \"Schematic positions for Walker A , Walker B and ABC-signature motifs on the Smc head domain are shown ( top row ) .\",\n \"ATP binding to the Walker A domain is blocked in Smc ( K37I ) ‘ ( 1 ) ’ .\",\n \"ATP-dependent engagement of two Smc heads is abolished in the Smc ( S1090R ) mutant ‘ ( 2 ) ’ .\",\n \"The E1118Q mutation strongly reduces ATP hydrolysis ‘ ( 3 ) ’ .\",\n \"( B ) Smc ATPase mutations abolish chromosomal loading of Smc–ScpAB .\",\n \"In-gel fluorescence detection of Smc-HT of input and eluate fractions from a representative chromosome entrapment assay performed with strains BSG1782 and BSG1784-6 .\",\n \"Protein extracts ( 10% of input ) were loaded next to samples subjected to the entrapment assay .\",\n \"Selected cross-linked species of Smc-HT are labeled ( top panel ) .\",\n \"Detection of cross-linked species of DnaN by immunoblotting was used as internal assay control ( bottom panel ) .\",\n \"The following figure supplement is available: Figure 3—figure supplement 1: ATPase mutants of Smc–ScpAB . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 00810 . 7554/eLife . 06659 . 009Figure 3—figure supplement 1 . ATPase mutants of Smc-ScpAB .\",\n \"( A ) Upper panel shows Smc expression levels of strains with mutant smc alleles ( BSG1002 , 1007 , 1008 , 1045 , 1046 , 1047 and 1074 ) determined by immunoblotting using anti-Smc antibodies .\",\n \"Lower panel shows Coomassie staining of a SDS-PAGE gel loaded with the same whole cell extract samples to control for equal protein extraction efficiency .\",\n \"( B ) Overnight cultures of strains ( BSG1002 , 1007 , 1008 , 1045 , 1046 , 1047 ) were spotted on SMG and NA as described before ( see Figure 2—figure supplement 1 ) .\",\n \"Smc locus was without mutation ( ‘WT’ ) , a smc deletion ( ‘Δsmc’ ) or mutations in the Smc ATPase domain .\",\n \"( C ) Smc ATPase mutations show slightly decreased levels of cross-linking species ‘e’ and increased levels of ‘d’ .\",\n \"Image is identical to Figure 3B , with higher contrast . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 009 What other factors might be required for the loading of condensin onto DNA ?\",\n \"The ScpB subunit forms homodimers that bind in an asymmetric manner to the central region of a single ScpA monomer .\",\n \"It thus is in close proximity of the Smc ATPase domains .\",\n \"Together with ScpA it putatively plays a role in the regulation of the Smc ATPase activity ( Kamada et al . , 2013 ) .\",\n \"Its precise molecular function , however , is not clear yet .\",\n \"To test whether ScpB is involved in the association of Smc–ScpA rings with chromosomes we combined the cysteine mutations in Smc and ScpA with an scpB in-frame deletion ( Figure 4—figure supplement 1 ) .\",\n \"Ring formation was only mildly affected by the absence of ScpB as judged by BMOE cross-linking and in-gel fluorescence detection ( Figure 4A ) ( Bürmann et al . , 2013 ) .\",\n \"However , Smc complexes lacking ScpB subunits failed to entrap chromosomes altogether demonstrating that ScpB is absolutely required for loading of prokaryotic condensin onto chromosomal DNA . 10 . 7554/eLife . 06659 . 010Figure 4 . ScpB and ParB are essential for efficient DNA entrapment by Smc complexes .\",\n \"( A ) Deletion of scpB eliminates loading of chromosomal DNA into Smc complexes .\",\n \"In-gel fluorescence detection of Smc-HT in input and eluate fractions is shown from chromosome entrapment assays performed with strains BSG1782 ( ‘WT’ ) and BSG1850 ( ‘ΔscpB’ ) ( top panel ) .\",\n \"DnaN was used as internal control ( bottom panel ) .\",\n \"( B ) Several parB mutations interfere with efficient chromosomal loading of Smc–ScpAB .\",\n \"Input and eluate fractions from chromosome entrapment assays with strains BSG1782 , BSG1783 and BSG1960-3 were analysed by in-gel fluorescence detection of Smc-HT ( top panel ) .\",\n \"DnaN was used as internal control ( middle panel ) .\",\n \"Immunoblotting using polyclonal rabbit anti-ParB antiserum confirms near-normal expression of mutant ParB proteins ( bottom panel ) .\",\n \"The following figure supplement is available: Figure 4—figure supplement 1: Growth of smc , parB double mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01010 . 7554/eLife . 06659 . 011Figure 4—figure supplement 1 . Growth of smc ( Cys ) mutants . Overnight cultures of strains ( BSG1002 , BSG1007 , BSG1782 , BSG1850 and BSG1783 ) were serially diluted and spotted as described before ( Figure 2—figure supplement 1 ) .\",\n \"Strains were wild-type ( ‘WT’ ) , harboured the smc deletion ( ‘Δ’ ) or cysteine mutations for cross-linking the Smc-ScpA ring ( ‘Cys’ ) .\",\n \"In addition , scpB ( ‘ΔscpB’ ) or parB ( ‘ΔparB’ ) were deleted where indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 011 ParB proteins—bound to parS sites—are crucial for efficient targeting of Smc–ScpAB to a large region of the chromosome near the replication origin ( Gruber and Errington , 2009; Sullivan et al . , 2009; Minnen et al . , 2011 ) .\",\n \"ParB might act by simply increasing the local concentration of Smc–ScpAB around oriC either before or after its loading onto the chromosome .\",\n \"Alternatively , ParB bound to parS sites might be more directly involved in the loading reaction itself , for example , as catalytic factor , and its absence might thus affect levels of chromosomal condensin .\",\n \"To test this , we performed the chromosome entrapment assay with cells lacking the parB gene .\",\n \"Intriguingly , the levels of Smc–ScpAB entrapping chromosomal DNA were strongly reduced in the parB null mutant as judged by the limited retention of Smc–ScpA species in agarose plugs ( Figure 4B ) .\",\n \"Thus , ParB protein likely promotes the entrapment of chromosomal DNA by Smc–ScpAB .\",\n \"This strongly suggests that most condensin is loaded onto the chromosome at parS sites , where ParB protein is bound .\",\n \"In all other parts of the chromosome entrapment of DNA fibers by Smc–ScpAB might be very inefficient .\",\n \"The cysteine bearing smc allele causes growth defects when combined with ΔparB ( Figure 4—figure supplement 1 ) .\",\n \"Therefore , we cannot formally exclude the possibility that the decreased loading of Smc observed in ΔparB are due to the cysteine modifications in Smc and that chromosomal loading of wild-type Smc is not or much less affected by parB deletion .\",\n \"Previously , two parB point mutations ( N112S and R149G ) , which prevent the formation of Smc-GFP foci , have been isolated in B . subtilis ( Gruber and Errington , 2009 ) .\",\n \"We found that these mutations strongly impair loading of Smc onto the chromosome in the entrapment assay similar to ΔparB ( Figure 4B ) .\",\n \"The R149G mutation is positioned on the helix-turn-helix motif of ParB and might thus directly affect binding to parS sites ( Leonard et al . , 2004 ) .\",\n \"The N112S mutation , however , is located in another highly conserved region , which has been implicated in the ‘spreading’ of ParB protein from parS sequences into adjacent DNA ( Leonard et al . , 2004; Graham et al . , 2014 ) .\",\n \"The spreading of ParB along several kb of DNA is a feature conserved in plasmid and chromosome derived ParB proteins , however , the underlying mechanism is only poorly understood ( Rodionov et al . , 1999 ) .\",\n \"It might possibly involve the formation of a large nucleoprotein complex ( Broedersz et al . , 2014 ) .\",\n \"Several other mutants of ParB ( including B . subtilis ParB G77S and R80A ) have been reported to be defective in spreading from parS sites ( Breier and Grossman , 2007; Graham et al . , 2014 ) .\",\n \"Intriguingly , also these mutations resulted in largely reduced levels of Smc on the chromosome in our entrapment assay , being comparable to the levels found in a parB deletion mutant ( Figure 4B ) .\",\n \"This implies that ParB spreading from parS sites or formation of large nucleoprotein complexes might be essential for loading of DNA into the Smc ring by ParB .\",\n \"These findings are consistent with the observation that formation of Smc-GFP foci near the origin of replication are affected by the G77S mutation ( Sullivan et al . , 2009 ) .\",\n \"In summary , these results demonstrate that several factors—including ScpB protein , a ParB/parS nucleoprotein complex and the Smc ATPase cycle—are required to promote efficient loading of condensin rings onto the chromosome .\",\n \"Smc proteins and fragments thereof exhibit affinity for single- and double-stranded DNA in vitro ( Chiu et al . , 2004; Hirano and Hirano , 2006; Soh et al . , 2015 ) .\",\n \"The physical contacts with DNA might occur once condensin has been successfully loaded onto chromosomes and thus be a permanent feature of chromosomal Smc–ScpAB .\",\n \"Alternatively , the direct association with DNA might be restricted to certain intermediates in the chromosomal loading reaction .\",\n \"To test for interactions between Smc–ScpAB and specific chromosomal DNA fragments , we have affinity-purified endogenous Smc–ScpAB from B . subtilis cell lysates using a short Avitag peptide fused to the C-terminus of the Smc protein , which gets biotinylated when the biotin ligase gene birA is co-expressed ( ‘Smc-Avitag’ ) .\",\n \"We then examined fractions for the co-purification of fragments of chromosomal DNA—generated by restriction digest with XbaI—using quantitative PCR with primer pairs specific for different parts of the chromosome .\",\n \"Since we worried that Smc–ScpAB might not be sufficiently stable in diluted cell extracts , we cross-linked the three ring interfaces in Smc–ScpAB using BMOE cross-linking of engineered pairs of cysteines .\",\n \"A small fraction of chromosomal DNA was reproducibly co-purified with wild-type Smc-Avitag , whereas the yield of co-purified DNA was significantly improved by the presence of cross-linkable cysteine residues in Smc–ScpAB ( Figure 5 ) .\",\n \"In both cases origin-proximal regions ( yyaD , parS-359 , dnaA and dnaN ) of the chromosome were more efficiently enriched than distal regions ( amyE , trnS and ter ) by the co-purification with Smc implying that the observed Smc-DNA contacts are dependent on chromosomal loading of Smc–ScpAB by ParB protein at parS sites and are thus physiologically relevant ( Figure 5 , Figure 5—figure supplement 1 ) ( Gruber and Errington , 2009 ) .\",\n \"The association of DNA with wild-type and BMOE cross-linked Smc–ScpAB was highly sensitive to washes with a salt solution ( 2M NaCl ) , suggesting that it was dependent on electrostatic contacts between DNA and protein .\",\n \"These DNA contacts are presumably formed by the Smc–ScpAB complex itself .\",\n \"Alternatively , albeit less likely , other chromosomal proteins physically bound to DNA could prevent the release of condensin from DNA by blocking its sliding towards DNA ends . 10 . 7554/eLife . 06659 . 012Figure 5 . A physical interaction of Smc–ScpAB rings and chromosomal DNA . Co-purification of chromosomal DNA fragments with native Smc–ScpAB .\",\n \"Cells of strains BSG1104-5 and BSG1107-8 were treated with the cross-linker BMOE prior to cell lysis .\",\n \"Strains carrying ( ‘+’ ) or lacking ( ‘−’ ) cysteine mutations ( ‘6xCys’ ) in the Smc-AviTag construct were expressed in presence ( ‘+’ ) or absence ( ‘−’ ) of the biotin ligase ( ‘BirA’ ) .\",\n \"Beads were washed in the presence of either a 150 mM ammonium acetate buffer ( ‘low salt’ ) or a 2 M sodium chloride buffer ( ‘high salt’ ) .\",\n \"The co-purification of DNA fragments with Smc-biotin on streptavidin beads was measured by quantitative PCR using primer pairs specific for genomic positions indicated on a representation of the circular B . subtilis genome .\",\n \"Mean values and standard deviations were calculated from two independent biological replicates .\",\n \"The following figure supplement is available: Figure 5—figure supplement 1: Chromatin immuno-precipitation of Smc . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01210 . 7554/eLife . 06659 . 013Figure 5—figure supplement 1 . Chromatin immuno-precipitation of Smc . All Smc variants used in Figure 5 behave similarly in standard ChIP experiments using anti-Smc antibodies .\",\n \"Cells of strains BSG1104-5 and 1107-8 were grown to mid-exponential phase and subjected to chromatin immunoprecipitation ( ChIP ) using polyclonal rabbit anti-Smc serum .\",\n \"Input and eluate DNA samples were analysed by quantitative PCR using specific primer pairs for the genomic positions indicated on a circular representation of the B . subtilis genome .\",\n \"Pull-down efficiency ( ChIP-DNA/input-DNA *100% ) was plotted for each primer pair .\",\n \"The lack and presence of the BirA biotin ligase or cysteines for cross-linking of Smc and ScpA proteins are denoted as ‘BirA−’ and ‘BirA+’ or ‘6xCys−’ and ‘6xCys+’ , respectively .\",\n \"It is noted that the highly transcribed tRNA locus , trnS , is highly enriched by Smc-Avitag using standard ChIP ( but not native ChIP , see Figure 5 ) , being consistent with the notion that enrichment of highly transcribed genes by ChIP is prone to artefacts due to non-uniform formaldehyde cross-linking . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 013\"\n ],\n [\n \"In many cases , it is challenging to measure the activity and outcome of biochemical processes in the living cell .\",\n \"Here , we report the establishment of a straight-forward method to determine the physical association of ring-shaped protein complexes with whole bacterial chromosomes .\",\n \"Two examples , the SMC condensin complex and the sliding clamp DnaN , document the significant potential of our simple entrapment approach .\",\n \"In principle , similar assays should also be possible with eukaryotic cells and for many other chromosomal proteins such as for example hexameric helicases and certain transcription factors .\",\n \"Furthermore , analogous procedures might be useful to address biological questions related to other denaturation-resistant cellular structures such as cell wall polymers ( e . g . , made up of peptidoglycan , chitin or cellulose ) .\",\n \"SMC–kleisin complexes are major governors of chromosome superstructure in most branches of the phylogenetic tree .\",\n \"The eukaryotic variants cohesin and condensin have been suggested to work as concatenases , which hold selected stretches of DNA together by simple embracement in their ring ( Haering et al . , 2008; Cuylen et al . , 2011 ) .\",\n \"Whether DNA entrapment is an ancestral and thus fundamentally conserved function of SMC–kleisin complexes , however , remained elusive so far .\",\n \"Furthermore , interaction studies developed for cohesin and condensin are based on small , artificial DNA substrates and might thus not necessarily reflect the mode of binding to native chromosomes .\",\n \"These assays also fall short of providing an estimate for the fraction of SMC complexes involved in interlocked associations with DNA and thus leave open the possibility that DNA entrapment might be an insignificant side reaction .\",\n \"Finally , it has not been tested under physiological conditions , whether the ATPase cycle is required for proper loading of DNA into any SMC–kleisin complex .\",\n \"To provide answers to these questions , we have established the chromosome entrapment assay to determine the association of prokaryotic condensin with native chromosomes .\",\n \"Our results clearly demonstrate that chromosomal DNA is loaded into condensin complexes in B . subtilis—in a manner that depends on the non-SMC subunit ScpB and at least one full cycle of Smc ATPase activity .\",\n \"The chromosome entrapment assay recovers about 10–20% of the fully cross-linked input material .\",\n \"This number probably understates the real proportion of chromosomally entrapped Smc complexes due to the loss of material during protein re-isolation from agarose plugs , due to possible adverse effects of cysteine mutations on Smc–ScpAB loading and because cysteine-maleimide linkages are vulnerable to hydrolytic reversal during and after the entrapment assay ( Kalia and Raines , 2007; Baldwin and Kiick , 2013 ) .\",\n \"Interestingly , a recent single-molecule tracking study in B . subtilis revealed two major populations of Smc: 80% of Smc-YFP proteins are displaying highly dynamic behavior on the nucleoid , whereas the other 20% ( and most ScpA-YFP protein ) are immobile and constrained within a small volume of the cell ( Kleine Borgmann et al . , 2013 ) .\",\n \"This immobile fraction possibly represents Smc–ScpAB complexes embracing origin proximal DNA after loading at parS sites as observed in the entrapment assay .\",\n \"Our results show that embracement of chromosomal DNA is a predominant feature of Smc–ScpAB , which has been evolutionarily retained in cohesin and likely all other SMC–kleisin complexes as well .\",\n \"Since the chromosome entrapment assay is based on the immobilization of intact replicating chromosomes , which possibly represent internally knotted and branched DNA structures , it is conceivable that Smc–ScpAB rings are linked to chromosomal DNA by non-topological capture of DNA loops , which themselves might be interlinked ( i . e . , knotted ) with other parts of the chromosome .\",\n \"Therefore , it remains to be determined whether DNA entrapment by Smc–ScpAB is of topological ( Figure 6A ) and/or non-topological ( Figure 6B ) nature . 10 . 7554/eLife . 06659 . 014Figure 6 . Models for entrapment of chromosomal DNA by Smc–ScpAB .\",\n \"( A ) Loop capture model .\",\n \"DNA loops might be pre-formed within ParB/parS nucleoprotein assemblies .\",\n \"Driven by ATP dependent engagement of Smc head domains Smc–ScpAB adopts a ring-like configuration .\",\n \"Occasional opening of the Smc hinge then allows capture of ParB-DNA loops within Smc–ScpAB .\",\n \"Subsequent ATP hydrolysis by Smc locks the hinge in a closed state and stabilizes the structure .\",\n \"( B ) Loop formation model .\",\n \"ParB/parS might serve as a landing platform for Smc–ScpAB allowing Smc–ScpAB in its ring-like conformation to guide DNA into its central cavity .\",\n \"Continuous extrusion of DNA through Smc–ScpAB then drives lengthwise condensation of chromosomes .\",\n \"Ring opening is not required in this model and DNA entrapment by Smc–ScpAB is thus strictly non-topological here .\",\n \"The following figure supplement is available: Figure 6—figure supplement 1: Quantitative Blotting of Smc protein and parB DNA . DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 01410 . 7554/eLife . 06659 . 015Figure 6—figure supplement 1 . Quantitative Blotting of Smc protein and parB DNA . To estimate Smc protein and parS-359 DNA abundance in B . subtilis cells , levels of Smc and parS-359 in extracts of wild-type cells were compared to defined amounts of purified Smc protein and parS-359 DNA spiked into equivalent extracts from cell harboring a tagged version of Smc and a modified parS-359 locus ( see panel C ) .\",\n \"The ratio of Smc protein ( see panel B ) to parS-359 DNA ( see panel A ) was calculated as average from three independent experiments ( biological replicates ) to be 61 ( ±12 SEM , standard error of mean ) Smc monomers per parS-359 DNA .\",\n \"Due to potential loss of chromosomal DNA during genomic DNA preparation this might be a slight over-estimation of the real number .\",\n \"( A ) Quantitative Southern blotting to estimate the number of molecules ( in fmol ) of the parS359 locus in a given number of cells .\",\n \"Genomic DNA from 52× cell culture equivalents ( 1 equivalent equals 0 . 025 ml*OD600 ) of wild type ( ‘WT’ , BSG1001 ) has been digested with the PstI restriction enzyme ( see panel C ) and loaded into the left lane .\",\n \"DNA loaded into the second and third lane is derived from mixtures of wild-type and tagged ( ‘parB-GFP’ , BSG2058 ) cells .\",\n \"Defined amounts of a purified PCR product covering the wild-type parS-359 locus have been spiked into cell extracts of the tagged strain .\",\n \"Digested DNA was detected using a Southern probe specific for the parS-359 locus .\",\n \"( B ) Quantitative Western blot to estimate the number of molecules of the Smc protein ( in fmol ) in a given number of cells .\",\n \"Cultures were identical to the ones used in ( A ) .\",\n \"Whole cell protein extract corresponding to 4 culture equivalents of wild-type cells ( ‘WT’ , BSG1001 ) was loaded into the left lane .\",\n \"Other lanes were loaded with samples derived from wild-type and tagged cells or mixtures thereof with or without spiked-in purified Smc protein as indicated in the figure panel .\",\n \"Smc protein was detected by immunoblotting using a polyclonal rabbit anti-Smc antibody .\",\n \"( C ) Protein and DNA spike-in was performed in extracts derived from cells ( BSG2058 ) with modifications at the parS-359 locus ( blue filled circle ) and the smc gene ( purple filled circle ) .\",\n \"PstI restriction sites ( in red ) in the parB gene ( light blue box ) and the neighbouring yyaC gene ( grey box ) generate a fragment of 1270 bp in size in case of the wild-type locus .\",\n \"From parB-gfp cells ( BSG2058 ) , however , a bigger DNA fragment of 3147 bp in size is generated .\",\n \"Strain BSG2058 also harboured a modified smc gene , which is fused to a halotag cassette ( pink box ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 06659 . 015 Smc–ScpAB plays a crucial role in the segregation of replication origins in B . subtilis cells ( Gruber , 2014; Wang et al . , 2014 ) , presumably by organizing nascent sister chromosomes so that their spatial overlap and entanglement is minimized .\",\n \"It is tempting to speculate that ParB/parS not only enriches Smc in the vicinity of the replication origin but also sets up lengthwise compaction of chromosomes by presenting DNA of a certain topology to the Smc–ScpAB complex .\",\n \"Consistent with this notion , we found that several parB mutants , which are defective in the ability to form large nucleoprotein complexes and to spread from parS sites , fail to promote loading of Smc–ScpAB onto the chromosome .\",\n \"Thus , Smc–ScpAB might capture , stabilize and expand structures–such as DNA loops or coils–that are pre-formed within larger ParB/parS nucleoprotein assemblies ( Figure 6A ) .\",\n \"Alternatively , ParB/parS might serve as an elaborate landing platform on the chromosome , where Smc–ScpAB initiates with the help of ParB the lengthwise compaction of chromosomes by forming and extruding loops of DNA ( Figure 6B ) .\",\n \"Extrusion of DNA might involve the translocation of Smc–ScpAB along DNA fibers made up of naked or ParB coated DNA .\",\n \"These models are not mutually exclusive .\",\n \"A reasonable first step towards understanding the architecture of Smc/ParB/parS assemblies might be the investigation of physical interactions between ParB/parS and Smc–ScpAB and their functional interconnection with the Smc ATPase cycle .\",\n \"In the future , Smc/ParB/parS structures could serve as a relatively simple paradigm for chromosome organization by the more intricately regulated cohesin and condensin complexes in eukaryotes .\",\n \"ParB serves a supplemental—albeit important—role in the Smc loading process .\",\n \"In contrast , Smc functionality and its loading onto DNA in vivo is critically dependent on the ScpB subunit and the ATPase cycle .\",\n \"It remains unclear though what the exact role of ScpB and ATP hydrolysis in the entrapment of DNA might be .\",\n \"If loading indeed depends on transient opening of a DNA entry gate , the open state would likely represent an energetically unfavorable reaction intermediate ( Figure 6A ) .\",\n \"Timely opening would require energy input as well as tight regulation .\",\n \"We have recently demonstrated that the ATP dependent engagement of Smc head domains—together with DNA binding to the Smc hinge domain—can transform the configuration of the Smc coiled coil from a rod to a more open ring-like conformation ( Soh et al . , 2015 ) .\",\n \"Hydrolysis of ATP and/or ScpB binding could drive a subsequent conformational change that might open the SMC–kleisin ring ( Figure 6A ) .\",\n \"Alternatively , ScpB and/or ATP hydrolysis might stabilize Smc–ScpAB once loaded onto DNA or promote Smc's sliding along DNA to allow efficient extrusion of DNA loops by the Smc–ScpAB ring ( Figure 6B ) .\",\n \"Deletion or mutation of the parB gene results in a clear drop in the levels of chromosomally bound condensin ( ∼5–10 fold less ) in our chromosome entrapment assay ( Figure 4B , Figure 2—figure supplement 2C ) .\",\n \"In addition to this loading defect , also the specific recruitment of Smc–ScpAB towards the replication origin is lost in the absence of ParB ( Gruber and Errington , 2009; Sullivan et al . , 2009 ) .\",\n \"Thus , in parB mutants only a very small proportion of cellular Smc–ScpAB is bound to chromosomes within the replication origin region , where it presumably performs its essential function by promoting the separation of nascent sister chromosomes ( Gruber et al . , 2014; Wang et al . , 2014 ) .\",\n \"Nevertheless , defects in chromosome segregation are rather mild in parB mutants when compared to mutants of smc .\",\n \"Using quantitative blotting of Smc protein and replication origin DNA from cell extracts , we have estimated the average number of Smc protein to be around 30 dimers per replication origin in a fast growing population of cells ( Figure 6—figure supplement 1 ) .\",\n \"Assuming that all Smc complexes entrap chromosomal DNA in wild-type cells , only three to six Smc dimers ( 10–20% of total ) are loaded onto the chromosome in parB mutants according to our measurements .\",\n \"Thus , a handful of Smc–ScpAB complexes , which are presumably randomly distributed over the chromosome , appears to be capable of supporting near-normal chromosome segregation under these conditions—when chromosome segregation is already compromised by the loss of the parABS system .\",\n \"Few Smc–ScpAB therefore seem to be able to provide enough organization to the replication origin region and the remainder of the chromosome to prevent lethal accumulation of inter-linked sister chromosomes .\",\n \"It is conceivable that individual Smc–ScpAB complexes are able to organize large chunks of a bacterial chromosome , possible by forming giant loops of DNA .\",\n \"Alternatively , Smc activity might be needed only at a limited number of defined locations on the B . subtilis chromosome and/or for very short periods of time .\",\n \"However , when levels of functional Smc dimers are in addition reduced for example by hypomorphic mutations in the smc gene itself , the loss of ParB protein becomes lethal ( Gruber and Errington , 2009 ) .\",\n \"This work reveals the mode of association of Smc–ScpAB with bacterial chromosomes , highlights its striking evolutionary conservation and demonstrates the involvement of the SMC ATPase cycle in chromosomal loading .\",\n \"Future work must address the underlying biochemical mechanisms to get basic insight into the architectural role of SMC in chromosome biology .\"\n ],\n [\n \"Genetic modifications at smc , scpAB , parB and dnaN loci were generated via double cross-over recombination in strains derived from B . subtilis 1A700 or B . subtilis 168ED .\",\n \"Genotypes of strains used in this study are listed in Supplementary file 1 .\",\n \"Cells were transformed with plasmids or B . subtilis genomic DNA using a 2-step starvation protocol as previously described ( Hamoen et al . , 2002; Bürmann et al . , 2013 ) .\",\n \"Transformants were selected by growth on nutrient agar ( NA ) plates ( Oxoid , UK ) supplemented with antibiotics as required: 5 µg ml−1 kanamycin , 80 µg ml−1 spectinomycin , 10 µg ml−1 tetracycline , 5 µg ml−1 chloramphenicol , 1 µg ml−1 erythromycin and 25 µg ml−1 lincomycin .\",\n \"Strains displaying a condensin null phenotype were selected on SMG medium instead: SMM salt solution ( 2 g l−1 ammonium sulphate , 14 g l−1 dipotassium hydrogen phosphate , 6 g l−1 potassium dihydrogen phosphate , 1 g l−1 trisodium citrate , 0 . 2 g l−1 magnesium sulphate , 6 g l−1 potassium hydrogen phosphate ) supplemented with 5 g l−1 glucose , 20 mg l−1 tryptophan and 1 g l−1 glutamate with the respective antibiotics .\",\n \"Strains were single-colony purified and grown in the absence of antibiotics for experiments .\",\n \"Cells were pre-grown in a 96-well plate in SMG medium for 24 hr at 37°C .\",\n \"Overnight cultures were diluted 9^2-fold ( high density spots ) or 9^5-fold ( low density spots ) and spotted onto NA or SMG agar plates .\",\n \"Plates were incubated at 37°C for 12 hr on NA or 24 hr on SMG agar .\",\n \"Cells were grown in either LB Miller medium ( 10 g l−1 tryptone , 5 g l−1 yeast extract , 10 g l−1 sodium chloride ) or SMG medium to mid-exponential phase at 37°C ( in LB Miller medium , OD600 of 0 . 4; in SMG medium , OD600 of 0 . 03 ) .\",\n \"Cells were harvested by centrifugation or vacuum filtration and washed in ice-cold PBS supplemented with 0 . 1% glycerol ( ‘PBSG’ ) .\",\n \"Cell aliquots ( corresponding to 1 ml at an OD600 of 1 . 25 ) were re-suspended in PBSG and incubated with the cross-linker BMOE ( bis-maleimidoethane , Applichem , Germany ) at a concentration of 1 mM ( diluted from a 20 mM stock solution in DMSO ) .\",\n \"After a 10 min incubation on ice the reaction was quenched by addition of 2-mercaptoethanol ( ‘2-ME’ ) to a final concentration of 28 mM .\",\n \"For the preparation of protein extracts ( ‘input’ ) a mixture of following components was added to an aliquot of cells: 400 units ready-lyse lysozyme ( Epicentre , Madison , WI ) , 12 . 5 units benzonase ( Sigma-Aldrich , St . Louis , MO ) and a protease-inhibitor cocktail ( Sigma ) .\",\n \"In addition , 1 µM HT Oregon Green substrate ( Promega , Madison , WI ) was added to cell suspensions with HaloTag bearing alleles .\",\n \"Samples were then incubated for 20 min at 37°C protected from light .\",\n \"Finally , the samples were heated to 70°C for 5 min in LDS Sample Buffer ( NuPage , Thermo Scientific , Waltham , MA ) containing 200 mM DTT and loaded onto a SDS-PAGE gel ( see below ) .\",\n \"Gels with Oregon Green labeled samples were scanned on a Typhoon scanner ( GE Healthcare , UK ) with Cy3-DIGE filter setup .\",\n \"Cells were grown , cross-linked and quenched as described above .\",\n \"Lysozyme stock solution , protease inhibitor and HT substrate were added to an aliquot of cells at concentrations given above .\",\n \"The cell suspension was mixed immediately in a 1:1 ratio with a 2% solution of Megabase agarose ( BioRad , Hercules , CA ) or low-melt agarose ( BioRad ) and casted into 100 µl agarose plugs using plug molds ( BioRad ) .\",\n \"Agarose plugs were incubated for 20 min at 37°C , protected from light , and then loaded into the wells of a 6% SDS-PAGE Tris-glycine gel .\",\n \"The polyacrylamide mini-gel was run for 60 min at 25 mA protected from light .\",\n \"Agarose plugs were then re-extracted from the gel and transferred into 1 . 5 ml Eppendorf tubes .\",\n \"1 ml of Wash Buffer ( ‘WB’: 0 . 01 mM EDTA , 0 . 5 mM Tris , 0 . 5 mM MgCl2 , 0 . 01% SDS ) was added per agarose plug .\",\n \"Plugs were incubated for 10 min with gentle agitation protected from light .\",\n \"This step was repeated once .\",\n \"Wash buffer was then discarded and replaced by 100 µl fresh WB supplemented with 50 units of benzonase ( Sigma ) .\",\n \"Plugs were incubated at 37°C for 30 min .\",\n \"Plugs were melted at 85°C for 2 min under vigorous agitation .\",\n \"The samples were frozen at −80°C and stored overnight .\",\n \"Samples were then thawed , centrifuged for 10 min at 4°C and 14 , 000×g and transferred to a 0 . 45 µm Cellulose acetate spin column ( Costar , Tewksbury , MA ) and spun for 1 min at 10 , 000×g .\",\n \"The flow-through was concentrated in a Speed Vac ( Thermo Scientific , no heating , 2 . 5 hr running time ) .\",\n \"The concentrated sample was resuspended in LDS Sample Buffer ( NuPage ) containing 200 mM DTT and heated for 3 min at 70°C .\",\n \"Samples were loaded onto Tris-acetate gels ( 3–8% Novex , Thermo Scientific ) and run for 2 . 5 hr at 35 mA per gel at 4°C .\",\n \"For DnaN detection Bis-Tris gels ( 8–12% Novex ) were run for 1 hr at 200 V at room temperature .\",\n \"Gels were either scanned on a Typhoon scanner ( FLA 9000 , GE Healthcare ) with Cy3-DIGE filter setup or immuno-blotted using antibodies against DnaN or Smc ( see below ) .\",\n \"For cleavage of ScpA ( TEVs ) or degradation of chromosomal DNA 15 units of His-TEV protease or 12 . 5 units of benzonase , respectively , was added before casting agarose plugs .\",\n \"B . subtilis strains containing smc-tev-avitag alleles were grown to OD600 of 0 . 4 in 100 ml LB Miller at 37°C .\",\n \"Part of the culture was fixed with formaldehyde and subjected to chromatin immunoprecipitation ( ChIP ) as described by ( Gruber and Errington , 2009 ) using a rabbit anti-Smc antiserum .\",\n \"In parallel , 10 ml of the culture were mixed with ice , harvested by centrifugation and washed in cold PBSG .\",\n \"Cells were resuspended in 200 µl PBSG and treated with 0 . 5 mM BMOE for 10 min on ice .\",\n \"The reaction was quenched with 14 mM 2-ME and cells were washed once in CutSmart buffer ( New England Biolabs , Ipswich , MA ) .\",\n \"Cells were resuspended in 200 µl CutSmart containing 10 kU Ready-Lyse lysozyme ( Epicentre ) , 40 U XbaI ( New England Biolabs ) and a protease inhibitor cocktail ( Sigma ) .\",\n \"The suspension was incubated for 15 min at 37°C before addition of 1800 µl buffer LS ( 10 mM Tris/HCl , 150 mM NH4OAc , 1 mM EDTA , 6 mM 2-ME , 0 . 05% Tween-20 , 0 . 01% NaN3 , final pH 7 . 9 at 23°C ) .\",\n \"Lysates were centrifuged for 5 min at 20 , 000×g .\",\n \"Subsequently , 1400 µl of the extract were incubated with 100 µl Dynabeads Streptavidin C1 for 30 min at room temperature .\",\n \"Beads were washed once in buffer LS , then split , resuspended either in buffer LS or in buffer HS ( 10 mM Tris/HCl , 2 M NaCl , 1 mM EDTA , 6 mM 2-ME , 0 . 05% Tween-20 , 0 . 01% NaN3 , final pH 7 . 9 at 23°C ) and incubated for 15 min at room temperature .\",\n \"Beads were washed twice with buffer LS , and protein/DNA complexes were eluted for 1 hr at 22°C by incubation with 350 µl LS containing TEV protease and 1 mM DTT .\",\n \"DNA from input and eluate fractions was purified by treatment with 0 . 5 mg/ml Proteinase K for 1 hr at 55°C followed by phenol/chloroform extraction .\",\n \"Samples were analysed by quantitative PCR using the second derivative maximum of a four parameter logistic model similar to the method described by ( Zhao and Fernald , 2005 ) .\",\n \"After gels were scanned for in-gel fluorescence detection , they were immediately transferred onto a PVDF membrane ( Immobilon-P , Merck Millipore , Germany ) using semi-dry transfer .\",\n \"Membranes were blocked with 3 . 5% ( wt/vol ) milk powder in PBS with 0 . 1% Tween 20 .\",\n \"Rabbit polyclonal sera against B . subtilis DnaN ( Lenhart et al . , 2013 ) , B . subtilis Smc ( this paper ) and B . subtilis ParB ( this paper ) were used as primary antibodies for immunoblotting at dilutions of 1:5000 each .\",\n \"The membrane was developed with HRP-coupled secondary antibodies and chemiluminescence ( SuperSignal West Femto , Thermo Scientific ) and visualized on a LAS-3000 scanner ( FujiFilm , Germany ) .\",\n \"To estimate DnaN cross-linking kinetics ( Figure 1—figure supplement 1A ) samples were grown as described above .\",\n \"An aliquot of cells was incubated with the cross-linker BMOE ( 1 mM ) for the indicated length of time before the reaction was quenched with 2-ME ( 28 mM ) .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Smc–ScpAB forms elongated , annular structures that promote chromosome segregation , presumably by compacting and resolving sister DNA molecules .","The mechanistic basis for its action , however , is only poorly understood .","Here , we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc–ScpAB ring .","To do so , we have chemically cross-linked the three ring interfaces in Smc–ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions .","Exclusively species of Smc–ScpA , which were previously cross-linked into covalent rings , remained associated with chromosomal DNA .","DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted , implying that most Smc–ScpAB is loaded onto the chromosome at parS sites near the replication origin .","We furthermore report a physical interaction between native Smc–ScpAB and chromosomal DNA fragments ."],"string":"[\n \"Smc–ScpAB forms elongated , annular structures that promote chromosome segregation , presumably by compacting and resolving sister DNA molecules .\",\n \"The mechanistic basis for its action , however , is only poorly understood .\",\n \"Here , we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc–ScpAB ring .\",\n \"To do so , we have chemically cross-linked the three ring interfaces in Smc–ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions .\",\n \"Exclusively species of Smc–ScpA , which were previously cross-linked into covalent rings , remained associated with chromosomal DNA .\",\n \"DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted , implying that most Smc–ScpAB is loaded onto the chromosome at parS sites near the replication origin .\",\n \"We furthermore report a physical interaction between native Smc–ScpAB and chromosomal DNA fragments .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["The genome of any living organism holds all the genetic information that the organism needs to live and grow .","This information is written in the sequence of the organism's DNA , and is often divided into sub-structures called chromosomes .","Different species have different sized genomes , but even bacteria with some of the smallest genomes still contain DNA molecules that are thousand times longer than the length of their cells .","DNA molecules must thus be highly compacted in order to fit inside the cells .","DNA compaction is particularly important during cell division , when the DNA is being equally distributed to the newly formed cells .","In plants , animals and all other eukaryotes , large protein complexes known as condensin and cohesin play a major role in compacting , and then separating , the cell's chromosomes .","Many bacteria also have condensin-like complexes .","At the core of all these complexes are pairs of so-called SMC proteins .","However , it is not clear how these SMC proteins direct chromosomes to become highly compacted when cells are dividing .","Wilhelm et al . have now developed two new approaches to investigate how SMC proteins associate with bacterial DNA .","These approaches were then used to study how SMC proteins coordinate the compaction of chromosomes in a bacterium called Bacillus subtilis .","The experiments revealed that SMC proteins are in direct physical contact with the bacterial chromosome , and that bacterial DNA fibers are physically captured within a ring structure formed by the SMC proteins .","Wilhelm et al . suggest that these new findings , and recent technological advances , have now set the stage for future studies to gain mechanistic insight into these protein complexes that organize and segregate chromosomes ."],"string":"[\n \"The genome of any living organism holds all the genetic information that the organism needs to live and grow .\",\n \"This information is written in the sequence of the organism's DNA , and is often divided into sub-structures called chromosomes .\",\n \"Different species have different sized genomes , but even bacteria with some of the smallest genomes still contain DNA molecules that are thousand times longer than the length of their cells .\",\n \"DNA molecules must thus be highly compacted in order to fit inside the cells .\",\n \"DNA compaction is particularly important during cell division , when the DNA is being equally distributed to the newly formed cells .\",\n \"In plants , animals and all other eukaryotes , large protein complexes known as condensin and cohesin play a major role in compacting , and then separating , the cell's chromosomes .\",\n \"Many bacteria also have condensin-like complexes .\",\n \"At the core of all these complexes are pairs of so-called SMC proteins .\",\n \"However , it is not clear how these SMC proteins direct chromosomes to become highly compacted when cells are dividing .\",\n \"Wilhelm et al . have now developed two new approaches to investigate how SMC proteins associate with bacterial DNA .\",\n \"These approaches were then used to study how SMC proteins coordinate the compaction of chromosomes in a bacterium called Bacillus subtilis .\",\n \"The experiments revealed that SMC proteins are in direct physical contact with the bacterial chromosome , and that bacterial DNA fibers are physically captured within a ring structure formed by the SMC proteins .\",\n \"Wilhelm et al . suggest that these new findings , and recent technological advances , have now set the stage for future studies to gain mechanistic insight into these protein complexes that organize and segregate chromosomes .\"\n]","isConversational":false},"year":{"kind":"string","value":"2015"}}},{"rowIdx":293,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["developmental biology","cell biology"],"string":"[\n \"developmental biology\",\n \"cell biology\"\n]","isConversational":false},"title":{"kind":"string","value":"Protein aggregates are associated with replicative aging without compromising protein quality control"},"id":{"kind":"string","value":"elife-06197-v1"},"sections":{"kind":"list like","value":[["Aging results in an increasing decline of the organism's fitness over time ( Lopez-Otin et al . , 2013 ) .","Remarkably , this process segregates asymmetrically during budding yeast division: the mother cell forms an aging lineage , whereas the daughters generated by these mothers rejuvenate to form eternal lineages , similar to the segregation of soma and germ lineages in metazoans .","Such lineage separation requires that the inheritance of factors that promote aging , such as defective/deleterious organelles , proteins , and DNA , is asymmetric during cell division ( Sinclair and Guarente , 1997; Aguilaniu et al . , 2003; Erjavec et al . , 2007; Henderson and Gottschling , 2008; Shcheprova et al . , 2008; Liu et al . , 2010; Zhou et al . , 2011; Hughes and Gottschling , 2012; Higuchi et al . , 2013; Clay et al . , 2014; Denoth Lippuner et al . , 2014; Henderson et al . , 2014; Higuchi-Sanabria et al . , 2014; Thayer et al . , 2014; Katajisto et al . , 2015 ) .","Therefore , how cells are able to recognize , sort , and coordinate the asymmetric segregation of aging factors and other fate determinants is an outstanding question in biology ( Neumuller and Knoblich , 2009 ) .","Protein aggregates and/or damaged proteins are a hallmark in the etiology of many human disorders associated with aging ( Hartl et al . , 2011; Wolff et al . , 2014 ) , and their presence correlates with aging of mitotically active yeast and drosophila stem cells ( Aguilaniu et al . , 2003; Erjavec et al . , 2007; Bufalino et al . , 2013; Coelho et al . , 2013 ) .","Studies on budding yeast have shown a correlation between the accumulation of protein aggregates and replicative aging by demonstrating that Hsp104-mediated protein disaggregation is required for full replicative life span ( Erjavec et al . , 2007 ) , and that over-expression of Mca1 , which counteracts the formation of stress- and age-associated protein aggregates ( Lee et al . , 2010; Hill et al . , 2014 ) , extends the life span of yeast mother cells ( Hill et al . , 2014 ) .","How cells respond to protein aggregation that occurs specifically during aging has remained elusive since most studies investigating the cellular responses to protein aggregation have relied on over-expression of non-native , aggregation prone proteins , proteostasis inhibitors , or other stressors , such as heat ( Kaganovich et al . , 2008; Liu et al . , 2010; Specht et al . , 2011; Zhou et al . , 2011; Malinovska et al . , 2012; Spokoini et al . , 2012; Winkler et al . , 2012; Escusa-Toret et al . , 2013; Zhou et al . , 2014 ) .","These studies have uncovered specific modes of cytosolic compartmentalization that take place when cells encounter proteotoxic stress .","For example , cells stressed with heat respond by forming multiple protein aggregates ( referred to as peripheral aggregates , stress foci , Q-bodies , or CytoQ ) at the surface of the ER ( Specht et al . , 2011; Spokoini et al . , 2012; Escusa-Toret et al . , 2013; Miller et al . , 2015; Zhou et al . , 2014; Wallace et al . , 2015 ) .","These structures , hereafter referred as Q-bodies , contain acutely misfolded proteins that are sorted between the nuclear and cytoplasmic degradation/deposit sites by the Hook family proteins Btn2 and Cur1 ( Malinovska et al . , 2012 ) , and coalesce together by the aid of small heat shock proteins; Hsp42 in budding yeast ( Specht et al . , 2011; Escusa-Toret et al . , 2013 ) , and Hsp16 in fission yeast ( Coelho et al . , 2014 ) .","Simultaneously , Q-bodies are being rapidly resolved by the protein disaggregase Hsp104 ( Parsell et al . , 1994; Specht et al . , 2011; Zhou et al . , 2011; Spokoini et al . , 2012; Escusa-Toret et al . , 2013 ) , together with other heat shock responsive chaperones such as Hsp70 and Hsp82 ( Escusa-Toret et al . , 2013 ) .","The formation of Q-bodies seems to aid stress survival , as the deletion of HSP42 resulted in defective tolerance of prolonged heat stress ( Escusa-Toret et al . , 2013 ) .","The asymmetric inheritance of Q-bodies by the mother cells is promoted by the geometry of the bud neck ( Zhou et al . , 2011 ) , tethering to mitochondria ( Zhou et al . , 2014 ) , and by actin cable-mediated retrograde transport , which is dependent of Hsp104 and Sir2 ( Liu et al . , 2010; Song et al . , 2014 ) .","Notably , Sir2 is also a key player in processes that underlie the asymmetric segregation of damaged mitochondria ( Higuchi et al . , 2013 ) and the accumulation of extrachromosomal DNA circles ( Sinclair and Guarente , 1997; Kaeberlein et al . , 1999 ) to the aging mother cell .","Prolonged Q-body-inducing stress ( heat or over-expression of thermolabile proteins ) combined with proteasome inhibition can lead to the formation of a dynamically exchanging deposit of ubiquitylated proteins named the juxtanuclear quality compartment , JUNQ ( Kaganovich et al . , 2008; Escusa-Toret et al . , 2013 ) .","This structure is regulated by the Upb3 deubiqutinase ( Oling et al . , 2014 ) , by proteosomal activity ( Andersson et al . , 2013 ) and by lipid droplets ( Moldavski et al . , 2015 ) , and it was also shown to appear during replicative aging ( Oling et al . , 2014 ) .","The faithful inheritance of this structure by the mother cell is dependent on its association with the nucleus ( Spokoini et al . , 2012 ) .","More recently , it was shown that the ‘JUNQ’ might actually reside inside the nucleus , and it was thus renamed as intranuclear quality control compartment , INQ ( Miller et al . , 2015 ) .","The JUNQ/INQ assembly is dependent on Btn2-aggregase ( Miller et al . , 2015 ) , a protein also found to be involved in prion curing ( Kryndushkin et al . , 2008 , 2012 ) .","Apart from the JUNQ/INQ structure , terminally aggregating proteins , such as the amyloidogenic prions Rnq1 and Ure2 , were shown to partition to an non-dynamic , vacuole-associated deposit called the insoluble protein deposit IPOD ( Kaganovich et al . , 2008; Tyedmers et al . , 2010b ) , which has remained less well characterized .","Despite this wealth of data , it remains unclear how these exogenous/stress-induced aggregation models relate to protein aggregation that takes place during physiological ‘healthy’ aging .","Particularly , it is unclear why/how protein aggregates arise during aging , how are they segregated during cell division and , importantly , what is their consequence to the protein quality control of the aging cell , as well as to the aging process itself .","To illuminate these aspects , we probed the role of protein aggregation during unperturbed replicative aging .","Our findings indicate that protein aggregation is a prevalent and highly coordinated event of early aging and is not solely associated with proteostasis deterioration .","Instead , we provide evidence that age-associated protein aggregation may initially benefit the cytosolic protein quality control , but eventually becomes involved with age-associated loss of fitness ."],["To address the role of protein aggregation in unperturbed , physiological aging , we analyzed microscopically the replicative age-associated protein aggregation landscape in budding yeast by visualizing different chaperone proteins that mark aberrantly folded and aggregated proteins .","By employing the Mother Enrichment Program ( MEP ) ( Lindstrom and Gottschling , 2009 ) ( Figure 1—figure supplement 1A ) , we harvested cells of different age and first analyzed the localization of endogenous GFP-tagged protein-disaggregase Hsp104 ( Parsell et al . , 1994; Glover and Lindquist , 1998 ) , a broad sensor for protein aggregates ( Figure 1A , Haslberger et al . , 2010 ) .","Interestingly , we found many cells displaying an aggregate ( typically a single bright Hsp104-labeled focus ) and this portion increased in a progressive , age-dependent manner such that >80% of cells that had undergone more than 6 divisions displayed such a structure ( Figure 1A , B ) , as previously reported ( Aguilaniu et al . , 2003; Erjavec et al . , 2007 ) .","Co-localization analysis with Hsp104 demonstrated that the Hsp70 proteins Ssa1 and Ssa2 , the small heat shock protein Hsp42 , and the Hsp40 protein Ydj1 readily localized to these aggregates , the Hsp26 was found to be enriched in only 15% of Hsp104-labeled foci , while no accumulation of Hsp40 protein Sis1 or the Hsp90 protein Hsp82 was detected ( Figure 1C , Figure 1—figure supplement 2A–C ) .","Importantly , age-dependent appearance of these structures was also detected in diploid cells , in other strain backgrounds ( W303 ) , independently of the MEP procedure , and when different fluorophores where used for tagging Hsp104 ( Figure 1—figure supplement 2D–H ) , indicating that their formation represents a general , age-dependent phenomenon of budding yeast cells . 10 . 7554/eLife . 06197 . 003Figure 1 . Replicative aging leads to the formation of age-associated protein deposit .","( A ) Representative images of cells expressing endogenous Hsp104 tagged with GFP .","The C-terminal tagging does not hamper Hsp104 disaggregation activity ( Specht et al . , 2011 ) .","Cells of different age were harvested using the Mother Enrichment Program ( MEP ) ( Lindstrom and Gottschling , 2009 ) and stained with calcofluor .","( B ) Percentage of cells of different age groups containing at least one Hsp104-focus , ( N = 135 to 472 cells per age group ) .","( C ) Fraction of Hsp104-mCherry foci that are enriched with the indicated chaperones ( N = 16 to 92 Hsp104-focus containing cells per strain , n . d . = not detected ) .","( D ) Representative frames of a movie of dividing cells expressing Hsp104-mCherry ( black ) .","Red arrowhead indicates an aggregate that is retained in the mother cell ( M ) .","This cell divided five times giving rise to four daughter cells that start to form aggregates after dividing 2–3 times ( blue arrows at 390 min ) .","( E ) Integrated density at newly forming age-associated protein deposits ( deposit/cytoplasm ) over time , ( N = 26 ) .","( F ) Fraction of divisions during which the age-associated protein deposit is asymmetrically inherited by the mother cell as a function of the age of the mother cell ( N = 66 to 306 divisions per age group from 2-3 independent experiments . The approximate mother cell age was estimated from separate bud scar analysis ) .","( G ) Representative micrographs of a dividing mother cell expressing Hsp104-GFP followed for 66 hr in a microfluidic chip ( Lee et al . , 2012 ) .","( H ) Proportion of cells having the indicated number of Hsp104 foci .","Hsp104 foci containing cells that could be followed >10 consecutive divisions were quantified for the number of Hsp104-foci/cell at each time point , ( N = 44 ) .","( I ) Total time spent with and without an Hsp104-focus for cells undergoing >10 consecutive divisions , starting with a focus , ( N = 44 ) .","( J ) Fluorescent recovery after photobleaching ( FRAP ) analysis of Hsp104-mCherry turnover at age-associated protein deposit .","The mCherry signal at the age-associated protein deposit was photobleached in HSP104-GFP/HSP104-mCherry diploid cells and the kinetics of recovery were monitored , using the GFP signal to localize the age-associated protein deposit over time .","( K ) Fitting of nine recovery curves showed that on average a large ( 59% ) fraction was immobile and the half-time recovery for the mobile fraction was 8 . 9 s , ( N = 9 ) .","Scale bars 5 μm .","Graphs display mean ± SEM , *p < 0 . 05 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 00310 . 7554/eLife . 06197 . 004Figure 1—figure supplement 1 . Schematic representation of the strategies used here to study aged cells .","( A ) Strategy to obtain old cells: cells were biotinylated and grown for ( x ) amount of time in YPD media containing estradiol , which activates the MEP ( Lindstrom and Gottschling , 2009 ) and results in daughter specific cell cycle arrest due to excision of two essential genes UBC9 and CDC20 .","After desired time , aged mother cells containing the cell wall-linked biotin coat were coupled to streptavidin microbeads , purified with affinity chromatography , and imaged under the microscope .","( B ) The microfluidic system ( Lee et al . , 2012 ) allows microscopic examination of trapped mother cells as they undergo repetitive divisions .","Cells are captured between a PDMS pad and a glass cover slip and subjected to constant media flow , which washes away the newly born daughter cells that are not captured between the pad due to their smaller size . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 00410 . 7554/eLife . 06197 . 005Figure 1—figure supplement 2 . Age-associated protein deposit formation is a general age-dependent phenomenon marked by a subset of chaperones .","( A–C )","Representative images of Hsp104-mCherry expressing cells co-tagged with ( A ) Ssa1-GFP ( B ) Hsp42-GFP , or ( C ) Hsp82 .","Age-associated protein deposit are depicted with a red arrowhead .","( D ) W303 cells expressing Hsp104-YFP .","( E ) S288C cells expressing Hsp104-mCherry or ( F ) Hsp104-GFP .","( G ) Diploid S288C cells expressing Hsp104-GFP .","( H ) Comparison of the relative amount of age-associated protein deposit containing cells in calcofluor treated MEP and non-MEP background cells vs non-calcofluor treated MEP strain background , ( N = 177–243 cells analyzed per genotype ) .","Scale bars: ( A–C ) 2 μm , ( D–G ) 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 005 To characterize the nature of these protein deposits further , we performed live-cell imaging by acquiring images every 15 min over several hours and covering the entire depth of the cell .","This showed that most mother cells started to form this aggregate already after budding three to four times ( Figure 1D , 390 min ) .","The fastest growth phase of the aggregate took place during the first half an hour following its detection , after which the Hsp104 signal intensity increased only marginally ( Figure 1D , E ) .","While the aggregates initially underwent occasional dissolution , they became stable within a few hours from nucleation ( Figure 1E ) .","Pedigree analysis of aggregate inheritance in cells of different age showed that they faithfully segregated to the aging mother cell ( 98% of divisions ) , irrespective of its age ( Figure 1D , F , Video 1 ) .","To further analyze the persistence and behavior of the deposits over the entire replicative life span of cells , we used a microscope-coupled microfluidic dissection platform ( Lee et al . , 2012 ) ( Figure 1—figure supplement 1B ) .","This showed that the aggregate was efficiently maintained as one compartment , and whenever new foci emerged , they typically merged soon after with the pre-existing deposit .","We quantified the number of Hsp104 foci ( 1 or >1 ) and its post-nucleation stability in cells that were tracked for at least 10 divisions after the aggregate had appeared .","These cells preferentially ( >80% of time ) displayed only a single Hsp104 focus ( Figure 1G–H ) , which was very stable , typically persisting until the last divisions of the cell ( Figure 1G , I ) .","Moreover , this structure was largely non-dynamic , displaying limited exchange of Hsp104 with the cytoplasm , as determined by fluorescent recovery after photobleaching ( FRAP ) analysis in diploid cells that expressed one GFP- and one mCherry-tagged copy of HSP104 ( Figure 1J ) .","We bleached and measured the recovery of the mCherry signal ( Figure 1J ) and by fitting nine recovery curves , found that on average a large fraction ( 59% ) of Hsp104 was immobile , while the mobile fraction displayed a half-time recovery rate of 8 . 9 s ( Figure 1K ) .","Together , these data show that a large majority of unstressed yeast cells develop a protein deposit early during replicative aging .","This deposit displays limited exchange with the cytoplasm ( assessed by Hsp104 ) , is efficiently maintained as a single compartment , and is faithfully inherited by the aging mother cell . 10 . 7554/eLife . 06197 . 006Video 1 . Age-associated protein deposit is a stable structure that is faithfully inherited by the aging mother cell lineage . Aged yeast cell expressing Hsp104-mCherry ( in green ) to mark protein aggregates and Cdc10-GFP ( in red ) to mark the mother bud interface undergoing four consecutive divisions . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 006 To better understand how the age-associated protein deposit forms and what are its physiological constituents , we selected two proteins containing glutamine- and/or asparagine-rich domains , Sup35 and Dcp2 , which are known to undergo conformational switches and to aggregate , and we monitored their localization in respect to the age-associated protein deposit .","Sup35 ( eRF3 ) is a translation termination factor that can undergo stable amyloid-like prion conversion from non-prion [psi–] to prion state [PSI+] ( Chernoff et al . , 1993; Ter-Avanesyan et al . , 1994; Wickner , 1994; Patino et al . , 1996; Paushkin et al . , 1996 ) .","Thus , we tested whether Sup35-GFP is targeted to the age-dependent aggregate and if the prion status plays a role in the targeting to and/or in the formation of the age-associated protein deposit .","Importantly , Sup35-GFP clearly accumulated into Hsp104-mCherry-labeled age-associated protein deposit in 33% of [PSI+] cells that contained such a deposit , whereas no accumulation was detected in the non-prion [psi−] cells ( Figure 2A–C ) .","As expected , the formation of this bright Sup35 focus was age-dependent ( Figure 2D ) , in line with earlier observations ( Derdowski et al . , 2010 ) , but interestingly the [PSI+] state did not have an overall effect on Hsp104-labeled deposit formation ( Figure 2E ) .","Similarly , the elimination of the [RNQ+] prion by deleting RNQ1 did not negatively influence the formation of the age-associated deposit ( data not shown ) .","Notably , time-lapse imaging showed that Sup35 was recruited into the pre-existing age-associated protein deposit ( marked by Hsp104-mCherry ) .","These Sup35-enriched age-associated protein deposits segregated asymmetrically towards the aging mother cell in 98% of mitoses ( Figure 2F , G ) .","These data provide evidence that the Hsp104-labeled focus is a bona fide deposit site for aggregating proteins , and that prion conversion of Sup35 promotes its gradual storage into the age-associated protein deposit , but does not potentiate its formation per se . 10 . 7554/eLife . 06197 . 007Figure 2 . Prion form of Sup35 is deposited to the age-associated protein deposit .","( A , B )","Co-localization of Sup35-GFP and Hsp104-mCherry in diploid [psi−] ( A ) and [PSI+] ( B ) cells , where a single locus of the respective gene was tagged with the fluorescent marker indicated .","White arrowhead indicates the age-associated protein deposit ( Hsp104 positive ) and the red arrowhead Sup35-aggregates not associated with the age-associated protein deposit .","( C ) Percentage of age-associated protein deposits in [psi–] and [PSI+] cells with enriched Sup35 , ( N = 130 ) .","( D ) Percentage of cells with large Sup35-foci in cells of indicated age groups ( N = 157–184 cells pre age group , average age young [psi–] 1 . 1 , young [PSI+] 1 . 1 , aged [psi–] 6 . 9 , aged [PSI+] 5 . 9 generations ) .","( E ) Percentage of [psi–] and [PSI+] cells of the indicated age group ( see D ) containing an age-associated protein deposit , ( N = 157–184 cells pre age group ) .","( F ) Time-lapse images of a [PSI+] cell co-expressing Sup35-GFP ( green ) Hsp104-mCherry ( red ) at the indicated time points .","Arrowheads point at the age-associated protein deposit as observed in the different channels .","The newborn daughters are indicated in the bottom row .","( G ) Percentage of divisions where the Sup35-GFP-labeled age-associated protein deposit is retained in the aging mother cell lineage , ( N = 204 divisions ) .","( H ) Fluorescent images of a cell co-expressing the P-body protein Dcp2 tagged with GFP and Hsp104 tagged with mCherry .","( I ) Percentages of cells of indicated age groups that contain the indicated fluorescent foci , ( N = 18–71 per group ) .","( J ) Time-lapse , fluorescent images of Dcp2-GFP and Hsp104-mCherry expressing cells at the indicated time points after switching the cells to 0 . 1% glucose .","Scale bars ( A , B , H , J ) 5 μm , ( F ) 2 μm .","Graphs display mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 007 In contrast , Dcp2 , a Q/N-rich component of the reversible P-body mRNP aggregates ( Reijns et al . , 2008 ) , did not accumulate into the age-associated deposit ( Figure 2H , I ) , even after induction of P-body formation by reducing the glucose level in the medium to 0 . 1% ( Decker et al . , 2007 ) ( 0/57 Hsp104 foci with Dcp2-GFP , Figure 2J ) .","Furthermore , we never observed age-associated protein deposits and P-bodies fusing with each other ( Figure 2J ) .","This suggests that P-bodies and age-associated protein deposits have different physicochemical properties , representing two distinct modes of cytosolic sub-compartmentalization ( Hyman et al . , 2014; Kroschwald et al . , 2015 ) .","Altogether , these data show that the substrates of the age-associated deposit are non-random and possibly amyloid-like , which might explain their irreversible nature .","During recent years , numerous distinct protein deposits , including Q-bodies , JUNQ/INQ , and the IPOD , have been discovered and thus we wanted to explore whether the age-associated protein deposit matches the identity of any of these structures .","We first looked at the behavior of Q-bodies , which assemble in response to heat-stress .","In stark contrast to the age-associated aggregates ( Figure 1G , I ) , Q-bodies induced upon heat stress ( 42°C , 30 min ) were transient and readily solubilized after the removal of the stress factor ( Figure 3A; Liu et al . , 2010; Escusa-Toret et al . , 2013; Wallace et al . , 2015 ) .","Comparative analysis of the localization of different chaperone proteins between Q-bodies and the age-associated protein deposit showed that all markers of the age-associated protein deposit were also found to accumulate in Q-bodies ( Figure 3B ) .","Conversely , we found chaperones such as Hsp82 and Btn2 localizing to Q-bodies ( Figure 3B ) albeit being excluded from the age-dependent protein deposit ( Figure 1C ) .","Together , the reversible nature and the difference in associated chaperones demonstrate that the Q-bodies and age-associated protein deposits can be distinguished . 10 . 7554/eLife . 06197 . 008Figure 3 . The age-associated protein deposit can be distinguished from Q-bodies , JUNQ/INQ , and IPOD .","( A ) Cells were heat shocked ( 42°C for 30 min ) to induce the formation of Q-bodies ( stress induced Hsp104-labeled aggregates ) and the kinetics of Q-bodies dissolution was followed with time-lapse microscopy of Hsp104-GFP .","( B ) Cells were imaged after heat shock ( 42°C for 30 min ) .","Arrowhead in 3D-projected images depicts co-localization of Hsp104 with age-associated deposit-resident ( Ssa1 , Hsp42 ) and non-resident ( Hsp82 , Btn2 ) markers in Q-bodies ( compare with Figure 1C ) .","( C ) The expression of JUNQ/INQ marker VHL-GFP was induced at the start of the imaging to follow its recognition by Hsp104-mCherry .","( D ) Quantification of VHL-GFP foci recognized by Hsp104-GFP within 30 min of their appearance ( N = 29 cells ) .","( E ) 3D-projected images of RPN4 deleted cells expressing Hsp82-GFP and Hsp104-mCherry .","Hsp82-GFP , which localizes to Q-bodies ( Figure 3A ) but not to age-associated protein deposits ( Figure 1C ) , co-localizes with one of the two Hsp104-mCherry foci in aged cell ( see fluorescence intensity line-scan over the two foci ) .","( F ) 3D-projected image of Btn2-GFP and Hsp104-mCherry expressing cell .","Btn2 is typically very low abundant and does not accumulate ( 98 . 6% of cases , N = 72 ) to the age-associated deposit ( white arrowhead ) .","( G ) Cell with a Btn2 focus , which typically ( 96 . 4% of cases , N = 30 ) did not display accumulation of Hsp104-mCherry ) .","( H ) Rnq1-overexpression was induced at the onset of imaging .","The panel shows the z-sections displaying the age-associated deposit ( white arrowhead in ‘merge’ ) .","( I ) Quantification of newly formed Rnq1-GFP foci that accumulate Hsp104-mCherry within 30 min of their appearance ( N = 53 cells ) .","( J ) Representative image of [PIN+ , PSI+] cell harboring GFP-tagged endogenous Rnq1 and mCherry-tagged Hsp104 .","Arrowhead indicates the age-associated protein deposit .","( K ) Example of a [PIN+ , PSI+] cell that displays an Rnq1-aggregate .","Arrowhead indicates Hsp104-labeled foci , of which only one has accumulated Rnq1 .","Scale bars: A-B , E , J-K 5 μm , C , G 2 μm .","Graphs display mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 008 We then examined whether the age-associated protein deposit would bear the characteristics of the JUNQ/INQ compartment , which is formed during prolonged stress and/or in response to proteasome inhibition .","To this end , we induced the GAL-promoter-driven expression of the JUNQ/INQ marker , the unstable proteasome substrate human von Hippel-Lindau tumor suppressor protein ( VHL ) ( Kaganovich et al . , 2008; Miller et al . , 2015 ) , and monitored the accumulation of Hsp104-mCherry into the newly forming VHL-foci .","VHL typically formed a single focus , which in 87% of the cases did not accumulate Hsp104-mCherry within 30 min after its appearance ( Figure 3C , D , N = 29 ) .","However , the VHL expression was often associated with overall increased Hsp104-mCherry expression over time , suggesting that its expression activates a stress response .","Hence , we visualized the JUNQ/INQ without over-expression of exogenous substrates .","To accomplish this , we used an Hsp82-GFP , Hsp104-mCherry expressing strain deleted of the RPN4 gene , leading to increased burden of proteasomal substrates , due to decreased amount of functional proteasomes ( Xie and Varshavsky , 2001 ) .","Indeed , many of rpn4Δ cells displayed numerous Hsp82-Hsp104 double-positive Q-body-like puncta ( data not shown ) .","In addition , we found cells that displayed two puncta: a Hsp104-Hsp42 double-positive JUNQ-like deposit , and a Hsp104-positive , Hsp82-negative deposit that fills the criteria of the age-associated deposit ( Figure 3E ) .","To consolidate this further , we investigated the co-localization of the endogenous JUNQ/INQ marker Btn2 ( Miller et al . , 2015 ) together with the age-associated protein deposit ( Hsp104 ) .","This analysis showed that in 98 . 6% of the cases , Btn2 did not localize to the age-associated Hsp104-foci ( Figure 1C and Figure 3F ) .","Moreover , from all thirty identified cells displaying endogenous Btn2 foci , we found only one case in which Hsp104 was enriched at this site ( Figure 3G , N = 1268 cells ) .","Together , these data suggest that the age-associated protein deposit and the JUNQ/INQ are discrete structures that may exist in parallel .","Finally , we monitored the resemblance between the age-associated protein deposit and IPOD .","To visualize cells during IPOD appearance , we imaged Hsp104-mCherry-expressing cells together with the canonical IPOD marker , the galactose-inducible Rnq1-GFP ( Kaganovich et al . , 2008 ) , immediately after placing cells to galactose-containing media .","This showed that the IPOD typically appeared as a single focus to which Hsp104 rapidly accumulated ( Figure 3H , I ) ( >98% of Rnq1-foci displayed accumulation of Hsp104 within 30 min after their appearance , N = 53 ) .","However , by dissecting the Rnq1-GFP appearance dynamics in cell with a pre-existing age-associated protein deposit ( marked by Hsp104-mCherry ) , we found that the aggregating Rnq1-GFP did not accumulate to the age-associated deposit ( see white arrowhead in Figure 3H ) , but rather it formed a new aggregate to which Hsp104 then strongly accumulated , while its intensity at the age-associated protein deposit declined ( Figure 3H ) .","To rule out possible artifacts induced by Rnq1-GFP over-expression , we also looked at the co-localization between the age-associated protein deposit and GFP-tagged endogenous Rnq1 in its prion [RNQ+]-state .","We analyzed altogether 117 Hsp104 foci-containing [PIN+ , PSI+] cells and found that in 98 . 3% of the cases , Rnq1-GFP did not accumulate to this deposit ( Figure 3J ) .","Within this cohort , we found two cells with an Rnq1-GFP-aggregate , which was in both cases enriched with Hsp104-mCherry ( Figure 3K ) .","However , both of the Rnq1-aggregate containing cells also displayed an additional Hsp104 focus to which Rnq1 had not accumulated ( Figure 3K ) .","Together with the results from the Rnq1 over-expression , these data suggest that Rnq1 aggregate is likely to represent a structure that is different from the majority of the age-associated protein deposits .","In sum , these data point out marked differences between the age-dependent protein deposit and the previously characterized Q-bodies , IPOD , and the JUNQ/INQ that could derive from context dependent ( stress vs aging ) differences in cellular responses to protein aggregation .","Appearance of protein aggregates has commonly been associated with defects in protein quality control ( Tyedmers et al . , 2010a ) .","Therefore , we wanted to elucidate if the formation of the age-associated protein deposit is a sign of defective protein quality control .","First , we examined whether aged cells display a decline in dealing with proteotoxic stress .","As shown before , young cells clear heat-induced protein aggregates ( Q-bodies ) rapidly after stress removal ( Figure 3A ) .","Hence , we first wanted to test if this recovery period is affected by the age of the cell ( Figure 4A ) .","We measured the clearance time of Q-bodies ( defined as two or more Hsp104 foci ) between young ( average age 0 . 4 generations , between 0 and 1 generations , N = 70 ) and aged cells ( average age 9 . 2 generations , between 6 and 19 generations , N = 50 ) following acute heat stress ( Figure 4B ) .","Surprisingly , no difference in the mean time of protein aggregate clearance was detected between these populations: young: 71 ±5 min old: 72 ±4 min , n . s . ) ( Figure 4C ) , suggesting that aged cells , despite having a protein deposit , are fit to cope with proteotoxic stress . 10 . 7554/eLife . 06197 . 009Figure 4 . Aged cells are not impaired in handling proteotoxic stress .","( A ) Experimental scheme .","( B ) Representative image of displaying the dynamics of recovery from heat shock induced proteotoxic stress ( Q-bodies ) between aged ( red star ) and young cell ( blue star ) .","( C ) Q-body clearance time of individual cells of the indicated age groups , ( old: average 9 . 2 generations , age between 6 and 19 , N = 50; young: 0 . 4 generations , age between 0 and 1 , N = 70 ) .","( D ) Experimental scheme: Hsp104-mCherry-expressing cells were captured on a temperature-controlled microfluidic device and imaged prior ( −50 min ) , during ( 30 min ) and after ( up to 300 min ) mild heat shock .","It is important to note , that the strength of the heat stress induced on the microfluidic platform is not comparable with Figure 3A , B and Figure 4A–C .","( E ) Single cell analysis of Q-body formation ( cells with >1 Hsp104-foci ) in cells with a pre-existing age-associated protein deposit ( red line , N = 53 ) , cells without a pre-existing deposit ( blue line , N = 97 ) , and cells that did not experience stress ( N = 82 ) .","Data from two independent experiments .","( F ) Transitions in the aggregate state were recorded 50 min before the heat stress and 300 min after the heat stress .","( G ) Quantification of transitions ( N = 32–77 per group , from two independent experiments ) .","White bars indicate cells that did not experience heat stress and red bars denote transitions in aggregate state in heat-stress experienced cells .","Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 009 We then used a temperature-controlled microfluidic device that enabled us to categorize cells depending on their pre-stress age-associated protein deposit-status , and monitor them under the microscope prior to , during and after undergoing acute proteotoxic stress conditions ( Figure 4D ) .","The duration of the Q-body response ( state in which cells display two or more Hsp104-foci ) was plotted over time and demonstrated that cells with and without a pre-existing age-associated deposit showed a comparable Q-body response when heat was applied , while cells that were not exposed to heat did not show similar Q-body response during this time period ( Figure 4E , N = 53–97 cells ) .","Interestingly , the deposit-containing cells responded slightly faster and to a lesser extend when compared to their clonal counterparts without a pre-existing protein deposit ( Figure 4E ) .","We then asked if exposure to proteotoxic stress ( Q-body state ) would promote the formation of age-associated protein deposits .","To this end , we compared the aggregate status of single cells ( Hsp104-mCherry: no/one/several puncta ) at time points 0 and 380 min , between stress-experienced ( between 50 and 80 min ) and non-stressed ( constantly at 30 °C ) cells ( Figure 4F , N = 32–77 per group ) .","This analysis showed that the majority of non-stressed cells ( 62 . 5% ) without a deposit at the beginning of the experiment displayed a single aggregate at the end of the experiment ( Figure 4G ) , in accordance with the appearance kinetics of age-associated deposit ( Figure 1B , D ) .","Surprisingly , when the cells without a prior aggregate encountered heat stress ( i . e . conditions that induce proteotoxic stress ) , a substantially smaller portion of them ( 36 . 4% ) displayed an aggregate at the end of the experiment ( Figure 4G ) .","Similarly , there was a slight increase of cells without an aggregate in the stress-encountered cohort ( 17 . 7% vs 6 . 8% ) among cells that displayed an age-associated deposit at the beginning of the experiment ( Figure 4G ) .","However , irrespectively of stress , cells with an age-associated deposit at the beginning of the experiment preferentially displayed a single deposit at the end of the experiment ( non-stressed 82% , stress-experienced 79% ) ( Figure 4G ) .","These results indicate that mild exposure to proteotoxic stress conditions counteract the formation of age-associated protein deposits and demonstrate that cells with an age-associated deposit prior to encountering the stress predominantly reverted back to this state .","Altogether , these data indicate that despite forming a protein deposit , aged cells are still fit to coping with acute proteotoxic stress and suggest that the formation of the age-dependent protein deposit is not due to the overload of the general quality control machinery .","The notion that aged cells with a protein deposit handled proteotoxic stress comparably to their young counterparts prompted us to test the effect of the age-associated deposit on the function of the ubiquitin–proteasome system ( UPS ) , which has an important role in yeast replicative aging ( Kruegel et al . , 2011 ) .","Pathological polyQ protein fragments have been shown to impair the UPS function both in yeast and mammalian cells ( Bennett et al . , 2005; Park et al . , 2013 ) , and UPS substrates have been shown to accumulate in aged cells ( Andersson et al . , 2013 ) .","On the other hand , stress-induced protein aggregates ( Q-bodies ) do not result from failed protein degradation , but may actually benefit the proteostasis of cells undergoing stress ( Escusa-Toret et al . , 2013 ) .","We therefore wanted to analyze the contribution of the age-associated deposit on the functionality of the UPS .","In order to selectively measure the effect of the age-associated protein deposit on UPS function , we made use of the auxin-inducible degron ( AID ) system ( Nishimura et al . , 2009 ) , which enabled us to measure the degradation rate of cytosolic ( AID-GFP ) and nuclear ( AID-GFP-NLS ) UPS substrates in vivo ( Figure 5A , B ) .","Cells co-expressing Hsp104-mCherry and AID-GFP were switched to auxin-containing media at the onset of imaging , and the GFP intensities were measured over time from neighboring cells containing or not an Hsp104-puncta .","The normalized values were pooled and fitted with a non-linear one-phase decay function .","Importantly , this showed that the decay rate of AID-GFP was significantly accelerated in cells with an age-associated deposit when compared to neighboring cells that did not contain an Hsp104-focus ( Figure 5C , D ) ( rate constant ( K ) : 0 . 155 ± 0 . 007 ( with ) vs 0 . 129 ± 0 . 006 ( without ) , p < 0 . 01 , N = 91 cells/category ) .","However , no difference was detected in the decay rate of nuclear GFP between cells with or without an age-associated protein deposit ( Figure 5E , F ) ( K: 0 . 075 ± 0 . 004 ( with ) vs 0 . 079 ± 0 . 005 ( without ) , n . s . ) , demonstrating that the effect of the age-associated protein deposit on the UPS function is specific to the cytosolic compartment .","Collectively , these results suggest that the age-associated deposit promotes cytosolic quality control by enabling more efficient clearance of degradation substrates . 10 . 7554/eLife . 06197 . 010Figure 5 . Presence of age-associated protein deposit promotes the function of cytosolic ubiquitin–proteasome system in vivo .","( A ) Schematic representation of the auxin-induced degron ( AID ) -system ( Nishimura et al . , 2009 ) .","Addition of auxin facilitates the recognition of the degron-motif in the target protein ( GFP ) by the exogenously expressed Arabidobsis thaliana E3 ligase SCF-Tir1 and subsequent ubiquitination and degradation by the endogenous ubiquitin–proteaome system ( UPS ) .","( B ) Representative images of cells expressing Hsp104-mCherry and AID-GFP taken immediately ( 0 min ) and 48 min after auxin-addition .","( C ) Examples of time frames GFP degradation in the absence of auxin ( upper panel ) in the presence of auxin in cells with ( middle ) or without ( below ) an age-associated protein deposit .","( D ) The decay rates of GFP in the indicated groups .","The error bars depict the normalized intensity values ( average ± SEM ) derived from 53 ( ctrl ) and 91 ( auxin added ) cells from three pooled replicates .","The solid lines indicated non-linear one-phase decay fit .","( E ) GFP-NLS decay in the representative groups .","( F ) The decay rates and the graph fitting of NLS-GFP in the indicated groups as in ( D ) .","N = 15 ( ctrl ) , 65–66 ( auxin added ) cells from three pooled replicates .","The error bars depict the normalized intensity values ( average ± SEM ) , while the solid lines indicate the non-linear one-phase decay fit . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 010 Next , we wanted to understand what are the assembly principles of the age-associated deposit .","Molecular chaperones generally recognize , refold , or sort aberrantly folded proteins , making them prime candidates to regulate the age-associated protein deposit assembly .","Thus , we addressed the contribution of chaperones localizing to the age-associated protein deposit to its formation .","Time-lapse microscopy showed that in 24 out of 25 cells , Hsp42 was at the age-associated protein deposit before Hsp104 and was the first protein we find to mark this structure ( Figure 6A ) .","Ssa1 and Hsp104 were recruited subsequently , appearing concurrently up to 120 min after appearance of the Hsp42-focus ( Figure 6A and data not shown ) .","To identify the role of these chaperones in the assembly of the age-associated protein deposit , we investigated the consequences of their deletion .","Interestingly , loss of the Hsp70 proteins Ssa1 and Ssa2 , which act synergistically with the disaggregase Hsp104 in protein refolding ( Glover and Lindquist , 1998; Kim et al . , 2013 ) , resulted in rapid formation of the Hsp104 focus ( Figure 6B , C ) and a similar , but milder effect was detected upon deletion of HSP26 .","In stark contrast , deletion of HSP42 abolished age-associated protein deposit formation ( Figure 6B , C ) , which parallels to its function in Q-body assembly ( Specht et al . , 2011; Escusa-Toret et al . , 2013 ) .","To probe the role of Hsp104 and Hsp42 further , we analyzed the consequences of their over-expression on age-associated protein deposit formation ( monitored by endogenous Hsp104-GFP ) .","Over-expression of Hsp42 caused precocious nucleation of age-associated protein deposit and promoted its growth ( Figure 6D–F ) .","On the contrary , a significantly smaller fraction of cells over-expressing Hsp104 contained an age-associated protein deposit and while age-associated deposits still occasionally formed , they were rapidly destabilized ( Figure 6D–F ) .","Collectively , these data suggest that these chaperones functionally oppose each other ( Figure 6G ) : the Hsp104/Hsp70 proteins prevent age-associated protein deposit formation possibly by refolding cytoplasmic substrates , whereas Hsp42 coordinates the collection of these substrates from the cytoplasm into the age-associated protein deposit ( Figure 6G ) . 10 . 7554/eLife . 06197 . 011Figure 6 . Identification of the roles of chaperones in age-associated protein deposit assembly .","( A ) Representative time-lapse images of cells expressing endogenously tagged Hsp42-GFP ( upper panel ) and Hsp104-mCherry ( middle panel ) .","Arrowhead depicts the age-associated protein deposit structure .","( B ) Representative images of aged Hsp104-GFP expressing wild-type , ssa1Δ , and hsp42Δ mutant cells .","( C ) Quantification of Hsp104-foci containing cells in indicated age groups , ( N = 317–1020 cells per genotype ) .","( D ) Time-lapse images of cells expressing endogenous Hsp104-GFP in wild-type , Hsp104 over-expressing , and Hsp42 over-expressing cells ( age-associated protein deposit depicted by a red arrowhead ) .","( E ) Quantification of percentage of cells with age-associated protein deposit in wild-type , Hsp104 over-expressing , and Hsp42 over-expressing cells of indicated age groups ( N = 432–1020 cells per genotype ) .","( F ) Quantification of endogenous Hsp104-GFP integrated density ( age-associated protein deposit/cytoplasm ) following its appearance in wild-type ( black line ) and Hsp42 over expressing cells ( red line ) , ( N = 19–23 ) .","( G ) A summarizing model of the pathway underlying age-associated protein deposit formation .","Scale bars 5 μm .","Graphs display mean ± SEM , n . s not significant , *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 011 To consolidate this model , we further investigated the interplay between the refolding machinery ( Hsp70/Hsp104 ) and the nucleation/growth promoting Hsp42 .","We used cells expressing Ssa1-GFP to visualize the age-associated protein deposit in the absence of Hsp104 ( Figure 7A ) .","Similarly to what we found for the deletion of SSA1 and SSA2 ( Figure 6B , C ) , the hsp104Δ mutant cells formed the age-associated protein deposit more rapidly during aging .","Again , the deletion of HSP42 had an opposite effect , hampering formation of the Ssa1-GFP focus ( Figure 7A , B ) .","Remarkably , age-associated protein deposit formation was severely impaired in the hsp104∆ hsp42∆ double-mutant cells .","These cells were crowded with punctate Ssa1-GFP ( speckles ) , which failed to coalesce into a single regular age-associated protein deposit ( Figure 7A , B ) .","The portion of cells that contained multiple Ssa1-GFP foci was significantly higher in hsp104Δ hsp42Δ double-mutant cells than in hsp104Δ single-mutant cells .","Interestingly , whereas >80% of wild-type and hsp104∆ mutant cells that contained an aggregate displayed a single Ssa1-GFP focus , the majority of hsp42∆ and hsp104∆ hsp42∆ mutant cells harbored several foci ( Figure 7C ) . 10 . 7554/eLife . 06197 . 012Figure 7 . The assembly of age-associated protein deposit is promoted by Hsp42 and counteracted by Hsp104/Hsp70 . ( A ) Representative aged wild-type , hsp104Δ , hsp42Δ and hsp104Δ and hsp42Δ double-mutant cells .","( B ) Quantification of the fraction of cells in indicated age groups that display Ssa1-GFP foci ( age-associated protein deposit ) , ( N = 363–493 cells per genotype ) .","( C ) Quantification of the portion of cells that display >1 Ssa1 foci from all Ssa1-foci containing cells , ( N = 363–493 cells per genotype ) .","( D ) Illustration of the experimental scheme of the reconstitution assay .","Cells that lack both HSP104 and HSP42 and display fragmented aggregate phenotype were mated with cells of the opposite mating type to reintroduce either Hsp104 and/or Hsp42 .","The resulting zygote was imaged with time-lapse microscopy and analyzed 5 min after fusion to score for aggregation phenotype ( analyzed by Ssa1-GFP ) .","( E ) Quantification of the Ssa1-GFP phenotype ( no foci , one focus , multiple foci ) 5 min post fusion , ( N = 73–100 fusion events per genotype ) .","Scale bars 5 μm .","Graphs display mean ± SEM , n . s not significant , **p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 012 To decisively dissect the individual contributions of Hsp104 and Hsp42 on age-associated protein deposit formation , we conceived an in vivo reconstitution assay where we could rapidly re-introduce either Hsp104 or Hsp42 to the aggregate-enriched hsp104Δ hsp42Δ double-mutant cells .","To accomplish this , we mated hsp104Δ hsp42Δ double-mutant cells with wild-type ( re-introducing both Hsp104 and Hsp42 ) , hsp104∆ single-mutant ( to reintroduce only Hsp42 ) , hsp42Δ single-mutant ( to reintroduce only Hsp104 ) , or with the hsp104 hsp42 double-mutant cells ( reintroducing none of the chaperones ) ( Figure 7D ) .","We monitored the deposit by imaging the fusing cells at 1-min intervals , using Ssa1-GFP as a reporter ( Figure 7D ) .","Importantly , simultaneous reintroduction of Hsp104 and Hsp42 was sufficient to clear the cytoplasm of Ssa1-GFP speckles already within 5 min ( Figure 7E , no foci ) .","In contrast , introducing Hsp42 alone resulted in rapid disappearance of cytoplasmic speckles with concurrent formation of a single Ssa1-GFP focus ( Figure 7E , one focus ) .","In contrary , reintroduction of only Hsp104 led to the slow and incomplete clearance of the Ssa-1 speckles , which was a reminiscent , but a milder phenotype as that observed upon conjugating the cell with hsp104Δ hsp42Δ double-mutant partner ( Figure 6E , multiple foci ) .","Collectively , this analysis supports the conclusion that Hsp42 functions to collect aggregates into the age-associated protein deposit structure , whereas Hsp104 functions to disaggregate/refold age-associated protein deposit destined substrates .","In the context of the age-associated protein deposit cargo Sup35 ( Figure 2 ) , it is interesting to note that Hsp42 over-expression promotes [PSI+] curing , while its deletion results in enhanced [PSI+] induction , further supporting its role as the depositor for the age-associated protein deposit ( Duennwald et al . , 2012 ) .","We then asked whether age-associated protein deposit formation ensures the asymmetric inheritance of protein aggregates by the aging lineage .","Examining its mitotic segregation ( monitored with Ssa1-GFP ) in 96 wild-type and 186 hsp104Δ mutant cell divisions demonstrated that the age-associated protein deposit was in both cases inherited by the aging mother cell with high fidelity ( in 98% of divisions; data not shown ) .","In strong contrast , Ssa1-GFP foci were frequently inherited by the daughter cells when both HSP104 and HSP42 were deleted ( Figure 8A , Video 2 ) .","By examining 61 mitotic events by time-lapse microscopy in the double-deleted cells , we observed Ssa1-GFP-foci relocating from the mother to the bud in 53 cases , but did not find any biased retrograde movement from the bud to the mother ( Fig . 8A , Video 2 and data not shown ) .","Quantification of the percentage of buds with protein deposits demonstrated that more than 65% of all hsp104Δ hsp42Δ double-mutant buds displayed at least one aggregate , compared to less than 4% in hsp104Δ and hsp42Δ single-mutant and in wild-type cells ( Figure 8B ) .","Altogether , these data provide evidence that collection of protein aggregates into a single deposit by Hsp42 promotes their asymmetric retention in the mother cell during cell division . 10 . 7554/eLife . 06197 . 013Figure 8 . Age-associated protein deposit formation establishes asymmetric inheritance of protein aggregates and correlates with replicative age .","( A ) Time-lapse imaging of Ssa1-GFP in hsp42Δ hsp104Δ double-mutant cells ( arrowheads denote Ssa1-GFP aggregates relocating from the mother cell to the bud ) .","( B ) Quantification of Ssa1-GFP foci found in the buds of mitotic yeast cells ( N = 216–434 buds per genotype ) .","( C ) Replicative aging experiments of wild-type ( black line , 27 generations ) , hsp104Δ mutant ( blue line , 21 . 5 generations ) , hsp42Δ mutant ( red line , 39 generations ) , and hsp104Δ and hsp42Δ double mutant ( purple , 23 generations ) single cells , ( N = 101–140 cells per genotype ) .","( D ) A schematic model for the age-associated protein deposit pathway: Hsp42 acts as a collector of protein aggregate seeds and promotes their deposition at the ER membrane ensuring their asymmetric inheritance by the aging lineage during mitosis .","These cytoplasmic seeds are subjected to Hsp104/Hsp70-dependent refolding , which is inactive ( blue rectangle ) at the site of the age-associated protein deposit assembly .","Scale bar 5 μm .","Graph displays mean ± SEM , n . s not significant , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 01310 . 7554/eLife . 06197 . 014Video 2 . The lack of the age-associated protein deposit assembly pathway results in the inheritance of the aggregates by the daughter cells . Expression of Ssa1-GFP was followed in hsp104Δ hsp42Δ double-mutant cell as it underwent cell division . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 014 Finally , we tested the significance of the age-associated protein deposit pathway and asymmetric protein aggregate segregation on replicative aging ( Figure 8C ) .","In accordance to previous work ( Erjavec et al . , 2007 ) , mutant hsp104∆ cells were short-lived compared to wild-type cells ( 21 . 5 vs 27 generations , p < 0 . 001 ) , supporting the idea that accelerated aggregate accumulation promotes aging .","Further supporting this notion , deletion of HSP42 extended the lifespan of the cells by more than 40% ( 39 generations , p < 0 . 001 ) .","The lifespan of the hsp104Δ hsp42Δ double-mutant cells was similar to that of the hsp104Δ single-mutant cells ( 23 vs 21 . 5 generations , respectively , ns . ) and significantly shorter than that of the wild-type ( p < 0 . 01 ) and hsp42Δ mutant cells ( p < 0 . 001 ) .","The over-expression of HSP42 had no effect on the lifespan ( data not shown ) , suggesting that the wild-type levels of Hsp42 are sufficient to provide its maximal effects on life span , which is consistent with the appearance of the deposit early in life span in the wild-type cells .","Together , these data imply that the Hsp104/Hsp70 system , which counteracts protein aggregation , is essential for longevity , while the Hsp42-dependent circuit promotes aging in the mother lineage , presumably by building the age-associated protein deposit and thereby establishing the asymmetric inheritance of age-associated protein aggregates ."],["Protein aggregation has been frequently associated with aging and aging-associated diseases .","Here , by using the budding yeast as a model for replicative aging , we identify an Hsp42-promoted and Hsp104/Hsp70-counteracted pathway that deposits age-associated protein aggregates and thereby ensures their biased segregation to the aging mother-lineage upon cell division ( see model in Figure 7D ) .","This might represent a common mechanism to drive asymmetric segregation of aberrant proteins in dividing cells , as stressed fission yeast cells utilize a similar Hsp16-dependent mechanism for asymmetric partitioning of misfolded proteins ( Coelho et al . , 2014 ) .","Intriguingly , our data suggest that depositing age-associated protein aggregates to the mother cells might be a mixed blessing .","On one hand , the age-associated deposit can promote spatial protein quality control ( Figures 4 , 5 ) and promote cell diversity as an asymmetrically dividing structure that may harbor protein conformation encoded epigenetic information , such as the prion fold of Sup35 ( Figure 2 ) .","At the same time , the long-term consequences of this pathway appear negative for the aging lineage ( Figure 8C ) .","Why the deposit-forming pathway ultimately becomes associated with age-dependent loss of fitness remains to be determined .","In this regard , it will be of great importance to elucidate how age-associated deposit relates to other aging pathways , such as those influencing the function of mitochondria ( McMurray and Gottschling , 2003; McFaline-Figueroa et al . , 2011 ) , vacuoles ( Hughes and Gottschling , 2012 ) and nuclear pores ( Shcheprova et al . , 2008; Denoth-Lippuner et al . , 2014; Webster et al . , 2014 ) , and how the deposit localizes relative to these organelles and their components ( Chong et al . , 2015 ) .","Protein deposit-containing cells efficiently coped with acute proteotoxic stress ( Figure 4 ) and displayed improved degradation of cytosolic UPS substrate during early- to mid-life span ( Figure 5 ) .","It is important to note , however , that our assays were conducted with relatively young cells that had recently acquired the protein deposit , and hence it is possible that UPS function starts to decline later during the aging process , as shown previously ( Andersson et al . , 2013 ) .","Curiously , the decline in proteostasis has been associated with protein aggregation pathologies such as Alzheimer's , Huntington's , and Parkinson's disease ( Vilchez et al . , 2014 ) .","The decreased degradation of cytosolic UPS substrates in a polyQ disease model was found to be due to the sequestration of the Hsp40 protein Sis1 ( Park et al . , 2013 ) —a protein that was not associated with the age-associated deposit ( Figure 1C ) .","It is thus plausible that the presence of the deposit might , for example , lead to increased levels of available ( substrate unbound ) Sis1 , thereby allowing more efficient nuclear import and degradation of cytosolic UPS substrates .","Altogether , these data suggest that there is a need to better differentiate between pathophysiological protein aggregation and ‘homeostatic’ protein aggregation that takes place during physiological aging , of which the latter might initially help to coordinate chaperones and UPS factors involved in protein quality control .","Related to this , the age-associated protein deposit did not clearly fill out the set criterion for any of the previously characterized cytosolic deposits , including Q-bodies , JUNQ/INQ , or IPOD , but rather seems to exist in parallel with the JUNQ and IPOD structures ( Figure 1 , Figure 3 ) —although , for example , the chaperones regulating these deposits were often shared between these structures .","This favors the notion that pathological aggregation processes ( mimicked e . g . , by VHL and Rnq1 over-expression ) can hijack endogenous proteostasis regulatory mechanisms , possibly underlying their harmful effects in cells ( Park et al . , 2013 ) .","Altogether , these apparent differences between the stress-induced , pathological , and aging-associated protein aggregates ( Figure 2 , Figure 3 ) demonstrates that protein aggregates are not all equal in their composition or in the way they are being recognized and dealt by the cell and reinforces the importance to discriminate between different aggregation models .","In the future , it will be important to identify the cargo deposited to the age-associated protein deposits .","In this aspect , it is interesting to note that many of the long-lived asymmetrically retained proteins that accumulate to mother cells as they age ( Thayer et al . , 2014 ) were among those localizing to the age-associated protein deposit ( including Hsp104 , Ssa1 , Ssa2 , and Hsp26 ) .","One example of an age-associated deposit resident protein was the prion-like Sup35 .","This storage rendered Sup35 to be inherited by the mother cells ( unlike a prion , analogous to a mnemon ( Caudron and Barral , 2013 ) ) ( Figure 2 ) , which is consistent with the size-dependent transmission model of Sup35-aggregates ( Derdowski et al . , 2010 ) .","However , aging does not enhance Sup35-dependent [PSI+] prion generation ( Shewmaker and Wickner , 2006 ) .","Together , these notions suggest that Sup35 cannot escape the age-associated deposit under normal growth conditions , perhaps owing to the rigid amyloid-like packing ( Saibil et al . , 2012 ) , being consistent with the durable and non-dynamic nature of these deposits .","However , this notion may come with a caveat: we found that the age-associated deposit could be , at least temporarily , disassembled by the heat shock response ( Figure 4 ) .","This suggests the captivating possibility that changes in the environment might trigger the spread from the age-associated deposit .","For cells facing fluctuating environments in the wild , the strategy to stochastically store prion-like proteins in aged cells might prove beneficial , as it could allow stress-dependent spread of the prion conformers from a subpopulation of aged-cells to their daughters , promoting faster adaptation to the changing environment ( Newby and Lindquist , 2013 ) .","Since asymmetric inheritance of damaged proteins and protein aggregates seems to be conserved in metazoan stem cells ( Rujano et al . , 2006; Bufalino et al . , 2013 ) that are also subjected to both replicative decline and segregation of lineages , we hypothesize that similar pathways that selectively segregate protein-aggregate based fate determinants asymmetrically during cell division are likely to be conserved and may contribute to cellular lineage specification across species ."],["Yeast strains used in this study are listed in Supplementary file 1 .","Strains were generated either manually ( Janke et al . , 2004 ) or derived from collections: ( http://web . uni-frankfurt . de/fb15/mikro/euroscarf/col_index . html or http://clones . lifetechnologies . com/cloneinfo . php ? clone=yeastgfp ) .","Plasmids ( pAG415-GDP ) over-expressing Hsp104 ( O-1360 ) and Hsp42 ( O-2252 ) , and the [PIN+][PSI+] ( yJW508 ) and [PIN+][psi–] ( yJW584 ) parental strains ( Osherovich and Weissman , 2001 ) were a generous gift from Simon Alberti ( Max Planck Institute of Molecular Cell Biology and Genetics Dresden , Germany ) .","The Mother Enrichment parental strains ( Lindstrom and Gottschling , 2009 ) were a kind gift from Dan Gottschling ( Fred Hutchinson Cancer Research Center , Seattle USA ) and the plasmids encoding VHL-GFP and Rnq1-GFP were provided by Judith Frydman ( via Addgene ) .","The integrative plasmids for the auxin-mediated degradation assays ( pADH1-OsTIR1-9myc , pADH1-eGFP-IAA17-NLS , pADH1-eGFP-IAA17 ) have been described in ( Nishimura et al . ) and were kindly provided by Matthias Peter ( ETH Zurich , Switzerland ) .","Cells subjected to imaging were re-inoculated from overnight cultures to O . D . 600 nm 0 . 05 in YPD and grown to O . D . 0 . 5–0 . 8 at 30°C , centrifuged 500 g for 5 min , resuspended in synthetic complete ( SC ) media ( -his ) , and mounted between a coverslip and an agar pad ( SC-his ) .","The over-expression of Rnq1-GFP and VHL-GFP was induced by switching cells to 2% galactose at the onset of imaging .","The auxin-mediated degradation assays were performed similarly as above by placing cells to 0 . 5 mM 3-Indoleacetic acid ( Sigma–Aldrich , #12886 ) at the onset of imaging .","The MEP was performed as described in ( Lindstrom and Gottschling , 2009 ) .","Briefly , cells were grown in log phase for 15 hr before coupling the cell wall with Sulfo-NHS-LC-Biotin ( Pierce ) .","Following 2 hour recovery period , the MEP was engaged by addition of estradiol to a 1 μM final concentration .","After desired incubation period , cells were coupled to uMACS Streptavidin MicroBeads ( Miltenyi Biotec ) and affinity purified with MACS separation columns ( Miltenyi Biotec ) .","Calcofluor staining was done by incubating cells with 5 μg/ml Fluorescent Brightener 28 ( Sigma–Aldrich ) for 1 min prior to centrifugation and resuspension to SC media .","The Q-bodies were induced by incubating cells in a water bath at +42 °C for 30 min , or by using a temperature-controlled microfluidic chamber ( see microfluidics ) .","For the mating experiments , cells ( 1 . 85×107 ) of the opposite mating type were centrifuged 500 g for 5 min and resuspended to 40 μl SC media , mixed and immediately imaged with 1-min intervals ( see microscopy for details ) .","Wide-field microscopy was performed at 30 °C with a DeltaVision microscope coupled to a coolSNAP CCD HQ2 camera ( Roper ) , 250W Xenon lamps and 100X/1 . 40 and 60x/1 . 42 NA Olympus oil immersion objectives .","Z-sectioning was performed with 500-nm spacing ( unless otherwise indicated ) , obtaining 9 or 11 ( live-cell imaging ) or 15 ( calcofluor stained cells ) stacks .","Images were deconvolved with Softworx software ( Applied Precision ) .","Microfluidics on the aging chip were imaged with a DeltaVision microscope every 45 min for total of 66 hr by obtaining seven z-sections with 0 . 6-μm spacing .","FRAP was done with temperature-controlled Zeiss LSM 510 microscope controlled with AIM LSM4 . 0 software , 63x 1 . 4NA Oil DIC Plan-Apochromat objective at 30°C , using DPSS and Argon lasers .","Diploid cells expressing HSp104-GFP/Hsp104mCherry were imaged every 5 s at three z-planes with 800-nm spacing five times before bleaching the mCherry signal at the age-associated protein deposit with 100% DPSS laser power , after which the recovery of mCherry at the age-associated protein deposit was monitored over the period of two minutes by orienting with the GFP-signal .","The temperature ramp experiments ( Figure 4 ) were carried out with ONIX microfluidic perfusion system equipped with a micro-incubator temperature controller CellASIC ) , using Y04C microfluidic plates ( CellASIC ) .","The temperature setup was the following: 30°C ( 50 min ) , 42°C ( 30 min ) 30°C ( 300 min ) and the cells were imaged every 10 min as described in ( microscopy ) .","The experiments were carried out in synthetic full medium with even flow rate of 2 psi .","Microfluidics on the aging chip was performed with 1 . 5 μl/min flow rate as described in ( Denoth-Lippuner et al . , 2014 ) .","All image analyses were performed with Image J software ( http://imagej . nih . gov/ij/ ) .","The aggregation foci were scored by eye from maximum intensity projected images ( spanning the entire volume of the cell ) and were defined as puncta that display high-intensity over the surrounding cytoplasmic background signal .","For age-associated protein deposit intensity measurements , the integrated density was measured at the site over time at the age-associated protein deposit from stacked , non-processed sum-projected videos and was then normalized to the cytoplasmic Hsp104-GFP intensity .","For the auxin-mediated degradation experiments , the 11-plane z-stack covering 5 . 5 μm was sum projected and an integrated density of a defined area ( 18 μm2 ( GFP ) or 16 μm2 ( GFP-NLS ) was measured over time from the region of interest ( ROI ) and the neighboring background region ( BG ) .","To distinguish between cells that contained an aggregate from those that did not contain an aggregate , cells were categorized based on the first frame Hsp104-mCherry signal into the two respective groups .","The ROI values were background subtracted [ROI ( t ) -BG ( t ) ] and normalized to the first value [ROI ( tx ) /ROI ( t1 ) ] .","The average of the pooled values was fitted with Prism5 software using non-linear one-phase decay .","For the FRAP analysis , raw data were background subtracted , corrected for acquisition-induced bleaching and normalized , and the curve was fitted from pooled values with Prism5 software using one-phase association non-linear fitting .","The FRAP data where the aggregate structure was lost from the focal plane during recovery period image acquisition were discarded .","Lifespan of virgin daughters was analyzed on YPD plates using Zeiss Axioscope 40 microdissection microscope as previously described in ( Denoth-Lippuner et al . , 2014 ) .","All statistical analyses and graph preparations were done with Prism5 software .","The error bars represent ±SEM from experimental triplicates with independent clones and statistical analyses were conducted with one-way ANOVA using Newman–Keuls post test , t-test , or Gehan-Breslow-Wilcoxon test ."]],"string":"[\n [\n \"Aging results in an increasing decline of the organism's fitness over time ( Lopez-Otin et al . , 2013 ) .\",\n \"Remarkably , this process segregates asymmetrically during budding yeast division: the mother cell forms an aging lineage , whereas the daughters generated by these mothers rejuvenate to form eternal lineages , similar to the segregation of soma and germ lineages in metazoans .\",\n \"Such lineage separation requires that the inheritance of factors that promote aging , such as defective/deleterious organelles , proteins , and DNA , is asymmetric during cell division ( Sinclair and Guarente , 1997; Aguilaniu et al . , 2003; Erjavec et al . , 2007; Henderson and Gottschling , 2008; Shcheprova et al . , 2008; Liu et al . , 2010; Zhou et al . , 2011; Hughes and Gottschling , 2012; Higuchi et al . , 2013; Clay et al . , 2014; Denoth Lippuner et al . , 2014; Henderson et al . , 2014; Higuchi-Sanabria et al . , 2014; Thayer et al . , 2014; Katajisto et al . , 2015 ) .\",\n \"Therefore , how cells are able to recognize , sort , and coordinate the asymmetric segregation of aging factors and other fate determinants is an outstanding question in biology ( Neumuller and Knoblich , 2009 ) .\",\n \"Protein aggregates and/or damaged proteins are a hallmark in the etiology of many human disorders associated with aging ( Hartl et al . , 2011; Wolff et al . , 2014 ) , and their presence correlates with aging of mitotically active yeast and drosophila stem cells ( Aguilaniu et al . , 2003; Erjavec et al . , 2007; Bufalino et al . , 2013; Coelho et al . , 2013 ) .\",\n \"Studies on budding yeast have shown a correlation between the accumulation of protein aggregates and replicative aging by demonstrating that Hsp104-mediated protein disaggregation is required for full replicative life span ( Erjavec et al . , 2007 ) , and that over-expression of Mca1 , which counteracts the formation of stress- and age-associated protein aggregates ( Lee et al . , 2010; Hill et al . , 2014 ) , extends the life span of yeast mother cells ( Hill et al . , 2014 ) .\",\n \"How cells respond to protein aggregation that occurs specifically during aging has remained elusive since most studies investigating the cellular responses to protein aggregation have relied on over-expression of non-native , aggregation prone proteins , proteostasis inhibitors , or other stressors , such as heat ( Kaganovich et al . , 2008; Liu et al . , 2010; Specht et al . , 2011; Zhou et al . , 2011; Malinovska et al . , 2012; Spokoini et al . , 2012; Winkler et al . , 2012; Escusa-Toret et al . , 2013; Zhou et al . , 2014 ) .\",\n \"These studies have uncovered specific modes of cytosolic compartmentalization that take place when cells encounter proteotoxic stress .\",\n \"For example , cells stressed with heat respond by forming multiple protein aggregates ( referred to as peripheral aggregates , stress foci , Q-bodies , or CytoQ ) at the surface of the ER ( Specht et al . , 2011; Spokoini et al . , 2012; Escusa-Toret et al . , 2013; Miller et al . , 2015; Zhou et al . , 2014; Wallace et al . , 2015 ) .\",\n \"These structures , hereafter referred as Q-bodies , contain acutely misfolded proteins that are sorted between the nuclear and cytoplasmic degradation/deposit sites by the Hook family proteins Btn2 and Cur1 ( Malinovska et al . , 2012 ) , and coalesce together by the aid of small heat shock proteins; Hsp42 in budding yeast ( Specht et al . , 2011; Escusa-Toret et al . , 2013 ) , and Hsp16 in fission yeast ( Coelho et al . , 2014 ) .\",\n \"Simultaneously , Q-bodies are being rapidly resolved by the protein disaggregase Hsp104 ( Parsell et al . , 1994; Specht et al . , 2011; Zhou et al . , 2011; Spokoini et al . , 2012; Escusa-Toret et al . , 2013 ) , together with other heat shock responsive chaperones such as Hsp70 and Hsp82 ( Escusa-Toret et al . , 2013 ) .\",\n \"The formation of Q-bodies seems to aid stress survival , as the deletion of HSP42 resulted in defective tolerance of prolonged heat stress ( Escusa-Toret et al . , 2013 ) .\",\n \"The asymmetric inheritance of Q-bodies by the mother cells is promoted by the geometry of the bud neck ( Zhou et al . , 2011 ) , tethering to mitochondria ( Zhou et al . , 2014 ) , and by actin cable-mediated retrograde transport , which is dependent of Hsp104 and Sir2 ( Liu et al . , 2010; Song et al . , 2014 ) .\",\n \"Notably , Sir2 is also a key player in processes that underlie the asymmetric segregation of damaged mitochondria ( Higuchi et al . , 2013 ) and the accumulation of extrachromosomal DNA circles ( Sinclair and Guarente , 1997; Kaeberlein et al . , 1999 ) to the aging mother cell .\",\n \"Prolonged Q-body-inducing stress ( heat or over-expression of thermolabile proteins ) combined with proteasome inhibition can lead to the formation of a dynamically exchanging deposit of ubiquitylated proteins named the juxtanuclear quality compartment , JUNQ ( Kaganovich et al . , 2008; Escusa-Toret et al . , 2013 ) .\",\n \"This structure is regulated by the Upb3 deubiqutinase ( Oling et al . , 2014 ) , by proteosomal activity ( Andersson et al . , 2013 ) and by lipid droplets ( Moldavski et al . , 2015 ) , and it was also shown to appear during replicative aging ( Oling et al . , 2014 ) .\",\n \"The faithful inheritance of this structure by the mother cell is dependent on its association with the nucleus ( Spokoini et al . , 2012 ) .\",\n \"More recently , it was shown that the ‘JUNQ’ might actually reside inside the nucleus , and it was thus renamed as intranuclear quality control compartment , INQ ( Miller et al . , 2015 ) .\",\n \"The JUNQ/INQ assembly is dependent on Btn2-aggregase ( Miller et al . , 2015 ) , a protein also found to be involved in prion curing ( Kryndushkin et al . , 2008 , 2012 ) .\",\n \"Apart from the JUNQ/INQ structure , terminally aggregating proteins , such as the amyloidogenic prions Rnq1 and Ure2 , were shown to partition to an non-dynamic , vacuole-associated deposit called the insoluble protein deposit IPOD ( Kaganovich et al . , 2008; Tyedmers et al . , 2010b ) , which has remained less well characterized .\",\n \"Despite this wealth of data , it remains unclear how these exogenous/stress-induced aggregation models relate to protein aggregation that takes place during physiological ‘healthy’ aging .\",\n \"Particularly , it is unclear why/how protein aggregates arise during aging , how are they segregated during cell division and , importantly , what is their consequence to the protein quality control of the aging cell , as well as to the aging process itself .\",\n \"To illuminate these aspects , we probed the role of protein aggregation during unperturbed replicative aging .\",\n \"Our findings indicate that protein aggregation is a prevalent and highly coordinated event of early aging and is not solely associated with proteostasis deterioration .\",\n \"Instead , we provide evidence that age-associated protein aggregation may initially benefit the cytosolic protein quality control , but eventually becomes involved with age-associated loss of fitness .\"\n ],\n [\n \"To address the role of protein aggregation in unperturbed , physiological aging , we analyzed microscopically the replicative age-associated protein aggregation landscape in budding yeast by visualizing different chaperone proteins that mark aberrantly folded and aggregated proteins .\",\n \"By employing the Mother Enrichment Program ( MEP ) ( Lindstrom and Gottschling , 2009 ) ( Figure 1—figure supplement 1A ) , we harvested cells of different age and first analyzed the localization of endogenous GFP-tagged protein-disaggregase Hsp104 ( Parsell et al . , 1994; Glover and Lindquist , 1998 ) , a broad sensor for protein aggregates ( Figure 1A , Haslberger et al . , 2010 ) .\",\n \"Interestingly , we found many cells displaying an aggregate ( typically a single bright Hsp104-labeled focus ) and this portion increased in a progressive , age-dependent manner such that >80% of cells that had undergone more than 6 divisions displayed such a structure ( Figure 1A , B ) , as previously reported ( Aguilaniu et al . , 2003; Erjavec et al . , 2007 ) .\",\n \"Co-localization analysis with Hsp104 demonstrated that the Hsp70 proteins Ssa1 and Ssa2 , the small heat shock protein Hsp42 , and the Hsp40 protein Ydj1 readily localized to these aggregates , the Hsp26 was found to be enriched in only 15% of Hsp104-labeled foci , while no accumulation of Hsp40 protein Sis1 or the Hsp90 protein Hsp82 was detected ( Figure 1C , Figure 1—figure supplement 2A–C ) .\",\n \"Importantly , age-dependent appearance of these structures was also detected in diploid cells , in other strain backgrounds ( W303 ) , independently of the MEP procedure , and when different fluorophores where used for tagging Hsp104 ( Figure 1—figure supplement 2D–H ) , indicating that their formation represents a general , age-dependent phenomenon of budding yeast cells . 10 . 7554/eLife . 06197 . 003Figure 1 . Replicative aging leads to the formation of age-associated protein deposit .\",\n \"( A ) Representative images of cells expressing endogenous Hsp104 tagged with GFP .\",\n \"The C-terminal tagging does not hamper Hsp104 disaggregation activity ( Specht et al . , 2011 ) .\",\n \"Cells of different age were harvested using the Mother Enrichment Program ( MEP ) ( Lindstrom and Gottschling , 2009 ) and stained with calcofluor .\",\n \"( B ) Percentage of cells of different age groups containing at least one Hsp104-focus , ( N = 135 to 472 cells per age group ) .\",\n \"( C ) Fraction of Hsp104-mCherry foci that are enriched with the indicated chaperones ( N = 16 to 92 Hsp104-focus containing cells per strain , n . d . = not detected ) .\",\n \"( D ) Representative frames of a movie of dividing cells expressing Hsp104-mCherry ( black ) .\",\n \"Red arrowhead indicates an aggregate that is retained in the mother cell ( M ) .\",\n \"This cell divided five times giving rise to four daughter cells that start to form aggregates after dividing 2–3 times ( blue arrows at 390 min ) .\",\n \"( E ) Integrated density at newly forming age-associated protein deposits ( deposit/cytoplasm ) over time , ( N = 26 ) .\",\n \"( F ) Fraction of divisions during which the age-associated protein deposit is asymmetrically inherited by the mother cell as a function of the age of the mother cell ( N = 66 to 306 divisions per age group from 2-3 independent experiments . The approximate mother cell age was estimated from separate bud scar analysis ) .\",\n \"( G ) Representative micrographs of a dividing mother cell expressing Hsp104-GFP followed for 66 hr in a microfluidic chip ( Lee et al . , 2012 ) .\",\n \"( H ) Proportion of cells having the indicated number of Hsp104 foci .\",\n \"Hsp104 foci containing cells that could be followed >10 consecutive divisions were quantified for the number of Hsp104-foci/cell at each time point , ( N = 44 ) .\",\n \"( I ) Total time spent with and without an Hsp104-focus for cells undergoing >10 consecutive divisions , starting with a focus , ( N = 44 ) .\",\n \"( J ) Fluorescent recovery after photobleaching ( FRAP ) analysis of Hsp104-mCherry turnover at age-associated protein deposit .\",\n \"The mCherry signal at the age-associated protein deposit was photobleached in HSP104-GFP/HSP104-mCherry diploid cells and the kinetics of recovery were monitored , using the GFP signal to localize the age-associated protein deposit over time .\",\n \"( K ) Fitting of nine recovery curves showed that on average a large ( 59% ) fraction was immobile and the half-time recovery for the mobile fraction was 8 . 9 s , ( N = 9 ) .\",\n \"Scale bars 5 μm .\",\n \"Graphs display mean ± SEM , *p < 0 . 05 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 00310 . 7554/eLife . 06197 . 004Figure 1—figure supplement 1 . Schematic representation of the strategies used here to study aged cells .\",\n \"( A ) Strategy to obtain old cells: cells were biotinylated and grown for ( x ) amount of time in YPD media containing estradiol , which activates the MEP ( Lindstrom and Gottschling , 2009 ) and results in daughter specific cell cycle arrest due to excision of two essential genes UBC9 and CDC20 .\",\n \"After desired time , aged mother cells containing the cell wall-linked biotin coat were coupled to streptavidin microbeads , purified with affinity chromatography , and imaged under the microscope .\",\n \"( B ) The microfluidic system ( Lee et al . , 2012 ) allows microscopic examination of trapped mother cells as they undergo repetitive divisions .\",\n \"Cells are captured between a PDMS pad and a glass cover slip and subjected to constant media flow , which washes away the newly born daughter cells that are not captured between the pad due to their smaller size . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 00410 . 7554/eLife . 06197 . 005Figure 1—figure supplement 2 . Age-associated protein deposit formation is a general age-dependent phenomenon marked by a subset of chaperones .\",\n \"( A–C )\",\n \"Representative images of Hsp104-mCherry expressing cells co-tagged with ( A ) Ssa1-GFP ( B ) Hsp42-GFP , or ( C ) Hsp82 .\",\n \"Age-associated protein deposit are depicted with a red arrowhead .\",\n \"( D ) W303 cells expressing Hsp104-YFP .\",\n \"( E ) S288C cells expressing Hsp104-mCherry or ( F ) Hsp104-GFP .\",\n \"( G ) Diploid S288C cells expressing Hsp104-GFP .\",\n \"( H ) Comparison of the relative amount of age-associated protein deposit containing cells in calcofluor treated MEP and non-MEP background cells vs non-calcofluor treated MEP strain background , ( N = 177–243 cells analyzed per genotype ) .\",\n \"Scale bars: ( A–C ) 2 μm , ( D–G ) 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 005 To characterize the nature of these protein deposits further , we performed live-cell imaging by acquiring images every 15 min over several hours and covering the entire depth of the cell .\",\n \"This showed that most mother cells started to form this aggregate already after budding three to four times ( Figure 1D , 390 min ) .\",\n \"The fastest growth phase of the aggregate took place during the first half an hour following its detection , after which the Hsp104 signal intensity increased only marginally ( Figure 1D , E ) .\",\n \"While the aggregates initially underwent occasional dissolution , they became stable within a few hours from nucleation ( Figure 1E ) .\",\n \"Pedigree analysis of aggregate inheritance in cells of different age showed that they faithfully segregated to the aging mother cell ( 98% of divisions ) , irrespective of its age ( Figure 1D , F , Video 1 ) .\",\n \"To further analyze the persistence and behavior of the deposits over the entire replicative life span of cells , we used a microscope-coupled microfluidic dissection platform ( Lee et al . , 2012 ) ( Figure 1—figure supplement 1B ) .\",\n \"This showed that the aggregate was efficiently maintained as one compartment , and whenever new foci emerged , they typically merged soon after with the pre-existing deposit .\",\n \"We quantified the number of Hsp104 foci ( 1 or >1 ) and its post-nucleation stability in cells that were tracked for at least 10 divisions after the aggregate had appeared .\",\n \"These cells preferentially ( >80% of time ) displayed only a single Hsp104 focus ( Figure 1G–H ) , which was very stable , typically persisting until the last divisions of the cell ( Figure 1G , I ) .\",\n \"Moreover , this structure was largely non-dynamic , displaying limited exchange of Hsp104 with the cytoplasm , as determined by fluorescent recovery after photobleaching ( FRAP ) analysis in diploid cells that expressed one GFP- and one mCherry-tagged copy of HSP104 ( Figure 1J ) .\",\n \"We bleached and measured the recovery of the mCherry signal ( Figure 1J ) and by fitting nine recovery curves , found that on average a large fraction ( 59% ) of Hsp104 was immobile , while the mobile fraction displayed a half-time recovery rate of 8 . 9 s ( Figure 1K ) .\",\n \"Together , these data show that a large majority of unstressed yeast cells develop a protein deposit early during replicative aging .\",\n \"This deposit displays limited exchange with the cytoplasm ( assessed by Hsp104 ) , is efficiently maintained as a single compartment , and is faithfully inherited by the aging mother cell . 10 . 7554/eLife . 06197 . 006Video 1 . Age-associated protein deposit is a stable structure that is faithfully inherited by the aging mother cell lineage . Aged yeast cell expressing Hsp104-mCherry ( in green ) to mark protein aggregates and Cdc10-GFP ( in red ) to mark the mother bud interface undergoing four consecutive divisions . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 006 To better understand how the age-associated protein deposit forms and what are its physiological constituents , we selected two proteins containing glutamine- and/or asparagine-rich domains , Sup35 and Dcp2 , which are known to undergo conformational switches and to aggregate , and we monitored their localization in respect to the age-associated protein deposit .\",\n \"Sup35 ( eRF3 ) is a translation termination factor that can undergo stable amyloid-like prion conversion from non-prion [psi–] to prion state [PSI+] ( Chernoff et al . , 1993; Ter-Avanesyan et al . , 1994; Wickner , 1994; Patino et al . , 1996; Paushkin et al . , 1996 ) .\",\n \"Thus , we tested whether Sup35-GFP is targeted to the age-dependent aggregate and if the prion status plays a role in the targeting to and/or in the formation of the age-associated protein deposit .\",\n \"Importantly , Sup35-GFP clearly accumulated into Hsp104-mCherry-labeled age-associated protein deposit in 33% of [PSI+] cells that contained such a deposit , whereas no accumulation was detected in the non-prion [psi−] cells ( Figure 2A–C ) .\",\n \"As expected , the formation of this bright Sup35 focus was age-dependent ( Figure 2D ) , in line with earlier observations ( Derdowski et al . , 2010 ) , but interestingly the [PSI+] state did not have an overall effect on Hsp104-labeled deposit formation ( Figure 2E ) .\",\n \"Similarly , the elimination of the [RNQ+] prion by deleting RNQ1 did not negatively influence the formation of the age-associated deposit ( data not shown ) .\",\n \"Notably , time-lapse imaging showed that Sup35 was recruited into the pre-existing age-associated protein deposit ( marked by Hsp104-mCherry ) .\",\n \"These Sup35-enriched age-associated protein deposits segregated asymmetrically towards the aging mother cell in 98% of mitoses ( Figure 2F , G ) .\",\n \"These data provide evidence that the Hsp104-labeled focus is a bona fide deposit site for aggregating proteins , and that prion conversion of Sup35 promotes its gradual storage into the age-associated protein deposit , but does not potentiate its formation per se . 10 . 7554/eLife . 06197 . 007Figure 2 . Prion form of Sup35 is deposited to the age-associated protein deposit .\",\n \"( A , B )\",\n \"Co-localization of Sup35-GFP and Hsp104-mCherry in diploid [psi−] ( A ) and [PSI+] ( B ) cells , where a single locus of the respective gene was tagged with the fluorescent marker indicated .\",\n \"White arrowhead indicates the age-associated protein deposit ( Hsp104 positive ) and the red arrowhead Sup35-aggregates not associated with the age-associated protein deposit .\",\n \"( C ) Percentage of age-associated protein deposits in [psi–] and [PSI+] cells with enriched Sup35 , ( N = 130 ) .\",\n \"( D ) Percentage of cells with large Sup35-foci in cells of indicated age groups ( N = 157–184 cells pre age group , average age young [psi–] 1 . 1 , young [PSI+] 1 . 1 , aged [psi–] 6 . 9 , aged [PSI+] 5 . 9 generations ) .\",\n \"( E ) Percentage of [psi–] and [PSI+] cells of the indicated age group ( see D ) containing an age-associated protein deposit , ( N = 157–184 cells pre age group ) .\",\n \"( F ) Time-lapse images of a [PSI+] cell co-expressing Sup35-GFP ( green ) Hsp104-mCherry ( red ) at the indicated time points .\",\n \"Arrowheads point at the age-associated protein deposit as observed in the different channels .\",\n \"The newborn daughters are indicated in the bottom row .\",\n \"( G ) Percentage of divisions where the Sup35-GFP-labeled age-associated protein deposit is retained in the aging mother cell lineage , ( N = 204 divisions ) .\",\n \"( H ) Fluorescent images of a cell co-expressing the P-body protein Dcp2 tagged with GFP and Hsp104 tagged with mCherry .\",\n \"( I ) Percentages of cells of indicated age groups that contain the indicated fluorescent foci , ( N = 18–71 per group ) .\",\n \"( J ) Time-lapse , fluorescent images of Dcp2-GFP and Hsp104-mCherry expressing cells at the indicated time points after switching the cells to 0 . 1% glucose .\",\n \"Scale bars ( A , B , H , J ) 5 μm , ( F ) 2 μm .\",\n \"Graphs display mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 007 In contrast , Dcp2 , a Q/N-rich component of the reversible P-body mRNP aggregates ( Reijns et al . , 2008 ) , did not accumulate into the age-associated deposit ( Figure 2H , I ) , even after induction of P-body formation by reducing the glucose level in the medium to 0 . 1% ( Decker et al . , 2007 ) ( 0/57 Hsp104 foci with Dcp2-GFP , Figure 2J ) .\",\n \"Furthermore , we never observed age-associated protein deposits and P-bodies fusing with each other ( Figure 2J ) .\",\n \"This suggests that P-bodies and age-associated protein deposits have different physicochemical properties , representing two distinct modes of cytosolic sub-compartmentalization ( Hyman et al . , 2014; Kroschwald et al . , 2015 ) .\",\n \"Altogether , these data show that the substrates of the age-associated deposit are non-random and possibly amyloid-like , which might explain their irreversible nature .\",\n \"During recent years , numerous distinct protein deposits , including Q-bodies , JUNQ/INQ , and the IPOD , have been discovered and thus we wanted to explore whether the age-associated protein deposit matches the identity of any of these structures .\",\n \"We first looked at the behavior of Q-bodies , which assemble in response to heat-stress .\",\n \"In stark contrast to the age-associated aggregates ( Figure 1G , I ) , Q-bodies induced upon heat stress ( 42°C , 30 min ) were transient and readily solubilized after the removal of the stress factor ( Figure 3A; Liu et al . , 2010; Escusa-Toret et al . , 2013; Wallace et al . , 2015 ) .\",\n \"Comparative analysis of the localization of different chaperone proteins between Q-bodies and the age-associated protein deposit showed that all markers of the age-associated protein deposit were also found to accumulate in Q-bodies ( Figure 3B ) .\",\n \"Conversely , we found chaperones such as Hsp82 and Btn2 localizing to Q-bodies ( Figure 3B ) albeit being excluded from the age-dependent protein deposit ( Figure 1C ) .\",\n \"Together , the reversible nature and the difference in associated chaperones demonstrate that the Q-bodies and age-associated protein deposits can be distinguished . 10 . 7554/eLife . 06197 . 008Figure 3 . The age-associated protein deposit can be distinguished from Q-bodies , JUNQ/INQ , and IPOD .\",\n \"( A ) Cells were heat shocked ( 42°C for 30 min ) to induce the formation of Q-bodies ( stress induced Hsp104-labeled aggregates ) and the kinetics of Q-bodies dissolution was followed with time-lapse microscopy of Hsp104-GFP .\",\n \"( B ) Cells were imaged after heat shock ( 42°C for 30 min ) .\",\n \"Arrowhead in 3D-projected images depicts co-localization of Hsp104 with age-associated deposit-resident ( Ssa1 , Hsp42 ) and non-resident ( Hsp82 , Btn2 ) markers in Q-bodies ( compare with Figure 1C ) .\",\n \"( C ) The expression of JUNQ/INQ marker VHL-GFP was induced at the start of the imaging to follow its recognition by Hsp104-mCherry .\",\n \"( D ) Quantification of VHL-GFP foci recognized by Hsp104-GFP within 30 min of their appearance ( N = 29 cells ) .\",\n \"( E ) 3D-projected images of RPN4 deleted cells expressing Hsp82-GFP and Hsp104-mCherry .\",\n \"Hsp82-GFP , which localizes to Q-bodies ( Figure 3A ) but not to age-associated protein deposits ( Figure 1C ) , co-localizes with one of the two Hsp104-mCherry foci in aged cell ( see fluorescence intensity line-scan over the two foci ) .\",\n \"( F ) 3D-projected image of Btn2-GFP and Hsp104-mCherry expressing cell .\",\n \"Btn2 is typically very low abundant and does not accumulate ( 98 . 6% of cases , N = 72 ) to the age-associated deposit ( white arrowhead ) .\",\n \"( G ) Cell with a Btn2 focus , which typically ( 96 . 4% of cases , N = 30 ) did not display accumulation of Hsp104-mCherry ) .\",\n \"( H ) Rnq1-overexpression was induced at the onset of imaging .\",\n \"The panel shows the z-sections displaying the age-associated deposit ( white arrowhead in ‘merge’ ) .\",\n \"( I ) Quantification of newly formed Rnq1-GFP foci that accumulate Hsp104-mCherry within 30 min of their appearance ( N = 53 cells ) .\",\n \"( J ) Representative image of [PIN+ , PSI+] cell harboring GFP-tagged endogenous Rnq1 and mCherry-tagged Hsp104 .\",\n \"Arrowhead indicates the age-associated protein deposit .\",\n \"( K ) Example of a [PIN+ , PSI+] cell that displays an Rnq1-aggregate .\",\n \"Arrowhead indicates Hsp104-labeled foci , of which only one has accumulated Rnq1 .\",\n \"Scale bars: A-B , E , J-K 5 μm , C , G 2 μm .\",\n \"Graphs display mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 008 We then examined whether the age-associated protein deposit would bear the characteristics of the JUNQ/INQ compartment , which is formed during prolonged stress and/or in response to proteasome inhibition .\",\n \"To this end , we induced the GAL-promoter-driven expression of the JUNQ/INQ marker , the unstable proteasome substrate human von Hippel-Lindau tumor suppressor protein ( VHL ) ( Kaganovich et al . , 2008; Miller et al . , 2015 ) , and monitored the accumulation of Hsp104-mCherry into the newly forming VHL-foci .\",\n \"VHL typically formed a single focus , which in 87% of the cases did not accumulate Hsp104-mCherry within 30 min after its appearance ( Figure 3C , D , N = 29 ) .\",\n \"However , the VHL expression was often associated with overall increased Hsp104-mCherry expression over time , suggesting that its expression activates a stress response .\",\n \"Hence , we visualized the JUNQ/INQ without over-expression of exogenous substrates .\",\n \"To accomplish this , we used an Hsp82-GFP , Hsp104-mCherry expressing strain deleted of the RPN4 gene , leading to increased burden of proteasomal substrates , due to decreased amount of functional proteasomes ( Xie and Varshavsky , 2001 ) .\",\n \"Indeed , many of rpn4Δ cells displayed numerous Hsp82-Hsp104 double-positive Q-body-like puncta ( data not shown ) .\",\n \"In addition , we found cells that displayed two puncta: a Hsp104-Hsp42 double-positive JUNQ-like deposit , and a Hsp104-positive , Hsp82-negative deposit that fills the criteria of the age-associated deposit ( Figure 3E ) .\",\n \"To consolidate this further , we investigated the co-localization of the endogenous JUNQ/INQ marker Btn2 ( Miller et al . , 2015 ) together with the age-associated protein deposit ( Hsp104 ) .\",\n \"This analysis showed that in 98 . 6% of the cases , Btn2 did not localize to the age-associated Hsp104-foci ( Figure 1C and Figure 3F ) .\",\n \"Moreover , from all thirty identified cells displaying endogenous Btn2 foci , we found only one case in which Hsp104 was enriched at this site ( Figure 3G , N = 1268 cells ) .\",\n \"Together , these data suggest that the age-associated protein deposit and the JUNQ/INQ are discrete structures that may exist in parallel .\",\n \"Finally , we monitored the resemblance between the age-associated protein deposit and IPOD .\",\n \"To visualize cells during IPOD appearance , we imaged Hsp104-mCherry-expressing cells together with the canonical IPOD marker , the galactose-inducible Rnq1-GFP ( Kaganovich et al . , 2008 ) , immediately after placing cells to galactose-containing media .\",\n \"This showed that the IPOD typically appeared as a single focus to which Hsp104 rapidly accumulated ( Figure 3H , I ) ( >98% of Rnq1-foci displayed accumulation of Hsp104 within 30 min after their appearance , N = 53 ) .\",\n \"However , by dissecting the Rnq1-GFP appearance dynamics in cell with a pre-existing age-associated protein deposit ( marked by Hsp104-mCherry ) , we found that the aggregating Rnq1-GFP did not accumulate to the age-associated deposit ( see white arrowhead in Figure 3H ) , but rather it formed a new aggregate to which Hsp104 then strongly accumulated , while its intensity at the age-associated protein deposit declined ( Figure 3H ) .\",\n \"To rule out possible artifacts induced by Rnq1-GFP over-expression , we also looked at the co-localization between the age-associated protein deposit and GFP-tagged endogenous Rnq1 in its prion [RNQ+]-state .\",\n \"We analyzed altogether 117 Hsp104 foci-containing [PIN+ , PSI+] cells and found that in 98 . 3% of the cases , Rnq1-GFP did not accumulate to this deposit ( Figure 3J ) .\",\n \"Within this cohort , we found two cells with an Rnq1-GFP-aggregate , which was in both cases enriched with Hsp104-mCherry ( Figure 3K ) .\",\n \"However , both of the Rnq1-aggregate containing cells also displayed an additional Hsp104 focus to which Rnq1 had not accumulated ( Figure 3K ) .\",\n \"Together with the results from the Rnq1 over-expression , these data suggest that Rnq1 aggregate is likely to represent a structure that is different from the majority of the age-associated protein deposits .\",\n \"In sum , these data point out marked differences between the age-dependent protein deposit and the previously characterized Q-bodies , IPOD , and the JUNQ/INQ that could derive from context dependent ( stress vs aging ) differences in cellular responses to protein aggregation .\",\n \"Appearance of protein aggregates has commonly been associated with defects in protein quality control ( Tyedmers et al . , 2010a ) .\",\n \"Therefore , we wanted to elucidate if the formation of the age-associated protein deposit is a sign of defective protein quality control .\",\n \"First , we examined whether aged cells display a decline in dealing with proteotoxic stress .\",\n \"As shown before , young cells clear heat-induced protein aggregates ( Q-bodies ) rapidly after stress removal ( Figure 3A ) .\",\n \"Hence , we first wanted to test if this recovery period is affected by the age of the cell ( Figure 4A ) .\",\n \"We measured the clearance time of Q-bodies ( defined as two or more Hsp104 foci ) between young ( average age 0 . 4 generations , between 0 and 1 generations , N = 70 ) and aged cells ( average age 9 . 2 generations , between 6 and 19 generations , N = 50 ) following acute heat stress ( Figure 4B ) .\",\n \"Surprisingly , no difference in the mean time of protein aggregate clearance was detected between these populations: young: 71 ±5 min old: 72 ±4 min , n . s . ) ( Figure 4C ) , suggesting that aged cells , despite having a protein deposit , are fit to cope with proteotoxic stress . 10 . 7554/eLife . 06197 . 009Figure 4 . Aged cells are not impaired in handling proteotoxic stress .\",\n \"( A ) Experimental scheme .\",\n \"( B ) Representative image of displaying the dynamics of recovery from heat shock induced proteotoxic stress ( Q-bodies ) between aged ( red star ) and young cell ( blue star ) .\",\n \"( C ) Q-body clearance time of individual cells of the indicated age groups , ( old: average 9 . 2 generations , age between 6 and 19 , N = 50; young: 0 . 4 generations , age between 0 and 1 , N = 70 ) .\",\n \"( D ) Experimental scheme: Hsp104-mCherry-expressing cells were captured on a temperature-controlled microfluidic device and imaged prior ( −50 min ) , during ( 30 min ) and after ( up to 300 min ) mild heat shock .\",\n \"It is important to note , that the strength of the heat stress induced on the microfluidic platform is not comparable with Figure 3A , B and Figure 4A–C .\",\n \"( E ) Single cell analysis of Q-body formation ( cells with >1 Hsp104-foci ) in cells with a pre-existing age-associated protein deposit ( red line , N = 53 ) , cells without a pre-existing deposit ( blue line , N = 97 ) , and cells that did not experience stress ( N = 82 ) .\",\n \"Data from two independent experiments .\",\n \"( F ) Transitions in the aggregate state were recorded 50 min before the heat stress and 300 min after the heat stress .\",\n \"( G ) Quantification of transitions ( N = 32–77 per group , from two independent experiments ) .\",\n \"White bars indicate cells that did not experience heat stress and red bars denote transitions in aggregate state in heat-stress experienced cells .\",\n \"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 009 We then used a temperature-controlled microfluidic device that enabled us to categorize cells depending on their pre-stress age-associated protein deposit-status , and monitor them under the microscope prior to , during and after undergoing acute proteotoxic stress conditions ( Figure 4D ) .\",\n \"The duration of the Q-body response ( state in which cells display two or more Hsp104-foci ) was plotted over time and demonstrated that cells with and without a pre-existing age-associated deposit showed a comparable Q-body response when heat was applied , while cells that were not exposed to heat did not show similar Q-body response during this time period ( Figure 4E , N = 53–97 cells ) .\",\n \"Interestingly , the deposit-containing cells responded slightly faster and to a lesser extend when compared to their clonal counterparts without a pre-existing protein deposit ( Figure 4E ) .\",\n \"We then asked if exposure to proteotoxic stress ( Q-body state ) would promote the formation of age-associated protein deposits .\",\n \"To this end , we compared the aggregate status of single cells ( Hsp104-mCherry: no/one/several puncta ) at time points 0 and 380 min , between stress-experienced ( between 50 and 80 min ) and non-stressed ( constantly at 30 °C ) cells ( Figure 4F , N = 32–77 per group ) .\",\n \"This analysis showed that the majority of non-stressed cells ( 62 . 5% ) without a deposit at the beginning of the experiment displayed a single aggregate at the end of the experiment ( Figure 4G ) , in accordance with the appearance kinetics of age-associated deposit ( Figure 1B , D ) .\",\n \"Surprisingly , when the cells without a prior aggregate encountered heat stress ( i . e . conditions that induce proteotoxic stress ) , a substantially smaller portion of them ( 36 . 4% ) displayed an aggregate at the end of the experiment ( Figure 4G ) .\",\n \"Similarly , there was a slight increase of cells without an aggregate in the stress-encountered cohort ( 17 . 7% vs 6 . 8% ) among cells that displayed an age-associated deposit at the beginning of the experiment ( Figure 4G ) .\",\n \"However , irrespectively of stress , cells with an age-associated deposit at the beginning of the experiment preferentially displayed a single deposit at the end of the experiment ( non-stressed 82% , stress-experienced 79% ) ( Figure 4G ) .\",\n \"These results indicate that mild exposure to proteotoxic stress conditions counteract the formation of age-associated protein deposits and demonstrate that cells with an age-associated deposit prior to encountering the stress predominantly reverted back to this state .\",\n \"Altogether , these data indicate that despite forming a protein deposit , aged cells are still fit to coping with acute proteotoxic stress and suggest that the formation of the age-dependent protein deposit is not due to the overload of the general quality control machinery .\",\n \"The notion that aged cells with a protein deposit handled proteotoxic stress comparably to their young counterparts prompted us to test the effect of the age-associated deposit on the function of the ubiquitin–proteasome system ( UPS ) , which has an important role in yeast replicative aging ( Kruegel et al . , 2011 ) .\",\n \"Pathological polyQ protein fragments have been shown to impair the UPS function both in yeast and mammalian cells ( Bennett et al . , 2005; Park et al . , 2013 ) , and UPS substrates have been shown to accumulate in aged cells ( Andersson et al . , 2013 ) .\",\n \"On the other hand , stress-induced protein aggregates ( Q-bodies ) do not result from failed protein degradation , but may actually benefit the proteostasis of cells undergoing stress ( Escusa-Toret et al . , 2013 ) .\",\n \"We therefore wanted to analyze the contribution of the age-associated deposit on the functionality of the UPS .\",\n \"In order to selectively measure the effect of the age-associated protein deposit on UPS function , we made use of the auxin-inducible degron ( AID ) system ( Nishimura et al . , 2009 ) , which enabled us to measure the degradation rate of cytosolic ( AID-GFP ) and nuclear ( AID-GFP-NLS ) UPS substrates in vivo ( Figure 5A , B ) .\",\n \"Cells co-expressing Hsp104-mCherry and AID-GFP were switched to auxin-containing media at the onset of imaging , and the GFP intensities were measured over time from neighboring cells containing or not an Hsp104-puncta .\",\n \"The normalized values were pooled and fitted with a non-linear one-phase decay function .\",\n \"Importantly , this showed that the decay rate of AID-GFP was significantly accelerated in cells with an age-associated deposit when compared to neighboring cells that did not contain an Hsp104-focus ( Figure 5C , D ) ( rate constant ( K ) : 0 . 155 ± 0 . 007 ( with ) vs 0 . 129 ± 0 . 006 ( without ) , p < 0 . 01 , N = 91 cells/category ) .\",\n \"However , no difference was detected in the decay rate of nuclear GFP between cells with or without an age-associated protein deposit ( Figure 5E , F ) ( K: 0 . 075 ± 0 . 004 ( with ) vs 0 . 079 ± 0 . 005 ( without ) , n . s . ) , demonstrating that the effect of the age-associated protein deposit on the UPS function is specific to the cytosolic compartment .\",\n \"Collectively , these results suggest that the age-associated deposit promotes cytosolic quality control by enabling more efficient clearance of degradation substrates . 10 . 7554/eLife . 06197 . 010Figure 5 . Presence of age-associated protein deposit promotes the function of cytosolic ubiquitin–proteasome system in vivo .\",\n \"( A ) Schematic representation of the auxin-induced degron ( AID ) -system ( Nishimura et al . , 2009 ) .\",\n \"Addition of auxin facilitates the recognition of the degron-motif in the target protein ( GFP ) by the exogenously expressed Arabidobsis thaliana E3 ligase SCF-Tir1 and subsequent ubiquitination and degradation by the endogenous ubiquitin–proteaome system ( UPS ) .\",\n \"( B ) Representative images of cells expressing Hsp104-mCherry and AID-GFP taken immediately ( 0 min ) and 48 min after auxin-addition .\",\n \"( C ) Examples of time frames GFP degradation in the absence of auxin ( upper panel ) in the presence of auxin in cells with ( middle ) or without ( below ) an age-associated protein deposit .\",\n \"( D ) The decay rates of GFP in the indicated groups .\",\n \"The error bars depict the normalized intensity values ( average ± SEM ) derived from 53 ( ctrl ) and 91 ( auxin added ) cells from three pooled replicates .\",\n \"The solid lines indicated non-linear one-phase decay fit .\",\n \"( E ) GFP-NLS decay in the representative groups .\",\n \"( F ) The decay rates and the graph fitting of NLS-GFP in the indicated groups as in ( D ) .\",\n \"N = 15 ( ctrl ) , 65–66 ( auxin added ) cells from three pooled replicates .\",\n \"The error bars depict the normalized intensity values ( average ± SEM ) , while the solid lines indicate the non-linear one-phase decay fit . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 010 Next , we wanted to understand what are the assembly principles of the age-associated deposit .\",\n \"Molecular chaperones generally recognize , refold , or sort aberrantly folded proteins , making them prime candidates to regulate the age-associated protein deposit assembly .\",\n \"Thus , we addressed the contribution of chaperones localizing to the age-associated protein deposit to its formation .\",\n \"Time-lapse microscopy showed that in 24 out of 25 cells , Hsp42 was at the age-associated protein deposit before Hsp104 and was the first protein we find to mark this structure ( Figure 6A ) .\",\n \"Ssa1 and Hsp104 were recruited subsequently , appearing concurrently up to 120 min after appearance of the Hsp42-focus ( Figure 6A and data not shown ) .\",\n \"To identify the role of these chaperones in the assembly of the age-associated protein deposit , we investigated the consequences of their deletion .\",\n \"Interestingly , loss of the Hsp70 proteins Ssa1 and Ssa2 , which act synergistically with the disaggregase Hsp104 in protein refolding ( Glover and Lindquist , 1998; Kim et al . , 2013 ) , resulted in rapid formation of the Hsp104 focus ( Figure 6B , C ) and a similar , but milder effect was detected upon deletion of HSP26 .\",\n \"In stark contrast , deletion of HSP42 abolished age-associated protein deposit formation ( Figure 6B , C ) , which parallels to its function in Q-body assembly ( Specht et al . , 2011; Escusa-Toret et al . , 2013 ) .\",\n \"To probe the role of Hsp104 and Hsp42 further , we analyzed the consequences of their over-expression on age-associated protein deposit formation ( monitored by endogenous Hsp104-GFP ) .\",\n \"Over-expression of Hsp42 caused precocious nucleation of age-associated protein deposit and promoted its growth ( Figure 6D–F ) .\",\n \"On the contrary , a significantly smaller fraction of cells over-expressing Hsp104 contained an age-associated protein deposit and while age-associated deposits still occasionally formed , they were rapidly destabilized ( Figure 6D–F ) .\",\n \"Collectively , these data suggest that these chaperones functionally oppose each other ( Figure 6G ) : the Hsp104/Hsp70 proteins prevent age-associated protein deposit formation possibly by refolding cytoplasmic substrates , whereas Hsp42 coordinates the collection of these substrates from the cytoplasm into the age-associated protein deposit ( Figure 6G ) . 10 . 7554/eLife . 06197 . 011Figure 6 . Identification of the roles of chaperones in age-associated protein deposit assembly .\",\n \"( A ) Representative time-lapse images of cells expressing endogenously tagged Hsp42-GFP ( upper panel ) and Hsp104-mCherry ( middle panel ) .\",\n \"Arrowhead depicts the age-associated protein deposit structure .\",\n \"( B ) Representative images of aged Hsp104-GFP expressing wild-type , ssa1Δ , and hsp42Δ mutant cells .\",\n \"( C ) Quantification of Hsp104-foci containing cells in indicated age groups , ( N = 317–1020 cells per genotype ) .\",\n \"( D ) Time-lapse images of cells expressing endogenous Hsp104-GFP in wild-type , Hsp104 over-expressing , and Hsp42 over-expressing cells ( age-associated protein deposit depicted by a red arrowhead ) .\",\n \"( E ) Quantification of percentage of cells with age-associated protein deposit in wild-type , Hsp104 over-expressing , and Hsp42 over-expressing cells of indicated age groups ( N = 432–1020 cells per genotype ) .\",\n \"( F ) Quantification of endogenous Hsp104-GFP integrated density ( age-associated protein deposit/cytoplasm ) following its appearance in wild-type ( black line ) and Hsp42 over expressing cells ( red line ) , ( N = 19–23 ) .\",\n \"( G ) A summarizing model of the pathway underlying age-associated protein deposit formation .\",\n \"Scale bars 5 μm .\",\n \"Graphs display mean ± SEM , n . s not significant , *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 011 To consolidate this model , we further investigated the interplay between the refolding machinery ( Hsp70/Hsp104 ) and the nucleation/growth promoting Hsp42 .\",\n \"We used cells expressing Ssa1-GFP to visualize the age-associated protein deposit in the absence of Hsp104 ( Figure 7A ) .\",\n \"Similarly to what we found for the deletion of SSA1 and SSA2 ( Figure 6B , C ) , the hsp104Δ mutant cells formed the age-associated protein deposit more rapidly during aging .\",\n \"Again , the deletion of HSP42 had an opposite effect , hampering formation of the Ssa1-GFP focus ( Figure 7A , B ) .\",\n \"Remarkably , age-associated protein deposit formation was severely impaired in the hsp104∆ hsp42∆ double-mutant cells .\",\n \"These cells were crowded with punctate Ssa1-GFP ( speckles ) , which failed to coalesce into a single regular age-associated protein deposit ( Figure 7A , B ) .\",\n \"The portion of cells that contained multiple Ssa1-GFP foci was significantly higher in hsp104Δ hsp42Δ double-mutant cells than in hsp104Δ single-mutant cells .\",\n \"Interestingly , whereas >80% of wild-type and hsp104∆ mutant cells that contained an aggregate displayed a single Ssa1-GFP focus , the majority of hsp42∆ and hsp104∆ hsp42∆ mutant cells harbored several foci ( Figure 7C ) . 10 . 7554/eLife . 06197 . 012Figure 7 . The assembly of age-associated protein deposit is promoted by Hsp42 and counteracted by Hsp104/Hsp70 . ( A ) Representative aged wild-type , hsp104Δ , hsp42Δ and hsp104Δ and hsp42Δ double-mutant cells .\",\n \"( B ) Quantification of the fraction of cells in indicated age groups that display Ssa1-GFP foci ( age-associated protein deposit ) , ( N = 363–493 cells per genotype ) .\",\n \"( C ) Quantification of the portion of cells that display >1 Ssa1 foci from all Ssa1-foci containing cells , ( N = 363–493 cells per genotype ) .\",\n \"( D ) Illustration of the experimental scheme of the reconstitution assay .\",\n \"Cells that lack both HSP104 and HSP42 and display fragmented aggregate phenotype were mated with cells of the opposite mating type to reintroduce either Hsp104 and/or Hsp42 .\",\n \"The resulting zygote was imaged with time-lapse microscopy and analyzed 5 min after fusion to score for aggregation phenotype ( analyzed by Ssa1-GFP ) .\",\n \"( E ) Quantification of the Ssa1-GFP phenotype ( no foci , one focus , multiple foci ) 5 min post fusion , ( N = 73–100 fusion events per genotype ) .\",\n \"Scale bars 5 μm .\",\n \"Graphs display mean ± SEM , n . s not significant , **p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 012 To decisively dissect the individual contributions of Hsp104 and Hsp42 on age-associated protein deposit formation , we conceived an in vivo reconstitution assay where we could rapidly re-introduce either Hsp104 or Hsp42 to the aggregate-enriched hsp104Δ hsp42Δ double-mutant cells .\",\n \"To accomplish this , we mated hsp104Δ hsp42Δ double-mutant cells with wild-type ( re-introducing both Hsp104 and Hsp42 ) , hsp104∆ single-mutant ( to reintroduce only Hsp42 ) , hsp42Δ single-mutant ( to reintroduce only Hsp104 ) , or with the hsp104 hsp42 double-mutant cells ( reintroducing none of the chaperones ) ( Figure 7D ) .\",\n \"We monitored the deposit by imaging the fusing cells at 1-min intervals , using Ssa1-GFP as a reporter ( Figure 7D ) .\",\n \"Importantly , simultaneous reintroduction of Hsp104 and Hsp42 was sufficient to clear the cytoplasm of Ssa1-GFP speckles already within 5 min ( Figure 7E , no foci ) .\",\n \"In contrast , introducing Hsp42 alone resulted in rapid disappearance of cytoplasmic speckles with concurrent formation of a single Ssa1-GFP focus ( Figure 7E , one focus ) .\",\n \"In contrary , reintroduction of only Hsp104 led to the slow and incomplete clearance of the Ssa-1 speckles , which was a reminiscent , but a milder phenotype as that observed upon conjugating the cell with hsp104Δ hsp42Δ double-mutant partner ( Figure 6E , multiple foci ) .\",\n \"Collectively , this analysis supports the conclusion that Hsp42 functions to collect aggregates into the age-associated protein deposit structure , whereas Hsp104 functions to disaggregate/refold age-associated protein deposit destined substrates .\",\n \"In the context of the age-associated protein deposit cargo Sup35 ( Figure 2 ) , it is interesting to note that Hsp42 over-expression promotes [PSI+] curing , while its deletion results in enhanced [PSI+] induction , further supporting its role as the depositor for the age-associated protein deposit ( Duennwald et al . , 2012 ) .\",\n \"We then asked whether age-associated protein deposit formation ensures the asymmetric inheritance of protein aggregates by the aging lineage .\",\n \"Examining its mitotic segregation ( monitored with Ssa1-GFP ) in 96 wild-type and 186 hsp104Δ mutant cell divisions demonstrated that the age-associated protein deposit was in both cases inherited by the aging mother cell with high fidelity ( in 98% of divisions; data not shown ) .\",\n \"In strong contrast , Ssa1-GFP foci were frequently inherited by the daughter cells when both HSP104 and HSP42 were deleted ( Figure 8A , Video 2 ) .\",\n \"By examining 61 mitotic events by time-lapse microscopy in the double-deleted cells , we observed Ssa1-GFP-foci relocating from the mother to the bud in 53 cases , but did not find any biased retrograde movement from the bud to the mother ( Fig . 8A , Video 2 and data not shown ) .\",\n \"Quantification of the percentage of buds with protein deposits demonstrated that more than 65% of all hsp104Δ hsp42Δ double-mutant buds displayed at least one aggregate , compared to less than 4% in hsp104Δ and hsp42Δ single-mutant and in wild-type cells ( Figure 8B ) .\",\n \"Altogether , these data provide evidence that collection of protein aggregates into a single deposit by Hsp42 promotes their asymmetric retention in the mother cell during cell division . 10 . 7554/eLife . 06197 . 013Figure 8 . Age-associated protein deposit formation establishes asymmetric inheritance of protein aggregates and correlates with replicative age .\",\n \"( A ) Time-lapse imaging of Ssa1-GFP in hsp42Δ hsp104Δ double-mutant cells ( arrowheads denote Ssa1-GFP aggregates relocating from the mother cell to the bud ) .\",\n \"( B ) Quantification of Ssa1-GFP foci found in the buds of mitotic yeast cells ( N = 216–434 buds per genotype ) .\",\n \"( C ) Replicative aging experiments of wild-type ( black line , 27 generations ) , hsp104Δ mutant ( blue line , 21 . 5 generations ) , hsp42Δ mutant ( red line , 39 generations ) , and hsp104Δ and hsp42Δ double mutant ( purple , 23 generations ) single cells , ( N = 101–140 cells per genotype ) .\",\n \"( D ) A schematic model for the age-associated protein deposit pathway: Hsp42 acts as a collector of protein aggregate seeds and promotes their deposition at the ER membrane ensuring their asymmetric inheritance by the aging lineage during mitosis .\",\n \"These cytoplasmic seeds are subjected to Hsp104/Hsp70-dependent refolding , which is inactive ( blue rectangle ) at the site of the age-associated protein deposit assembly .\",\n \"Scale bar 5 μm .\",\n \"Graph displays mean ± SEM , n . s not significant , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 01310 . 7554/eLife . 06197 . 014Video 2 . The lack of the age-associated protein deposit assembly pathway results in the inheritance of the aggregates by the daughter cells . Expression of Ssa1-GFP was followed in hsp104Δ hsp42Δ double-mutant cell as it underwent cell division . DOI: http://dx . doi . org/10 . 7554/eLife . 06197 . 014 Finally , we tested the significance of the age-associated protein deposit pathway and asymmetric protein aggregate segregation on replicative aging ( Figure 8C ) .\",\n \"In accordance to previous work ( Erjavec et al . , 2007 ) , mutant hsp104∆ cells were short-lived compared to wild-type cells ( 21 . 5 vs 27 generations , p < 0 . 001 ) , supporting the idea that accelerated aggregate accumulation promotes aging .\",\n \"Further supporting this notion , deletion of HSP42 extended the lifespan of the cells by more than 40% ( 39 generations , p < 0 . 001 ) .\",\n \"The lifespan of the hsp104Δ hsp42Δ double-mutant cells was similar to that of the hsp104Δ single-mutant cells ( 23 vs 21 . 5 generations , respectively , ns . ) and significantly shorter than that of the wild-type ( p < 0 . 01 ) and hsp42Δ mutant cells ( p < 0 . 001 ) .\",\n \"The over-expression of HSP42 had no effect on the lifespan ( data not shown ) , suggesting that the wild-type levels of Hsp42 are sufficient to provide its maximal effects on life span , which is consistent with the appearance of the deposit early in life span in the wild-type cells .\",\n \"Together , these data imply that the Hsp104/Hsp70 system , which counteracts protein aggregation , is essential for longevity , while the Hsp42-dependent circuit promotes aging in the mother lineage , presumably by building the age-associated protein deposit and thereby establishing the asymmetric inheritance of age-associated protein aggregates .\"\n ],\n [\n \"Protein aggregation has been frequently associated with aging and aging-associated diseases .\",\n \"Here , by using the budding yeast as a model for replicative aging , we identify an Hsp42-promoted and Hsp104/Hsp70-counteracted pathway that deposits age-associated protein aggregates and thereby ensures their biased segregation to the aging mother-lineage upon cell division ( see model in Figure 7D ) .\",\n \"This might represent a common mechanism to drive asymmetric segregation of aberrant proteins in dividing cells , as stressed fission yeast cells utilize a similar Hsp16-dependent mechanism for asymmetric partitioning of misfolded proteins ( Coelho et al . , 2014 ) .\",\n \"Intriguingly , our data suggest that depositing age-associated protein aggregates to the mother cells might be a mixed blessing .\",\n \"On one hand , the age-associated deposit can promote spatial protein quality control ( Figures 4 , 5 ) and promote cell diversity as an asymmetrically dividing structure that may harbor protein conformation encoded epigenetic information , such as the prion fold of Sup35 ( Figure 2 ) .\",\n \"At the same time , the long-term consequences of this pathway appear negative for the aging lineage ( Figure 8C ) .\",\n \"Why the deposit-forming pathway ultimately becomes associated with age-dependent loss of fitness remains to be determined .\",\n \"In this regard , it will be of great importance to elucidate how age-associated deposit relates to other aging pathways , such as those influencing the function of mitochondria ( McMurray and Gottschling , 2003; McFaline-Figueroa et al . , 2011 ) , vacuoles ( Hughes and Gottschling , 2012 ) and nuclear pores ( Shcheprova et al . , 2008; Denoth-Lippuner et al . , 2014; Webster et al . , 2014 ) , and how the deposit localizes relative to these organelles and their components ( Chong et al . , 2015 ) .\",\n \"Protein deposit-containing cells efficiently coped with acute proteotoxic stress ( Figure 4 ) and displayed improved degradation of cytosolic UPS substrate during early- to mid-life span ( Figure 5 ) .\",\n \"It is important to note , however , that our assays were conducted with relatively young cells that had recently acquired the protein deposit , and hence it is possible that UPS function starts to decline later during the aging process , as shown previously ( Andersson et al . , 2013 ) .\",\n \"Curiously , the decline in proteostasis has been associated with protein aggregation pathologies such as Alzheimer's , Huntington's , and Parkinson's disease ( Vilchez et al . , 2014 ) .\",\n \"The decreased degradation of cytosolic UPS substrates in a polyQ disease model was found to be due to the sequestration of the Hsp40 protein Sis1 ( Park et al . , 2013 ) —a protein that was not associated with the age-associated deposit ( Figure 1C ) .\",\n \"It is thus plausible that the presence of the deposit might , for example , lead to increased levels of available ( substrate unbound ) Sis1 , thereby allowing more efficient nuclear import and degradation of cytosolic UPS substrates .\",\n \"Altogether , these data suggest that there is a need to better differentiate between pathophysiological protein aggregation and ‘homeostatic’ protein aggregation that takes place during physiological aging , of which the latter might initially help to coordinate chaperones and UPS factors involved in protein quality control .\",\n \"Related to this , the age-associated protein deposit did not clearly fill out the set criterion for any of the previously characterized cytosolic deposits , including Q-bodies , JUNQ/INQ , or IPOD , but rather seems to exist in parallel with the JUNQ and IPOD structures ( Figure 1 , Figure 3 ) —although , for example , the chaperones regulating these deposits were often shared between these structures .\",\n \"This favors the notion that pathological aggregation processes ( mimicked e . g . , by VHL and Rnq1 over-expression ) can hijack endogenous proteostasis regulatory mechanisms , possibly underlying their harmful effects in cells ( Park et al . , 2013 ) .\",\n \"Altogether , these apparent differences between the stress-induced , pathological , and aging-associated protein aggregates ( Figure 2 , Figure 3 ) demonstrates that protein aggregates are not all equal in their composition or in the way they are being recognized and dealt by the cell and reinforces the importance to discriminate between different aggregation models .\",\n \"In the future , it will be important to identify the cargo deposited to the age-associated protein deposits .\",\n \"In this aspect , it is interesting to note that many of the long-lived asymmetrically retained proteins that accumulate to mother cells as they age ( Thayer et al . , 2014 ) were among those localizing to the age-associated protein deposit ( including Hsp104 , Ssa1 , Ssa2 , and Hsp26 ) .\",\n \"One example of an age-associated deposit resident protein was the prion-like Sup35 .\",\n \"This storage rendered Sup35 to be inherited by the mother cells ( unlike a prion , analogous to a mnemon ( Caudron and Barral , 2013 ) ) ( Figure 2 ) , which is consistent with the size-dependent transmission model of Sup35-aggregates ( Derdowski et al . , 2010 ) .\",\n \"However , aging does not enhance Sup35-dependent [PSI+] prion generation ( Shewmaker and Wickner , 2006 ) .\",\n \"Together , these notions suggest that Sup35 cannot escape the age-associated deposit under normal growth conditions , perhaps owing to the rigid amyloid-like packing ( Saibil et al . , 2012 ) , being consistent with the durable and non-dynamic nature of these deposits .\",\n \"However , this notion may come with a caveat: we found that the age-associated deposit could be , at least temporarily , disassembled by the heat shock response ( Figure 4 ) .\",\n \"This suggests the captivating possibility that changes in the environment might trigger the spread from the age-associated deposit .\",\n \"For cells facing fluctuating environments in the wild , the strategy to stochastically store prion-like proteins in aged cells might prove beneficial , as it could allow stress-dependent spread of the prion conformers from a subpopulation of aged-cells to their daughters , promoting faster adaptation to the changing environment ( Newby and Lindquist , 2013 ) .\",\n \"Since asymmetric inheritance of damaged proteins and protein aggregates seems to be conserved in metazoan stem cells ( Rujano et al . , 2006; Bufalino et al . , 2013 ) that are also subjected to both replicative decline and segregation of lineages , we hypothesize that similar pathways that selectively segregate protein-aggregate based fate determinants asymmetrically during cell division are likely to be conserved and may contribute to cellular lineage specification across species .\"\n ],\n [\n \"Yeast strains used in this study are listed in Supplementary file 1 .\",\n \"Strains were generated either manually ( Janke et al . , 2004 ) or derived from collections: ( http://web . uni-frankfurt . de/fb15/mikro/euroscarf/col_index . html or http://clones . lifetechnologies . com/cloneinfo . php ? clone=yeastgfp ) .\",\n \"Plasmids ( pAG415-GDP ) over-expressing Hsp104 ( O-1360 ) and Hsp42 ( O-2252 ) , and the [PIN+][PSI+] ( yJW508 ) and [PIN+][psi–] ( yJW584 ) parental strains ( Osherovich and Weissman , 2001 ) were a generous gift from Simon Alberti ( Max Planck Institute of Molecular Cell Biology and Genetics Dresden , Germany ) .\",\n \"The Mother Enrichment parental strains ( Lindstrom and Gottschling , 2009 ) were a kind gift from Dan Gottschling ( Fred Hutchinson Cancer Research Center , Seattle USA ) and the plasmids encoding VHL-GFP and Rnq1-GFP were provided by Judith Frydman ( via Addgene ) .\",\n \"The integrative plasmids for the auxin-mediated degradation assays ( pADH1-OsTIR1-9myc , pADH1-eGFP-IAA17-NLS , pADH1-eGFP-IAA17 ) have been described in ( Nishimura et al . ) and were kindly provided by Matthias Peter ( ETH Zurich , Switzerland ) .\",\n \"Cells subjected to imaging were re-inoculated from overnight cultures to O . D . 600 nm 0 . 05 in YPD and grown to O . D . 0 . 5–0 . 8 at 30°C , centrifuged 500 g for 5 min , resuspended in synthetic complete ( SC ) media ( -his ) , and mounted between a coverslip and an agar pad ( SC-his ) .\",\n \"The over-expression of Rnq1-GFP and VHL-GFP was induced by switching cells to 2% galactose at the onset of imaging .\",\n \"The auxin-mediated degradation assays were performed similarly as above by placing cells to 0 . 5 mM 3-Indoleacetic acid ( Sigma–Aldrich , #12886 ) at the onset of imaging .\",\n \"The MEP was performed as described in ( Lindstrom and Gottschling , 2009 ) .\",\n \"Briefly , cells were grown in log phase for 15 hr before coupling the cell wall with Sulfo-NHS-LC-Biotin ( Pierce ) .\",\n \"Following 2 hour recovery period , the MEP was engaged by addition of estradiol to a 1 μM final concentration .\",\n \"After desired incubation period , cells were coupled to uMACS Streptavidin MicroBeads ( Miltenyi Biotec ) and affinity purified with MACS separation columns ( Miltenyi Biotec ) .\",\n \"Calcofluor staining was done by incubating cells with 5 μg/ml Fluorescent Brightener 28 ( Sigma–Aldrich ) for 1 min prior to centrifugation and resuspension to SC media .\",\n \"The Q-bodies were induced by incubating cells in a water bath at +42 °C for 30 min , or by using a temperature-controlled microfluidic chamber ( see microfluidics ) .\",\n \"For the mating experiments , cells ( 1 . 85×107 ) of the opposite mating type were centrifuged 500 g for 5 min and resuspended to 40 μl SC media , mixed and immediately imaged with 1-min intervals ( see microscopy for details ) .\",\n \"Wide-field microscopy was performed at 30 °C with a DeltaVision microscope coupled to a coolSNAP CCD HQ2 camera ( Roper ) , 250W Xenon lamps and 100X/1 . 40 and 60x/1 . 42 NA Olympus oil immersion objectives .\",\n \"Z-sectioning was performed with 500-nm spacing ( unless otherwise indicated ) , obtaining 9 or 11 ( live-cell imaging ) or 15 ( calcofluor stained cells ) stacks .\",\n \"Images were deconvolved with Softworx software ( Applied Precision ) .\",\n \"Microfluidics on the aging chip were imaged with a DeltaVision microscope every 45 min for total of 66 hr by obtaining seven z-sections with 0 . 6-μm spacing .\",\n \"FRAP was done with temperature-controlled Zeiss LSM 510 microscope controlled with AIM LSM4 . 0 software , 63x 1 . 4NA Oil DIC Plan-Apochromat objective at 30°C , using DPSS and Argon lasers .\",\n \"Diploid cells expressing HSp104-GFP/Hsp104mCherry were imaged every 5 s at three z-planes with 800-nm spacing five times before bleaching the mCherry signal at the age-associated protein deposit with 100% DPSS laser power , after which the recovery of mCherry at the age-associated protein deposit was monitored over the period of two minutes by orienting with the GFP-signal .\",\n \"The temperature ramp experiments ( Figure 4 ) were carried out with ONIX microfluidic perfusion system equipped with a micro-incubator temperature controller CellASIC ) , using Y04C microfluidic plates ( CellASIC ) .\",\n \"The temperature setup was the following: 30°C ( 50 min ) , 42°C ( 30 min ) 30°C ( 300 min ) and the cells were imaged every 10 min as described in ( microscopy ) .\",\n \"The experiments were carried out in synthetic full medium with even flow rate of 2 psi .\",\n \"Microfluidics on the aging chip was performed with 1 . 5 μl/min flow rate as described in ( Denoth-Lippuner et al . , 2014 ) .\",\n \"All image analyses were performed with Image J software ( http://imagej . nih . gov/ij/ ) .\",\n \"The aggregation foci were scored by eye from maximum intensity projected images ( spanning the entire volume of the cell ) and were defined as puncta that display high-intensity over the surrounding cytoplasmic background signal .\",\n \"For age-associated protein deposit intensity measurements , the integrated density was measured at the site over time at the age-associated protein deposit from stacked , non-processed sum-projected videos and was then normalized to the cytoplasmic Hsp104-GFP intensity .\",\n \"For the auxin-mediated degradation experiments , the 11-plane z-stack covering 5 . 5 μm was sum projected and an integrated density of a defined area ( 18 μm2 ( GFP ) or 16 μm2 ( GFP-NLS ) was measured over time from the region of interest ( ROI ) and the neighboring background region ( BG ) .\",\n \"To distinguish between cells that contained an aggregate from those that did not contain an aggregate , cells were categorized based on the first frame Hsp104-mCherry signal into the two respective groups .\",\n \"The ROI values were background subtracted [ROI ( t ) -BG ( t ) ] and normalized to the first value [ROI ( tx ) /ROI ( t1 ) ] .\",\n \"The average of the pooled values was fitted with Prism5 software using non-linear one-phase decay .\",\n \"For the FRAP analysis , raw data were background subtracted , corrected for acquisition-induced bleaching and normalized , and the curve was fitted from pooled values with Prism5 software using one-phase association non-linear fitting .\",\n \"The FRAP data where the aggregate structure was lost from the focal plane during recovery period image acquisition were discarded .\",\n \"Lifespan of virgin daughters was analyzed on YPD plates using Zeiss Axioscope 40 microdissection microscope as previously described in ( Denoth-Lippuner et al . , 2014 ) .\",\n \"All statistical analyses and graph preparations were done with Prism5 software .\",\n \"The error bars represent ±SEM from experimental triplicates with independent clones and statistical analyses were conducted with one-way ANOVA using Newman–Keuls post test , t-test , or Gehan-Breslow-Wilcoxon test .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells .","In budding yeast , various aging factors segregate to the aging ( mother ) -lineage , with poorly understood consequences .","In this study , we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single , asymmetrically inherited deposit over the remaining lifespan .","Surprisingly , deposit formation was not associated with stress or general decline in proteostasis .","Rather , the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates .","Deposit formation was dependent on Hsp42 , which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery , including the prion Sup35 .","Importantly , loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell .","Together , these data suggest that protein aggregation is an early aging-associated differentiation event in yeast , having a two-faceted role in organismal fitness ."],"string":"[\n \"Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells .\",\n \"In budding yeast , various aging factors segregate to the aging ( mother ) -lineage , with poorly understood consequences .\",\n \"In this study , we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single , asymmetrically inherited deposit over the remaining lifespan .\",\n \"Surprisingly , deposit formation was not associated with stress or general decline in proteostasis .\",\n \"Rather , the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates .\",\n \"Deposit formation was dependent on Hsp42 , which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery , including the prion Sup35 .\",\n \"Importantly , loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell .\",\n \"Together , these data suggest that protein aggregation is an early aging-associated differentiation event in yeast , having a two-faceted role in organismal fitness .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Aging is a complex process .","Studies involving a single-celled organism called budding yeast are commonly used to investigate the factors that contribute to aging .","When these yeast cells divide , a small daughter cell buds out from a large mother cell .","A mother cell has a limited lifespan and produces a finite number of daughter cells and then dies ( a phenomenon referred to ‘replicative aging’ ) .","However , when a daughter cell forms , the daughter's age is reset to zero , giving it the full potential to produce new offspring .","Previous research on budding yeast has shown that damaged or aggregated proteins accumulate in the mother cells but not the daughter cells , and that this accumulation of proteins contributes to shortening the lifespan of the mother cell .","Furthermore , protein aggregation has also been associated with a number of age-related diseases in humans , including neurodegenerative disorders such as Alzheimer's and Parkinson's disease .","However , it remains unclear how cells respond to protein aggregation that occurs during aging .","Many studies that have previously investigated this question have relied on exposing cells to stressful conditions , such as high temperatures , in order to trigger proteins to aggregate .","But now , Saarikangas and Barral have studied how proteins aggregate under normal , unstressed conditions in budding yeast as they age .","The experiments revealed that most unstressed yeast cells develop a single deposit of aggregated proteins already during early aging .","This age-associated structure proved to be a different type of response than the protein aggregation that occurs during stress .","Furthermore , the deposit did not form as a consequence of the cell having obvious problems with folding its proteins , nor did the deposit hinder cells from coping with stressful conditions that trigger protein misfolding .","Rather , this deposit supported the ability of the cell to degrade defective proteins .","This suggests that the deposit represents an early adaptive response to aging , which might consequently provide aged cells some advantage over their younger counterparts .","Saarikangas and Barral also found that this protein deposit was always retained in the mother cell and not passed onto the daughters at cell division .","Further experiments showed that an enzyme called heat shock protein 42 was responsible for collecting target proteins and bring them together to form the single deposit .","Reducing the levels of this enzyme prevented the deposit from forming and extended the lifespan of the mother cells .","Thus , these findings suggest that mother cells collect harmful protein aggregates into a single deposit and prevent them from entering the daughter cells .","Further work is needed to understand how the deposit is preferentially retained within the mother cell ."],"string":"[\n \"Aging is a complex process .\",\n \"Studies involving a single-celled organism called budding yeast are commonly used to investigate the factors that contribute to aging .\",\n \"When these yeast cells divide , a small daughter cell buds out from a large mother cell .\",\n \"A mother cell has a limited lifespan and produces a finite number of daughter cells and then dies ( a phenomenon referred to ‘replicative aging’ ) .\",\n \"However , when a daughter cell forms , the daughter's age is reset to zero , giving it the full potential to produce new offspring .\",\n \"Previous research on budding yeast has shown that damaged or aggregated proteins accumulate in the mother cells but not the daughter cells , and that this accumulation of proteins contributes to shortening the lifespan of the mother cell .\",\n \"Furthermore , protein aggregation has also been associated with a number of age-related diseases in humans , including neurodegenerative disorders such as Alzheimer's and Parkinson's disease .\",\n \"However , it remains unclear how cells respond to protein aggregation that occurs during aging .\",\n \"Many studies that have previously investigated this question have relied on exposing cells to stressful conditions , such as high temperatures , in order to trigger proteins to aggregate .\",\n \"But now , Saarikangas and Barral have studied how proteins aggregate under normal , unstressed conditions in budding yeast as they age .\",\n \"The experiments revealed that most unstressed yeast cells develop a single deposit of aggregated proteins already during early aging .\",\n \"This age-associated structure proved to be a different type of response than the protein aggregation that occurs during stress .\",\n \"Furthermore , the deposit did not form as a consequence of the cell having obvious problems with folding its proteins , nor did the deposit hinder cells from coping with stressful conditions that trigger protein misfolding .\",\n \"Rather , this deposit supported the ability of the cell to degrade defective proteins .\",\n \"This suggests that the deposit represents an early adaptive response to aging , which might consequently provide aged cells some advantage over their younger counterparts .\",\n \"Saarikangas and Barral also found that this protein deposit was always retained in the mother cell and not passed onto the daughters at cell division .\",\n \"Further experiments showed that an enzyme called heat shock protein 42 was responsible for collecting target proteins and bring them together to form the single deposit .\",\n \"Reducing the levels of this enzyme prevented the deposit from forming and extended the lifespan of the mother cells .\",\n \"Thus , these findings suggest that mother cells collect harmful protein aggregates into a single deposit and prevent them from entering the daughter cells .\",\n \"Further work is needed to understand how the deposit is preferentially retained within the mother cell .\"\n]","isConversational":false},"year":{"kind":"string","value":"2015"}}},{"rowIdx":294,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience","genetics and genomics"],"string":"[\n \"neuroscience\",\n \"genetics and genomics\"\n]","isConversational":false},"title":{"kind":"string","value":"Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay"},"id":{"kind":"string","value":"elife-12245-v2"},"sections":{"kind":"list like","value":[["Macroautophagy , referred to hereafter as autophagy , is a cellular process by which proteins and organelles are degraded and recycled through sequestration within autophagosomes and delivery to lysosomes ( Levine and Klionsky , 2004 ) .","The autophagy pathway is highly conserved and required for organismal development and function .","Defects in autophagy are associated with diseases including cancer , metabolic disruption , and neurodegenerative disorder ( Choi et al . , 2013; Cuervo and Wong , 2014; Frake et al . , 2015 ) .","Patients with mutations in any of the non-redundant core autophagy-related ( ATG ) genes have not previously been reported .","Ataxia is a neurodegenerative disease caused by disruption of the cerebellum and Purkinje cells , which results in the lack of coordinated muscle movements .","Large phenotype diversity is present in individuals with ataxia , including age of onset , rate of progression , and other accompanying neurological and non-neurological features ( Jayadev and Bird , 2013 ) , with corresponding genotypic heterogeneity ( Sandford and Burmeister , 2014 ) .","Even within the more defined phenotype of childhood ataxia with developmental delay , there are a large number of associated genes , such that similar phenotypic features alone are often insufficient information for an accurate diagnosis ( Burns et al . , 2014; De Michele and Filla , 2012; Jayadev and Bird , 2013 ) .","Identification of genetic causes of childhood ataxia is important for understanding disease pathogenesis and for possible future treatment development .","Whole exome sequencing has been successfully utilized to identify known and novel genetic mutations responsible for ataxia ( Burns et al . , 2014; Fogel et al . , 2014 ) .","Identification of candidate genes can be further verified through additional molecular analysis and utilization of specific and general animal models .","Here we identified a novel mutation in a core autophagy gene , ATG5 , in two children with ataxia , and demonstrate a reduction in autophagic response , also reproducing the phenotype in yeast and fly models ."],["Two Turkish siblings presented with ataxia and developmental delay in childhood , as previously described ( Yapici and Eraksoy , 2005 ) .","We performed linkage analysis on both affected siblings , their unaffected siblings , and their unaffected mother , using a model of remote parental consanguinity and identified a single broad ( >14 Mbp ) peak with LOD score 3 . 16 on chromosome 6q21 , between 102 and 116 Mb ( Figure 1 ) .","Whole exome sequencing identified a homozygous missense mutation , hg19 chr6:106 , 727 , 648 T>A , corresponding to E122D in ATG5 ( Figure 2A ) as the only damaging mutation within the genetically identified chromosomal linkage interval .","The mutation was Sanger verified and found absent from variant databases and from Turkish controls . 10 . 7554/eLife . 12245 . 003Figure 1 . Linkage analysis in consanguineous family with two siblings with ataxia , mental retardation and developmental delay maps defect to chromosomal interval containing ATG5 . Remote consanguinity was detected between parents of two previously described siblings having ataxia ( Yapici and Eraksoy , 2005 ) , illustrated here as third cousins .","SNP and linkage results for chromosome 6 ( B ) are illustrated below the pedigree ( A ) .","The shared homozygous region lies between rs4334996 and rs1204817 , encompassing ATG5 at 106 . 6 Mb .","Father ( 501 ) ’s alleles were inferred , 0 denotes unknown alleles .","Affected siblings , 601 and 602 , are denoted by black squares and unaffected family members by open symbols .","The proximal boundary is defined by a recombination event between rs1547384 and rs4334996 in affected individual 602 , while the distal boundary is defined as an ancestral recombination event ( lack of homozygosity , dark green ) between rs1204817 and rs648248 .","Orange arrows indicate the position of ATG5 . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00310 . 7554/eLife . 12245 . 004Figure 2 . The primary sequence of ATG5 , including the mutant E122 residue , as well as the protein structure is highly conserved across eukaryotic species .","( A ) Amino acid sequence alignment between ATG5 orthologs from human ( HsaATG5 ) , mouse ( MmuAtg5 ) , Drosophila melanogaster ( DmeAtg5 ) and Saccharomyces cerevisiae ( SceAtg5 ) was constructed at GenomeNet ( Kyoto University Bioinformatics Center ) through CLUSTALW and rendered in Genedoc v . 2 . 7 using default settings .","E122 in human ATG5 and E141 in yeast Atg5 , which are homologous residues , are indicated by red arrows .","( B ) Location of E122 residue is highlighted in yellow on the crystal structure of a human ATG12 ( residues 53–140 ) –ATG5 -ATG16L1 ( residues 11–43 ) complex ( PDB ID: 4NAW ) .","ATG5 is shown in cyan , ATG16L1 in magenta , and ATG12 in green ( Otomo et al . , 2013 ) .","( C ) Location of the E141 residue in yeast Atg5 , which corresponds to the E122 in human ATG5 , is indicated in yellow on the crystal structure of a yeast Atg12 ( 100–186 ) –Atg5 -Atg16 ( 1–46 ) complex , colored as for the human counterparts as in panel B ( PDB ID: 3W1S ) ( Noda et al . , 2013 ) .","( D ) Superimposition of crystal structure of ATG5E122D-ATG16L1 with ATG5WT-ATG16L1 ( PDB: 4TQ0 ) ( Kim et al . , 2015a ) .","Close-up view of ATG5 structure around WT ( E ) and E122D ( F ) mutation . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 004 ATG5 plays a role in elongation of the phagophore and its subsequent maturation into the complete autophagosome .","ATG12 is a ubiquitin-like protein that covalently binds ATG5 ( Mizushima et al . , 1998a ) , and this conjugate noncovalently binds ATG16L1 .","Crystal structure of the resulting ATG12–ATG5-ATG16L1 complex indicated that E122 is located in the vicinity of the ATG12–ATG5 interaction surface ( Figure 2B ) ; hence , we predicted that the mutation in ATG5 could affect the conjugation of ATG12 .","Comparison of protein isolated from control lymphoblastoid cell lines ( LCL ) and of affected subjects revealed a severe reduction of the ATG12–ATG5 conjugate in the mutant cells under basal conditions ( Figure 3A ) , suggesting that the E122D mutation may have impaired autophagy by inhibiting conjugation between ATG12 and ATG5 . 10 . 7554/eLife . 12245 . 005Figure 3 . Cells from ataxia patients with ATG5E122D/E122D mutation exhibit autophagy defects .","( A ) Decreased expression of ATG12–ATG5 conjugates in cells from ataxia patients with ATG5E122D/E122D mutation .","ATG5 immunoblotting ( IB ) of ATG12–ATG5 conjugates of LCLs from individuals whose ATG5 genotype corresponds to wild type ( A to I ) or E122D ( J and K ) .","( B ) Decreased autophagic flux in ATG5E122D/E122D LCL cells .","A subset of LCLs from ( A ) were treated with 0 . 1 μM bafilomycin A1 ( Baf ) for the indicated hours and analyzed by IB .","LC3-II is an autophagosome marker , and LC3-I is a precursor for LC3-II .","Baf inhibits lysosomal degradation of LC3-II .","Actin is shown as a loading control .","( C ) Decreased autophagic flux and increased expression of SQSTM1 , an autophagy substrate , in ATG5E122D/E122D LCL cells .","A subset of LCLs from ( A ) were treated with 250 nM Torin 1 or 0 . 1 μM Baf , for 2 hr and analyzed by IB .","Torin 1 is an autophagic flux activator . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 005 The ATG12–ATG5-ATG16L1 complex functions in part as an E3 ligase to facilitate the conjugation of LC3 to phosphatidylethanolamine , generating LC3-II ( Fujita et al . , 2008; Hanada et al . , 2007 ) .","Compared to control cells , LCLs from patients with the E122D mutation exposed to bafilomycin A1 showed a substantial reduction in LC3-II accumulation under basal conditions ( Figure 3B ) , suggesting a possible decrease in E3 activity and subsequent attenuation of basal autophagic flux .","The patient LCLs were also unable to upregulate their autophagic flux in response to Torin 1 ( Figure 3C ) , which is a strong inducer of autophagy ( Thoreen et al . , 2009 ) .","ATG5E122D LCLs also showed elevated levels of SQSTM1/p62 , an autophagy receptor and substrate , further indicating disruption of basal autophagy ( Figure 3C ) .","To examine the effect of the ATG5E122D mutation on formation of the ATG12–ATG5-ATG16L1 complex , we expressed the recombinant human proteins in insect Hi5 cells and analyzed the complexes by affinity isolation .","We could detect the ATG12–ATG5 complex when both wild-type proteins were co-expressed , but we could only detect a minimal amount of the ATG12–ATG5E122D complex ( Figure 4A ) .","Although overexpression of human ATG5WT in HEK293 cells or Drosophila tissues resulted in efficient covalent conjugation with overexpressed human ATG12 ( Figure 4B and C ) or endogenous Drosophila Atg12 ( Figure 4D ) , mutant ATG5E122D was dramatically impaired in this process ( Figure 4B , C and D ) .","Interestingly , expression levels of ATG5WT and ATG5E122D monomers were comparable to each other , indicating that the mutation affects the conjugation process , rather than the stability of proteins .","This was consistent with the structural location of ATG5 E122 adjacent to the surface that interacts with ATG12 ( Figure 2B ) .","To confirm that the mutation does not overtly alter the structure of ATG5 or binding to ATG16L1 , we analyzed formation of the noncovalent ATG5-ATG16L1 complex using constructs containing a TEV protease site .","Both wild-type and mutant ATG5 protein were efficiently co-precipitated with ATG16L1 ( Figure 4E ) .","Indeed , the co-crystal structure of a human ATG5E122D-ATG16L1 complex ( Figure 2 and Table","1 ) superimposes well with the previously determined structure of the WT proteins ( Figure 2D ) , with the major obvious difference being replacement of the side-chain ( Figure 2E and F ) .","Thus , it appears that the E122D mutation interferes with the ATG12–ATG5 conjugation process , but not with ATG5 folding or binding of ATG16L1 . 10 . 7554/eLife . 12245 . 006Figure 4 . E122D mutation interferes with formation of the ATG12–ATG5 conjugate .","( A ) Coomassie Blue-stained SDS-PAGE gel following glutathione affinity purification from lysates of Hi5 cells infected with baculoviruses expressing GST-ATG12 and either WT or E122D mutant ATG5 .","( B and C )","HEK293 cells expressing the indicated proteins were analyzed by IB .","( D ) Drosophila whole bodies expressing the indicated transgenes under the control of Tub-Gal4 were analyzed by IB .","( E ) Lysates from Hi5 cells expressing the indicated proteins were subjected to His/Ni-NTA purification and subsequent TEV protease treatment .","Proteins were analyzed by Coomassie Blue staining . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00610 . 7554/eLife . 12245 . 007Table 1 . Crystallography data collection and refinement statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 007Data collectionBeam lineAPS 24-ID-CSpace groupC2Unit cell parametersa , b , c ( Å ) 217 . 1 , 84 . 5 , 151 . 9α , β , γ ( ° ) 90 , 133 . 8 , 90Resolution ( Å ) ( highest shell ) 50–3 . 0 ( 3 . 12-3 . 0 ) Wavelength ( Å ) 0 . 9792Number of measured reflections179 , 310Number of unique refections39 , 496Overall Rsym0 . 057 ( 0 . 645 ) Completeness ( % ) 98 . 9 ( 99 . 3 ) Overall I/σI14 . 3 ( 2 . 3 ) Multiplicity4 . 5RefinementResolution ( Å ) 50–3 . 0Rwork/Rfree0 . 198/0 . 244rmsd bond lengths ( Å ) 0 . 008rmsd bond angles ( ° ) 0 . 994Number of protein atoms9403Ramachandran statisticsPreferred ( % ) 97 . 69Allowed ( % ) 2 . 22Disallowed ( % ) 0 . 09 ATG5 is a highly conserved protein , and sequence alignment demonstrated that E122 corresponds to yeast E141 ( Figure 2A and C ) .","We extended our analysis of the effect of the mutation on autophagy activity , by taking advantage of the yeast system .","To test whether autophagy was affected by the Atg5 mutation in yeast , we initially relied on the GFP-Atg8 processing assay ( Shintani and Klionsky , 2004 ) .","During autophagy a population of Atg8 is continuously transported to the vacuole inside of autophagosomes .","Tagging the N terminus of Atg8 with GFP makes it possible to monitor autophagy flux because Atg8 is rapidly degraded inside the vacuole whereas GFP is relatively resistant to vacuolar hydrolases; the generation of free GFP is an indication of autophagic activity .","We observed a consistent decrease in autophagy activity with the Atg5E141D mutant relative to Atg5WT following autophagy induction by starvation ( Figure 5A ) .","Atg8 , or GFP-Atg8 , does not measure autophagic cargo per se ( Klionsky , 2016 ) , and the amount of GFP-Atg8 processing only corresponds to the inner surface of the autophagosome .","To corroborate the effects observed through the GFP-Atg8 processing assay we examined autophagy using the quantitative Pho8∆60 assay ( Noda and Klionsky , 2008 ) .","Pho8∆60 is an altered form of a phosphatase that is only delivered to the vacuole via autophagy; subsequent proteolytic processing generates an active form of the hydrolase .","After 4 and 6 hr of starvation , yeast cells expressing the plasmid-based Atg5E141D mutant showed a significant decrease in autophagy levels compared to cells expressing Atg5WT ( Figure 5B ) , and similar results were obtained when the WT and mutant ATG5 genes were integrated back into the chromosomal ATG5 locus ( Figure 5C ) . 10 . 7554/eLife . 12245 . 008Figure 5 . E141D mutation of yeast Atg5 attenuates autophagy .","( A-D )","Yeast cells were grown in SMD to mid-log phase and nitrogen starved for the indicated times .","( A ) WLY176 atg5∆ yeast cells expressed GFP-Atg8 through its endogenous promoter and plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector .","Protein extracts were analyzed for GFP-Atg8 processing by western blot .","The ratio of free GFP to Dpm1 ( loading control ) is presented below the blots , and quantification is presented on the right ( Student’s t test , n=4; *p < 0 . 05 ) ; the value for Atg5WT at 6 hr was set to 1 . 0 and other values were normalized .","( B ) WLY176 atg5∆ yeast cells expressed either plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector .","Protein extracts were used to measure autophagy through the Pho8Δ60 assay ( Student’s t test , n=6; *p < 0 . 05 ) .","( C ) WLY176 cells with genomic integrated Atg5WT or Atg5E141D were used to generate protein extracts and autophagy was monitored through the Pho8Δ60 assay ( Student’s t test , n=3; *p < 0 . 05 ) .","( D ) WLY176 atg5∆ yeast cells expressing plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector were used to generate protein extracts .","The ratio of Atg8–PE to total Atg8 is presented below the blots based on western blot using antiserum to Atg8 .","Dpm1 was used as a loading control .","( E ) MKO ATG3 ( YCY137 ) cells were co-transformed with pATG8∆R-ATG7-ATG10 ( 414 ) , and either pATG5WT-HA-ATG12 ( 416 ) , pATG5E141D-HA-ATG12 ( 416 ) , pATG5WT-HA-ATG12-ATG16 ( 416 ) , or pATG5E141D-HA-ATG12-ATG16 ( 416 ) .","Overnight cultures were diluted to OD=0 . 02 in SMD -Ura -Trp .","The cells were incubated at 30°C for 18 hr to mid-log phase before they were shifted to SD-N for nitrogen starvation .","Samples at the corresponding time points were collected , TCA precipitated and subsequently analyzed by western blot .","S . E . , short exposure; L . E . , long exposure . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 008 To determine the reason for reduced autophagic activity we tested the effects of the Atg5E141D mutant on Atg8 lipidation .","As shown by the ratio of Atg8–PE:total Atg8 , cells expressing Atg5E141D displayed a decrease in Atg8–PE conjugation at 30 and 60 min of starvation compared to cells expressing Atg5WT ( Figure 5D ) .","We extended this analysis using the in vivo reconstitution of Atg8–PE conjugation as described previously ( Cao et al . , 2008 ) .","In brief , we examined Atg8 lipidation in a multiple-knockout ( MKO ) strain in which 23 ATG genes are deleted , when expressing only the E1 , E2 and E3-like conjugation enzymes of the autophagy machinery .","Atg8∆R that lacks the C-terminal arginine was used in the assay to bypass the initial activation step initiated by Atg4; due to the absence of Atg4 , there is no cleavage of Atg8–PE from the membrane , resulting in stabilization of this form of the protein .","We found that the Atg5E141D mutant was significantly defective in Atg8–PE conjugation compared to the cells with Atg5WT when Atg16 was not present ( Figure 5E ) .","Atg16 is not required mechanistically for Atg8 conjugation , but its presence increases the efficiency of this process and may dictate the site of conjugation ( Cao et al . , 2008; Hanada et al . , 2007 ) .","Thus , the presence of Atg16 may partially mask the Atg8 lipidation defects of the Atg5E141D mutant , and this may explain why the E122D/E141D mutation induces a hypomorphic rather than a complete null phenotype .","To further characterize the effect of the E122D mutation on the development of ataxia , we generated Drosophila melanogaster knockouts for Atg5 ( Figure 6A ) , and reconstituted the Atg5-null mutant flies with transgenes expressing wild-type ( WT ) or E122D human ATG5 ( Figure 6B-D ) .","Unlike mouse models , Atg5-null flies are viable , although they exhibit severe mobility defects after adult eclosion as demonstrated by a negative geotaxis assay ( Figure 6E and I , and Video 1 ) , similar to Atg7 null mutant flies ( Juhasz et al . , 2007 ) .","These mobility defects were substantially restored by expression of ATG5WT ( Figure 6F and I , and Video 2 ) , suggesting that the molecular function of ATG5 is conserved between human and Drosophila .","However , Atg5-null mutant flies expressing ATG5E122D were still defective in mobility although slightly better than Atg5-null controls ( Figure 6G–I , and Videos 3 and 4 ) , demonstrating again that ATG5 activity is compromised but not eliminated by the E122D mutation .","ATG5E122D was also inferior to ATG5WT in suppressing Ref ( 2 ) P ( fly p62/SQSTM1 ) accumulation ( Figure 6J and K ) and cell death ( Figure 6L and M ) in the brain of Atg5-null mutant flies . 10 . 7554/eLife . 12245 . 009Figure 6 . Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by ATG5E122D .","( A ) Genomic organization of the Atg5 locus and the Atg5-null mutant ( Atg55cc5 ) .","Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1 . 5 kb residues that eliminate more than 85% of Atg5-coding sequences including the translation start site .","Open boxes , untranslated exons; closed boxes , protein-coding exons .","Scale bar , relative length of 1 kb genomic span .","( B ) Schematic representation of how ATG5 transgenic flies were made .","Plasmid which can express wild-type or E122D-mutated human ATG5 was inserted into an identical genomic location ( the attP site ) through phiC31-mediated recombination ( Bateman et al . , 2006; Bischof et al . , 2007; Venken et al . , 2006 ) .","The scheme was adapted from a previous publication ( Kim and Lee , 2015 ) .","( C ) Genetic scheme of how ATG5 transgenes were placed into the Atg5-null mutant flies .","Atg5 , UAS-ATG5 and Tub-Gal4 loci are on the X-chromosome , second chromosome and third chromosome , respectively .","( D ) Whole flies of indicated genotypes were analyzed by IB .","( E to H )","Photographs of the vials containing 2-week-old adult male flies of indicated genotypes taken at 3 sec after negative geotaxis induction: ( E ) Atg5-null flies exhibit severely impaired mobility .","( F ) Ataxic phenotype of Atg5-null flies is complemented by human ATG5WT expression .","( G and H )","Human ATG5E122D is less capable than human ATG5WT in suppressing the fly ataxia phenotype .","( I ) Quantification of the climbing speeds of 2-week-old adult male flies ( n≥20 ) of the indicated genotype .","Climbing speed is presented as mean ± standard deviation ( n=5 ) .","P values were calculated using the Student’s t test ( ***p<0 . 001 ) .","( J ) Drosophila heads from two-weeks-old flies of the indicated genotypes were analyzed by IB .","( K ) Ref ( 2 ) P [p62] is an autophagy substrate .","Relative protein expression was measured by densitometry and presented in a bar graph ( mean ± standard error; n=4 ) .","( L ) Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) of Drosophila brain ( middle layer of the medial compartment ) .","( M ) TUNEL-positive cells per field were quantified and presented in a bar graph ( mean ± standard error; n≥5 ) .","K and M: P values were calculated using the Student’s t test ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00910 . 7554/eLife . 12245 . 010Video 1 . Climbing assay in 2 weeks-old wild-type flies ( left ) and Atg5-null flies ( right ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01010 . 7554/eLife . 12245 . 011Video 2 . Climbing assay in 2 weeks-old Atg5-null flies ( left ) and Atg5-null flies expressing ATG5WT ( right ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01110 . 7554/eLife . 12245 . 012Video 3 . Climbing assay in 2 weeks-old Atg5-null flies ( left ) and Atg5-null flies expressing ATG5E122D ( right ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01210 . 7554/eLife . 12245 . 013Video 4 . Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT ( left ) or ATG5E122D ( right ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 013"],["In summary , we demonstrate that the homozygous E122D mutation of ATG5 , a unique mutation found in two human subjects with ataxia , results in reduced conjugation to ATG12 and in an overall decrease in autophagy activity .","The homologous mutation in yeast also interferes with autophagy , and the ataxia phenotype was replicated in a fly model .","Based on these results we propose that this ATG5 mutation , and the consequent disruption in autophagy activity , is the cause of the ataxic phenotype and disturbance of the cerebellum in the affected siblings .","This hypothesis is in agreement with previously characterized mouse models , in which neuron-specific knockout of Atg5 results in ataxia-like phenotypes ( Hara et al . , 2006; Nishiyama et al . , 2007 ) .","By contrast , mice with complete knockout of Atg5 die shortly after birth , demonstrating that autophagy is essential for mammalian survival ( Kuma et al . , 2004 ) .","Our results indicate that E122D is a partial loss-of-function allele that impairs but does not completely abolish ATG5 activity .","Although the overall structure of the E122D mutant ATG5 superimposes well with the wild-type protein , the mutation causes a striking decrease in the level of ATG12–ATG5 conjugate that is formed when the C terminus of ATG12 is covalently linked to Lys130 of ATG5 .","We speculate that the E122D mutation causes subtle changes in the conformational dynamics that propagate to Lys130 , which is less than 10 Å away , resulting in less ATG12–ATG5 , which in turn leads to reduced LC3/Atg8 conjugation .","In neurons , which are among the cells most dependent on autophagy for tissue homeostasis ( Button et al . , 2015 ) , the residual function of the E122D allele is inadequate , resulting in predominantly neurological symptoms in the two patients .","Since homozygous mutations with complete loss-of-function have not been reported , we predict that individuals carrying such mutations , similar to Atg5-null mice ( Kuma et al . , 2004 ) , might not be viable .","Autophagy is quickly gaining importance for its roles in preventing neurodegeneration .","WDR45 is a redundant , non-core autophagy gene , one of four mammalian homologs to Atg18 , and mutations in WDR45 cause SENDA , static encephalopathy of childhood with neurodegeneration in adulthood ( Haack et al . , 2012; Saitsu et al . , 2013 ) .","Autophagy appears to be critical in ataxia , whether mutant proteins evade autophagy processes or normal autophagy is disrupted .","Several ataxias are attributed to intranuclear or cytoplasmic aggregation of mutant proteins within the cell ( Matilla-Duenas et al . , 2014 ) .","These protein aggregates , in humans and in mouse models , not only evade autophagic sequestration but may even inhibit autophagy ( Alves et al . , 2014 ) , or lead to reduction in autophagy available for other proteins due to saturation .","Assessment of autophagy in patient cells may be used to refine and identify the genetic cause of a patient’s ataxia .","Further discovery of the role of autophagy in neurodegenerative diseases should be used to investigate therapies targeted at the autophagy process .","Many drugs enhance autophagy and their effects on a multitude of neurodegenerative diseases have been studied ( Sarkar et al . , 2009 ) .","Recently more studies have been conducted assessing the value of autophagy enhancers in ataxia models and patients .","Induction of autophagy through administration of Temsirolimus , a rapamycin ester , and lentiviral overexpression of BECN1 in SCA3 model mice increase autophagy and the clearance of mutant protein aggregates , and reduce the ataxic phenotype ( Menzies et al . , 2010; Nascimento-Ferreira et al . , 2013 ) .","In a single patient trial , trehalose treatment of patient fibroblasts increased autophagy and alleviated cellular pathogenic features by improving mitochondrial morphology and reducing free radicals in the cell ( Casarejos et al . , 2014; Sarkar et al . , 2007 ) .","Trehalose also showed success in trials involving models of SCA17 ( Chen et al . , 2015 ) .","Lithium , another inducer of autophagy , improved symptoms in a SCA1 mouse model ( Watase et al . , 2007 ) , but did not slow or reduce symptoms in a treatment trial in SCA2 patients ( Sacca et al . , 2015 ) .","An autophagy enhancer may be an appropriate treatment to test in the presented subjects , as autophagic flux is attenuated , but not completely abrogated , by the ATG5E122D/E122D mutation .","This study’s finding of the pathogenic human E122D mutation in ATG5 , a gene encoding part of the autophagy-controlling core machinery , is important and novel , but consistent with reports of neurodegenerative disorders in other autophagy-related genes ( Frake et al . , 2015 ) .","Our results suggest that other mutations in this and other ATG genes , which impair but do not completely abolish autophagy , may result in similar forms of ataxia , intellectual disability and developmental delay .","This study exemplifies the utility of exome sequencing in the identification of rare disease-causing variants , and supports the role of impaired autophagy in neurodegenerative disease .","In addition , we demonstrate the utility of a combined genetic , biochemical and cell biological analysis in multiple model systems to elucidate the underlying pathogenic mechanism of rare human diseases ."],["Study protocols including written informed consents have been approved by the University of Michigan Institutional Review Board and the Boğaziçi University Institutional Review Board for Research with Human Participants .","Two Turkish brothers , ages 5 and 7 in 2004 , presented with ataxia and developmental delay , as previously described ( Yapici and Eraksoy , 2005 ) .","Parents were initially reported to be unrelated , but recently suggested they might be remotely related .","Both patients were delayed in walking , had truncal ataxia and dysmetria , nystagmus , and lower IQ ( 68 and 70 ) .","MRI revealed cerebellar hypoplasia .","Follow-up examinations showed no progression of symptoms .","DNA was isolated from peripheral whole blood using the Qiagen ( Germantown , MD ) Gentra Puregene isolation kit .","Linkage analysis was performed using the genotype data generated with Illumina HumanOmniExpress-24 chip for the mother and the four sibs .","The Allegro module ( Gudbjartsson et al . , 2000 ) of easyLINKAGE software was used , assuming autosomal recessive inheritance and parents as third cousins .","No deletion or duplications common to just the two affected brothers were detected using cnvPartition plug-in in Illumina Genome Studio v . 1 . 02 software .","Exome sequencing was performed independently twice on one subject .","Capture for whole exome sequencing was performed with NimbleGen SeqCap EZ Exome Library v1 . 0 kit ( Roche , Indianapolis , IN ) .","Captured regions were sequenced with Illumina HiSeq2000 instruments .","Variants were filtered to remove common variants based on 1000 Genomes , Exome Sequencing Project , and Exome Aggregation Consortium databases , variants outside of identified linkage regions , variants not expected to change protein coding , and variants not following a recessive model of inheritance ( Exome Aggregation Consortium ( ExAC ) , 2015; Genomes Project Consortium et al . , 2012; NHLBI Go Exome Sequencing Project , 2015 ) .","PCR followed by Sanger sequencing was performed to validate the variant identified through exome sequencing and test for segregation within the family .","The variant of interest was further examined in two separate collections of a total of 500 Turkish samples , and found absent .","Lymphoblastoid cell lines ( LCL ) of both subjects were generated from heparinized whole blood samples and cultured as described ( Doyle , 1990 ) .","As they are made in house and cultured briefly , mycoplasma contamination risk is minimized .","We used a baculovirus/insect cell expression system to examine formation of the human ATG12–ATG5 conjugate in a heterologous system described previously ( Qiu et al . , 2013 ) .","Hi5 insect cells ( Invitrogen , Carlsbad , CA ) were infected with baculoviruses expressing human ATG7 , ATG10 , a GST-tagged version of ATG12 ( residues 53–140 , corresponding to the ubiquitin-like domain ) and a His-tagged version of either ATG5WT or ATG5E122D .","Three days post infection , lysates were subjected to glutathione affinity chromatography , and the GST-ATG12–His-ATG5 conjugate was detected by SDS-PAGE followed by Coomassie Blue staining .","To confirm that the baculoviruses produce protein , Hi5 cells were coinfected with baculoviruses expressing the His-tagged WT and mutant versions of ATG5 and the N-terminal domain of ATG16L1 ( residues 1–69 , as an MBP fusion ) .","At three days post infection , lysates were subjected to nickel affinity purification .","The ATG5-ATG16L1 complex formation was detected by SDS-PAGE and Coomassie Blue staining .","The complex containing ATG5E122D and the N-terminal domain of ATG16L1 ( residues 1–69 ) was expressed in Hi5 insect cells , and purified by nickel affinity , ion exchange , and size exclusion chromatography into a final buffer of 20 mM Tris , pH 8 . 5 , 50 mM NaCl , 10 mM DTT .","The complex was concentrated to 18 . 5 mg/ml , aliquoted , flash-frozen and stored at -80°C until further use .","Crystals were grown by the hanging drop vapor diffusion method by mixing purified protein 1:1 with reservoir solutions of 37 . 5 mM MES , pH 5 . 2–5 . 8 , 0 . 2 M sodium tartrate , and 11–13% polyethylene glycol 3350 .","Final crystals were obtained by micro-seeding with reservoir solution of 40 mM MES , pH 5 . 5 , 0 . 2 M sodium tartrate , 8 . 5% PEG3350 , 10 mM DTT .","Crystals were cryoprotected in reservoir solution supplemented with 25% xylitol , and flash frozen in liquid nitrogen prior to data collection .","Diffraction data were processed with XDS .","The structure was determined by molecular replacement using Phaser ( McCoy et al . , 2007 ) with the structure of the WT ATG5-ATG16L1 ( 1–69 ) ( PDB: 4TQ0 ) complex as a search model ( Kim et al . , 2015a ) .","Model construction and rebuilding were performed using Coot ( Emsley et al . , 2010 ) .","The structure was refined using Phenix ( Adams et al . , 2010 ) .","Diffraction data and refinement statistics are provided in Table 1 .","Cells or tissues were lysed in cell lysis buffer ( 20 mM Tris-HCl , pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 2 . 5 mM sodium pyrophosphate , 1 mM beta-glycerophosphate , 1 mM Na3VO4 , 1% Triton X-100 ) or RIPA buffer ( 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 1% sodium deoxycholate , 1% NP-40 , 0 . 1% SDS ) containing protease inhibitor cocktail ( Roche ) .","After being clarified with centrifugation , lysates were boiled in SDS sample buffer , separated by SDS-PAGE , transferred to polyvinylidene difluoride membranes and probed with the indicated antibodies .","ATG5 ( 12994 ) , LC3 ( 3868 ) and SQSTM1/p62 ( 5114 ) antibodies were purchased from Cell Signaling Technology .","Hemagglutinin ( HA , 3F10 ) antibody was from Roche .","Actin ( JLA20 ) and tubulin ( T5168 ) antibodies were from Developmental Studies Hybridoma Bank and Sigma , respectively .","Ref ( 2 ) P antibody was previously described ( Pircs et al . , 2012 ) .","Wild-type human ATG5-coding sequence was from Addgene #24922 ( deposited by Dr . Toren Finkel ) ( Lee et al . , 2008 ) .","The E122D mutation was introduced into ATG5 by PCR-based site-directed mutagenesis .","ATG5WT and ATG5E122D were cloned into the plasmid pLU-CMV-Flag .","The HA-ATG12-expressing plasmid was from Addgene #22950 ( deposited by Dr . Noboru Mizushima ) ( Mizushima et al . , 1998b ) .","HEK293 cells ( the 293 A substrain from Invitrogen , tested negative for mycoplasma by PCR ) were cultured in Dulbecco’s modified Eagle’s medium ( DMEM , Invitrogen ) containing 10% fetal bovine serum ( FBS ) and penicillin/streptomycin at 37°C in 5% CO2 .","For transient expression of proteins , HEK293 cells were transfected with purified plasmid constructs and polyethylenimine ( PEI , Sigma ) as previously described ( Horbinski et al . , 2001 ) .","Cells were harvested 24 hr after transfection for immunoblot analyses .","Saccharomyces cerevisiae strain WLY176 was used to generate an ATG5 knockout strain ( atg5∆ ) as previously described ( Gueldener et al . , 2002 ) .","The MKO strain YCY137 ( SEY6210 atg1∆ , 2∆ , 4∆ , 5∆ , 6∆ , 7∆ , 8∆ , 9∆ , 10∆ , 11∆ , 12∆ , 13∆ , 14∆ , 16∆ , 17∆ , 18∆ , 19∆ , 20∆ , 21∆ , 23∆ , 24∆ , 27∆ , 29∆ ) ( Cao et al . , 2008 ) , was used for in vivo reconstitution of Atg8 conjugation .","Site-directed mutagenesis was performed to generate ATG5 amplicons with the E141D mutation as previously described ( Liu and Naismith , 2008 ) .","A pRS406 empty plasmid was digested with Spel and SalI , and then ligated with a DNA fragment encoding either wild-type or mutant Atg5-PA .","atg5∆ was transformed with an empty pRS406 vector , or plasmids encoding Atg5-PA WT or Atg5-PA E141D .","Wild-type WLY176 colonies were transformed with empty pRS406 vector as a control .","Colonies were grown on SMD-URA medium and starved in nitrogen-deficient medium .","Pho8Δ60 and western blot analyses were performed as described previously ( Noda and Klionsky , 2008; Shintani and Klionsky , 2004 ) .","Quantification was performed using ImageJ software .","The pATG8∆R-ATG7-ATG10 ( 414 ) , pATG5 ( WT ) -HA-ATG12 ( 416 ) and pATG5 ( WT ) -HA-ATG12-ATG16 ( 416 ) plasmids were described previously ( Cao et al . , 2008 ) .","The pATG5 ( E141D ) -HA-ATG12 ( 416 ) and pATG5 ( E141D ) -HA-ATG12-ATG16 ( 416 ) plasmids were made by site-directed mutagenesis based on the wild-type constructs .","Atg5-null Drosophila flies ( Atg55cc5 ) were generated by CRISPR-Cas9-mediated genome editing , using a double gRNA approach , both targeting the same gene , as described ( Kondo and Ueda , 2013 ) .","The Atg55cc5 mutant was recovered by screening viable candidate lines for accumulation of the specific autophagy cargo Ref ( 2 ) P using western blots , followed by PCR and sequencing .","Atg55cc5 mutants have a deletion in X:7 , 322 , 242–7 , 323 , 717 residues ( Drosophila melanogaster R6 . 06 ) , which deletes five out of six exons of the Atg5 gene , eliminating more than 85% of protein-coding sequences including the translation start site ( Figure 6A ) .","The PhiC31 integrase-mediated site-specific transformation method was used to express human ATG5WT and ATG5E122D from an identical genomic locus ( Bateman et al . , 2006; Bischof et al . , 2007; Venken et al . , 2006 ) .","In brief , flag-tagged ATG5WT and ATG5E122D were cloned into a pUAST-attB vector ( Bischof et al . , 2007 ) and fully sequenced .","pUAST-attB-ATG5WT and pUAST-attB-ATG5E122D were microinjected into y1 M{vas-int . Dm}ZH-2A w*; M{3xP3-RFP . attP}ZH-51D flies and stable transformants were isolated by the presence of the mini-white+ marker ( Figure 6B ) .","The UAS-ATG5WT or UAS-ATG5E122D transgenes were crossed with a double balancer strain ( Bl/CyO; TM2/TM6B ) and then with +/CyO; Tub-Gal4/TM2 to be constructed as stable Tub>ATG5 lines ( UAS-ATG5/UAS-ATG5; Tub-Gal4/TM6B ) .","The Tub>ATG5 male flies were crossed with Atg55cc5/FM7 female flies to generate Atg5-null flies expressing human ATG5 transgenes .","Climbing assays and TUNEL staining were performed as previously described ( Kim et al . , 2015b ) ."]],"string":"[\n [\n \"Macroautophagy , referred to hereafter as autophagy , is a cellular process by which proteins and organelles are degraded and recycled through sequestration within autophagosomes and delivery to lysosomes ( Levine and Klionsky , 2004 ) .\",\n \"The autophagy pathway is highly conserved and required for organismal development and function .\",\n \"Defects in autophagy are associated with diseases including cancer , metabolic disruption , and neurodegenerative disorder ( Choi et al . , 2013; Cuervo and Wong , 2014; Frake et al . , 2015 ) .\",\n \"Patients with mutations in any of the non-redundant core autophagy-related ( ATG ) genes have not previously been reported .\",\n \"Ataxia is a neurodegenerative disease caused by disruption of the cerebellum and Purkinje cells , which results in the lack of coordinated muscle movements .\",\n \"Large phenotype diversity is present in individuals with ataxia , including age of onset , rate of progression , and other accompanying neurological and non-neurological features ( Jayadev and Bird , 2013 ) , with corresponding genotypic heterogeneity ( Sandford and Burmeister , 2014 ) .\",\n \"Even within the more defined phenotype of childhood ataxia with developmental delay , there are a large number of associated genes , such that similar phenotypic features alone are often insufficient information for an accurate diagnosis ( Burns et al . , 2014; De Michele and Filla , 2012; Jayadev and Bird , 2013 ) .\",\n \"Identification of genetic causes of childhood ataxia is important for understanding disease pathogenesis and for possible future treatment development .\",\n \"Whole exome sequencing has been successfully utilized to identify known and novel genetic mutations responsible for ataxia ( Burns et al . , 2014; Fogel et al . , 2014 ) .\",\n \"Identification of candidate genes can be further verified through additional molecular analysis and utilization of specific and general animal models .\",\n \"Here we identified a novel mutation in a core autophagy gene , ATG5 , in two children with ataxia , and demonstrate a reduction in autophagic response , also reproducing the phenotype in yeast and fly models .\"\n ],\n [\n \"Two Turkish siblings presented with ataxia and developmental delay in childhood , as previously described ( Yapici and Eraksoy , 2005 ) .\",\n \"We performed linkage analysis on both affected siblings , their unaffected siblings , and their unaffected mother , using a model of remote parental consanguinity and identified a single broad ( >14 Mbp ) peak with LOD score 3 . 16 on chromosome 6q21 , between 102 and 116 Mb ( Figure 1 ) .\",\n \"Whole exome sequencing identified a homozygous missense mutation , hg19 chr6:106 , 727 , 648 T>A , corresponding to E122D in ATG5 ( Figure 2A ) as the only damaging mutation within the genetically identified chromosomal linkage interval .\",\n \"The mutation was Sanger verified and found absent from variant databases and from Turkish controls . 10 . 7554/eLife . 12245 . 003Figure 1 . Linkage analysis in consanguineous family with two siblings with ataxia , mental retardation and developmental delay maps defect to chromosomal interval containing ATG5 . Remote consanguinity was detected between parents of two previously described siblings having ataxia ( Yapici and Eraksoy , 2005 ) , illustrated here as third cousins .\",\n \"SNP and linkage results for chromosome 6 ( B ) are illustrated below the pedigree ( A ) .\",\n \"The shared homozygous region lies between rs4334996 and rs1204817 , encompassing ATG5 at 106 . 6 Mb .\",\n \"Father ( 501 ) ’s alleles were inferred , 0 denotes unknown alleles .\",\n \"Affected siblings , 601 and 602 , are denoted by black squares and unaffected family members by open symbols .\",\n \"The proximal boundary is defined by a recombination event between rs1547384 and rs4334996 in affected individual 602 , while the distal boundary is defined as an ancestral recombination event ( lack of homozygosity , dark green ) between rs1204817 and rs648248 .\",\n \"Orange arrows indicate the position of ATG5 . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00310 . 7554/eLife . 12245 . 004Figure 2 . The primary sequence of ATG5 , including the mutant E122 residue , as well as the protein structure is highly conserved across eukaryotic species .\",\n \"( A ) Amino acid sequence alignment between ATG5 orthologs from human ( HsaATG5 ) , mouse ( MmuAtg5 ) , Drosophila melanogaster ( DmeAtg5 ) and Saccharomyces cerevisiae ( SceAtg5 ) was constructed at GenomeNet ( Kyoto University Bioinformatics Center ) through CLUSTALW and rendered in Genedoc v . 2 . 7 using default settings .\",\n \"E122 in human ATG5 and E141 in yeast Atg5 , which are homologous residues , are indicated by red arrows .\",\n \"( B ) Location of E122 residue is highlighted in yellow on the crystal structure of a human ATG12 ( residues 53–140 ) –ATG5 -ATG16L1 ( residues 11–43 ) complex ( PDB ID: 4NAW ) .\",\n \"ATG5 is shown in cyan , ATG16L1 in magenta , and ATG12 in green ( Otomo et al . , 2013 ) .\",\n \"( C ) Location of the E141 residue in yeast Atg5 , which corresponds to the E122 in human ATG5 , is indicated in yellow on the crystal structure of a yeast Atg12 ( 100–186 ) –Atg5 -Atg16 ( 1–46 ) complex , colored as for the human counterparts as in panel B ( PDB ID: 3W1S ) ( Noda et al . , 2013 ) .\",\n \"( D ) Superimposition of crystal structure of ATG5E122D-ATG16L1 with ATG5WT-ATG16L1 ( PDB: 4TQ0 ) ( Kim et al . , 2015a ) .\",\n \"Close-up view of ATG5 structure around WT ( E ) and E122D ( F ) mutation . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 004 ATG5 plays a role in elongation of the phagophore and its subsequent maturation into the complete autophagosome .\",\n \"ATG12 is a ubiquitin-like protein that covalently binds ATG5 ( Mizushima et al . , 1998a ) , and this conjugate noncovalently binds ATG16L1 .\",\n \"Crystal structure of the resulting ATG12–ATG5-ATG16L1 complex indicated that E122 is located in the vicinity of the ATG12–ATG5 interaction surface ( Figure 2B ) ; hence , we predicted that the mutation in ATG5 could affect the conjugation of ATG12 .\",\n \"Comparison of protein isolated from control lymphoblastoid cell lines ( LCL ) and of affected subjects revealed a severe reduction of the ATG12–ATG5 conjugate in the mutant cells under basal conditions ( Figure 3A ) , suggesting that the E122D mutation may have impaired autophagy by inhibiting conjugation between ATG12 and ATG5 . 10 . 7554/eLife . 12245 . 005Figure 3 . Cells from ataxia patients with ATG5E122D/E122D mutation exhibit autophagy defects .\",\n \"( A ) Decreased expression of ATG12–ATG5 conjugates in cells from ataxia patients with ATG5E122D/E122D mutation .\",\n \"ATG5 immunoblotting ( IB ) of ATG12–ATG5 conjugates of LCLs from individuals whose ATG5 genotype corresponds to wild type ( A to I ) or E122D ( J and K ) .\",\n \"( B ) Decreased autophagic flux in ATG5E122D/E122D LCL cells .\",\n \"A subset of LCLs from ( A ) were treated with 0 . 1 μM bafilomycin A1 ( Baf ) for the indicated hours and analyzed by IB .\",\n \"LC3-II is an autophagosome marker , and LC3-I is a precursor for LC3-II .\",\n \"Baf inhibits lysosomal degradation of LC3-II .\",\n \"Actin is shown as a loading control .\",\n \"( C ) Decreased autophagic flux and increased expression of SQSTM1 , an autophagy substrate , in ATG5E122D/E122D LCL cells .\",\n \"A subset of LCLs from ( A ) were treated with 250 nM Torin 1 or 0 . 1 μM Baf , for 2 hr and analyzed by IB .\",\n \"Torin 1 is an autophagic flux activator . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 005 The ATG12–ATG5-ATG16L1 complex functions in part as an E3 ligase to facilitate the conjugation of LC3 to phosphatidylethanolamine , generating LC3-II ( Fujita et al . , 2008; Hanada et al . , 2007 ) .\",\n \"Compared to control cells , LCLs from patients with the E122D mutation exposed to bafilomycin A1 showed a substantial reduction in LC3-II accumulation under basal conditions ( Figure 3B ) , suggesting a possible decrease in E3 activity and subsequent attenuation of basal autophagic flux .\",\n \"The patient LCLs were also unable to upregulate their autophagic flux in response to Torin 1 ( Figure 3C ) , which is a strong inducer of autophagy ( Thoreen et al . , 2009 ) .\",\n \"ATG5E122D LCLs also showed elevated levels of SQSTM1/p62 , an autophagy receptor and substrate , further indicating disruption of basal autophagy ( Figure 3C ) .\",\n \"To examine the effect of the ATG5E122D mutation on formation of the ATG12–ATG5-ATG16L1 complex , we expressed the recombinant human proteins in insect Hi5 cells and analyzed the complexes by affinity isolation .\",\n \"We could detect the ATG12–ATG5 complex when both wild-type proteins were co-expressed , but we could only detect a minimal amount of the ATG12–ATG5E122D complex ( Figure 4A ) .\",\n \"Although overexpression of human ATG5WT in HEK293 cells or Drosophila tissues resulted in efficient covalent conjugation with overexpressed human ATG12 ( Figure 4B and C ) or endogenous Drosophila Atg12 ( Figure 4D ) , mutant ATG5E122D was dramatically impaired in this process ( Figure 4B , C and D ) .\",\n \"Interestingly , expression levels of ATG5WT and ATG5E122D monomers were comparable to each other , indicating that the mutation affects the conjugation process , rather than the stability of proteins .\",\n \"This was consistent with the structural location of ATG5 E122 adjacent to the surface that interacts with ATG12 ( Figure 2B ) .\",\n \"To confirm that the mutation does not overtly alter the structure of ATG5 or binding to ATG16L1 , we analyzed formation of the noncovalent ATG5-ATG16L1 complex using constructs containing a TEV protease site .\",\n \"Both wild-type and mutant ATG5 protein were efficiently co-precipitated with ATG16L1 ( Figure 4E ) .\",\n \"Indeed , the co-crystal structure of a human ATG5E122D-ATG16L1 complex ( Figure 2 and Table\",\n \"1 ) superimposes well with the previously determined structure of the WT proteins ( Figure 2D ) , with the major obvious difference being replacement of the side-chain ( Figure 2E and F ) .\",\n \"Thus , it appears that the E122D mutation interferes with the ATG12–ATG5 conjugation process , but not with ATG5 folding or binding of ATG16L1 . 10 . 7554/eLife . 12245 . 006Figure 4 . E122D mutation interferes with formation of the ATG12–ATG5 conjugate .\",\n \"( A ) Coomassie Blue-stained SDS-PAGE gel following glutathione affinity purification from lysates of Hi5 cells infected with baculoviruses expressing GST-ATG12 and either WT or E122D mutant ATG5 .\",\n \"( B and C )\",\n \"HEK293 cells expressing the indicated proteins were analyzed by IB .\",\n \"( D ) Drosophila whole bodies expressing the indicated transgenes under the control of Tub-Gal4 were analyzed by IB .\",\n \"( E ) Lysates from Hi5 cells expressing the indicated proteins were subjected to His/Ni-NTA purification and subsequent TEV protease treatment .\",\n \"Proteins were analyzed by Coomassie Blue staining . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00610 . 7554/eLife . 12245 . 007Table 1 . Crystallography data collection and refinement statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 007Data collectionBeam lineAPS 24-ID-CSpace groupC2Unit cell parametersa , b , c ( Å ) 217 . 1 , 84 . 5 , 151 . 9α , β , γ ( ° ) 90 , 133 . 8 , 90Resolution ( Å ) ( highest shell ) 50–3 . 0 ( 3 . 12-3 . 0 ) Wavelength ( Å ) 0 . 9792Number of measured reflections179 , 310Number of unique refections39 , 496Overall Rsym0 . 057 ( 0 . 645 ) Completeness ( % ) 98 . 9 ( 99 . 3 ) Overall I/σI14 . 3 ( 2 . 3 ) Multiplicity4 . 5RefinementResolution ( Å ) 50–3 . 0Rwork/Rfree0 . 198/0 . 244rmsd bond lengths ( Å ) 0 . 008rmsd bond angles ( ° ) 0 . 994Number of protein atoms9403Ramachandran statisticsPreferred ( % ) 97 . 69Allowed ( % ) 2 . 22Disallowed ( % ) 0 . 09 ATG5 is a highly conserved protein , and sequence alignment demonstrated that E122 corresponds to yeast E141 ( Figure 2A and C ) .\",\n \"We extended our analysis of the effect of the mutation on autophagy activity , by taking advantage of the yeast system .\",\n \"To test whether autophagy was affected by the Atg5 mutation in yeast , we initially relied on the GFP-Atg8 processing assay ( Shintani and Klionsky , 2004 ) .\",\n \"During autophagy a population of Atg8 is continuously transported to the vacuole inside of autophagosomes .\",\n \"Tagging the N terminus of Atg8 with GFP makes it possible to monitor autophagy flux because Atg8 is rapidly degraded inside the vacuole whereas GFP is relatively resistant to vacuolar hydrolases; the generation of free GFP is an indication of autophagic activity .\",\n \"We observed a consistent decrease in autophagy activity with the Atg5E141D mutant relative to Atg5WT following autophagy induction by starvation ( Figure 5A ) .\",\n \"Atg8 , or GFP-Atg8 , does not measure autophagic cargo per se ( Klionsky , 2016 ) , and the amount of GFP-Atg8 processing only corresponds to the inner surface of the autophagosome .\",\n \"To corroborate the effects observed through the GFP-Atg8 processing assay we examined autophagy using the quantitative Pho8∆60 assay ( Noda and Klionsky , 2008 ) .\",\n \"Pho8∆60 is an altered form of a phosphatase that is only delivered to the vacuole via autophagy; subsequent proteolytic processing generates an active form of the hydrolase .\",\n \"After 4 and 6 hr of starvation , yeast cells expressing the plasmid-based Atg5E141D mutant showed a significant decrease in autophagy levels compared to cells expressing Atg5WT ( Figure 5B ) , and similar results were obtained when the WT and mutant ATG5 genes were integrated back into the chromosomal ATG5 locus ( Figure 5C ) . 10 . 7554/eLife . 12245 . 008Figure 5 . E141D mutation of yeast Atg5 attenuates autophagy .\",\n \"( A-D )\",\n \"Yeast cells were grown in SMD to mid-log phase and nitrogen starved for the indicated times .\",\n \"( A ) WLY176 atg5∆ yeast cells expressed GFP-Atg8 through its endogenous promoter and plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector .\",\n \"Protein extracts were analyzed for GFP-Atg8 processing by western blot .\",\n \"The ratio of free GFP to Dpm1 ( loading control ) is presented below the blots , and quantification is presented on the right ( Student’s t test , n=4; *p < 0 . 05 ) ; the value for Atg5WT at 6 hr was set to 1 . 0 and other values were normalized .\",\n \"( B ) WLY176 atg5∆ yeast cells expressed either plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector .\",\n \"Protein extracts were used to measure autophagy through the Pho8Δ60 assay ( Student’s t test , n=6; *p < 0 . 05 ) .\",\n \"( C ) WLY176 cells with genomic integrated Atg5WT or Atg5E141D were used to generate protein extracts and autophagy was monitored through the Pho8Δ60 assay ( Student’s t test , n=3; *p < 0 . 05 ) .\",\n \"( D ) WLY176 atg5∆ yeast cells expressing plasmid-based Atg5WT-PA , Atg5E141D-PA or an empty vector were used to generate protein extracts .\",\n \"The ratio of Atg8–PE to total Atg8 is presented below the blots based on western blot using antiserum to Atg8 .\",\n \"Dpm1 was used as a loading control .\",\n \"( E ) MKO ATG3 ( YCY137 ) cells were co-transformed with pATG8∆R-ATG7-ATG10 ( 414 ) , and either pATG5WT-HA-ATG12 ( 416 ) , pATG5E141D-HA-ATG12 ( 416 ) , pATG5WT-HA-ATG12-ATG16 ( 416 ) , or pATG5E141D-HA-ATG12-ATG16 ( 416 ) .\",\n \"Overnight cultures were diluted to OD=0 . 02 in SMD -Ura -Trp .\",\n \"The cells were incubated at 30°C for 18 hr to mid-log phase before they were shifted to SD-N for nitrogen starvation .\",\n \"Samples at the corresponding time points were collected , TCA precipitated and subsequently analyzed by western blot .\",\n \"S . E . , short exposure; L . E . , long exposure . DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 008 To determine the reason for reduced autophagic activity we tested the effects of the Atg5E141D mutant on Atg8 lipidation .\",\n \"As shown by the ratio of Atg8–PE:total Atg8 , cells expressing Atg5E141D displayed a decrease in Atg8–PE conjugation at 30 and 60 min of starvation compared to cells expressing Atg5WT ( Figure 5D ) .\",\n \"We extended this analysis using the in vivo reconstitution of Atg8–PE conjugation as described previously ( Cao et al . , 2008 ) .\",\n \"In brief , we examined Atg8 lipidation in a multiple-knockout ( MKO ) strain in which 23 ATG genes are deleted , when expressing only the E1 , E2 and E3-like conjugation enzymes of the autophagy machinery .\",\n \"Atg8∆R that lacks the C-terminal arginine was used in the assay to bypass the initial activation step initiated by Atg4; due to the absence of Atg4 , there is no cleavage of Atg8–PE from the membrane , resulting in stabilization of this form of the protein .\",\n \"We found that the Atg5E141D mutant was significantly defective in Atg8–PE conjugation compared to the cells with Atg5WT when Atg16 was not present ( Figure 5E ) .\",\n \"Atg16 is not required mechanistically for Atg8 conjugation , but its presence increases the efficiency of this process and may dictate the site of conjugation ( Cao et al . , 2008; Hanada et al . , 2007 ) .\",\n \"Thus , the presence of Atg16 may partially mask the Atg8 lipidation defects of the Atg5E141D mutant , and this may explain why the E122D/E141D mutation induces a hypomorphic rather than a complete null phenotype .\",\n \"To further characterize the effect of the E122D mutation on the development of ataxia , we generated Drosophila melanogaster knockouts for Atg5 ( Figure 6A ) , and reconstituted the Atg5-null mutant flies with transgenes expressing wild-type ( WT ) or E122D human ATG5 ( Figure 6B-D ) .\",\n \"Unlike mouse models , Atg5-null flies are viable , although they exhibit severe mobility defects after adult eclosion as demonstrated by a negative geotaxis assay ( Figure 6E and I , and Video 1 ) , similar to Atg7 null mutant flies ( Juhasz et al . , 2007 ) .\",\n \"These mobility defects were substantially restored by expression of ATG5WT ( Figure 6F and I , and Video 2 ) , suggesting that the molecular function of ATG5 is conserved between human and Drosophila .\",\n \"However , Atg5-null mutant flies expressing ATG5E122D were still defective in mobility although slightly better than Atg5-null controls ( Figure 6G–I , and Videos 3 and 4 ) , demonstrating again that ATG5 activity is compromised but not eliminated by the E122D mutation .\",\n \"ATG5E122D was also inferior to ATG5WT in suppressing Ref ( 2 ) P ( fly p62/SQSTM1 ) accumulation ( Figure 6J and K ) and cell death ( Figure 6L and M ) in the brain of Atg5-null mutant flies . 10 . 7554/eLife . 12245 . 009Figure 6 . Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by ATG5E122D .\",\n \"( A ) Genomic organization of the Atg5 locus and the Atg5-null mutant ( Atg55cc5 ) .\",\n \"Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1 . 5 kb residues that eliminate more than 85% of Atg5-coding sequences including the translation start site .\",\n \"Open boxes , untranslated exons; closed boxes , protein-coding exons .\",\n \"Scale bar , relative length of 1 kb genomic span .\",\n \"( B ) Schematic representation of how ATG5 transgenic flies were made .\",\n \"Plasmid which can express wild-type or E122D-mutated human ATG5 was inserted into an identical genomic location ( the attP site ) through phiC31-mediated recombination ( Bateman et al . , 2006; Bischof et al . , 2007; Venken et al . , 2006 ) .\",\n \"The scheme was adapted from a previous publication ( Kim and Lee , 2015 ) .\",\n \"( C ) Genetic scheme of how ATG5 transgenes were placed into the Atg5-null mutant flies .\",\n \"Atg5 , UAS-ATG5 and Tub-Gal4 loci are on the X-chromosome , second chromosome and third chromosome , respectively .\",\n \"( D ) Whole flies of indicated genotypes were analyzed by IB .\",\n \"( E to H )\",\n \"Photographs of the vials containing 2-week-old adult male flies of indicated genotypes taken at 3 sec after negative geotaxis induction: ( E ) Atg5-null flies exhibit severely impaired mobility .\",\n \"( F ) Ataxic phenotype of Atg5-null flies is complemented by human ATG5WT expression .\",\n \"( G and H )\",\n \"Human ATG5E122D is less capable than human ATG5WT in suppressing the fly ataxia phenotype .\",\n \"( I ) Quantification of the climbing speeds of 2-week-old adult male flies ( n≥20 ) of the indicated genotype .\",\n \"Climbing speed is presented as mean ± standard deviation ( n=5 ) .\",\n \"P values were calculated using the Student’s t test ( ***p<0 . 001 ) .\",\n \"( J ) Drosophila heads from two-weeks-old flies of the indicated genotypes were analyzed by IB .\",\n \"( K ) Ref ( 2 ) P [p62] is an autophagy substrate .\",\n \"Relative protein expression was measured by densitometry and presented in a bar graph ( mean ± standard error; n=4 ) .\",\n \"( L ) Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) of Drosophila brain ( middle layer of the medial compartment ) .\",\n \"( M ) TUNEL-positive cells per field were quantified and presented in a bar graph ( mean ± standard error; n≥5 ) .\",\n \"K and M: P values were calculated using the Student’s t test ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 00910 . 7554/eLife . 12245 . 010Video 1 . Climbing assay in 2 weeks-old wild-type flies ( left ) and Atg5-null flies ( right ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01010 . 7554/eLife . 12245 . 011Video 2 . Climbing assay in 2 weeks-old Atg5-null flies ( left ) and Atg5-null flies expressing ATG5WT ( right ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01110 . 7554/eLife . 12245 . 012Video 3 . Climbing assay in 2 weeks-old Atg5-null flies ( left ) and Atg5-null flies expressing ATG5E122D ( right ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 01210 . 7554/eLife . 12245 . 013Video 4 . Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT ( left ) or ATG5E122D ( right ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12245 . 013\"\n ],\n [\n \"In summary , we demonstrate that the homozygous E122D mutation of ATG5 , a unique mutation found in two human subjects with ataxia , results in reduced conjugation to ATG12 and in an overall decrease in autophagy activity .\",\n \"The homologous mutation in yeast also interferes with autophagy , and the ataxia phenotype was replicated in a fly model .\",\n \"Based on these results we propose that this ATG5 mutation , and the consequent disruption in autophagy activity , is the cause of the ataxic phenotype and disturbance of the cerebellum in the affected siblings .\",\n \"This hypothesis is in agreement with previously characterized mouse models , in which neuron-specific knockout of Atg5 results in ataxia-like phenotypes ( Hara et al . , 2006; Nishiyama et al . , 2007 ) .\",\n \"By contrast , mice with complete knockout of Atg5 die shortly after birth , demonstrating that autophagy is essential for mammalian survival ( Kuma et al . , 2004 ) .\",\n \"Our results indicate that E122D is a partial loss-of-function allele that impairs but does not completely abolish ATG5 activity .\",\n \"Although the overall structure of the E122D mutant ATG5 superimposes well with the wild-type protein , the mutation causes a striking decrease in the level of ATG12–ATG5 conjugate that is formed when the C terminus of ATG12 is covalently linked to Lys130 of ATG5 .\",\n \"We speculate that the E122D mutation causes subtle changes in the conformational dynamics that propagate to Lys130 , which is less than 10 Å away , resulting in less ATG12–ATG5 , which in turn leads to reduced LC3/Atg8 conjugation .\",\n \"In neurons , which are among the cells most dependent on autophagy for tissue homeostasis ( Button et al . , 2015 ) , the residual function of the E122D allele is inadequate , resulting in predominantly neurological symptoms in the two patients .\",\n \"Since homozygous mutations with complete loss-of-function have not been reported , we predict that individuals carrying such mutations , similar to Atg5-null mice ( Kuma et al . , 2004 ) , might not be viable .\",\n \"Autophagy is quickly gaining importance for its roles in preventing neurodegeneration .\",\n \"WDR45 is a redundant , non-core autophagy gene , one of four mammalian homologs to Atg18 , and mutations in WDR45 cause SENDA , static encephalopathy of childhood with neurodegeneration in adulthood ( Haack et al . , 2012; Saitsu et al . , 2013 ) .\",\n \"Autophagy appears to be critical in ataxia , whether mutant proteins evade autophagy processes or normal autophagy is disrupted .\",\n \"Several ataxias are attributed to intranuclear or cytoplasmic aggregation of mutant proteins within the cell ( Matilla-Duenas et al . , 2014 ) .\",\n \"These protein aggregates , in humans and in mouse models , not only evade autophagic sequestration but may even inhibit autophagy ( Alves et al . , 2014 ) , or lead to reduction in autophagy available for other proteins due to saturation .\",\n \"Assessment of autophagy in patient cells may be used to refine and identify the genetic cause of a patient’s ataxia .\",\n \"Further discovery of the role of autophagy in neurodegenerative diseases should be used to investigate therapies targeted at the autophagy process .\",\n \"Many drugs enhance autophagy and their effects on a multitude of neurodegenerative diseases have been studied ( Sarkar et al . , 2009 ) .\",\n \"Recently more studies have been conducted assessing the value of autophagy enhancers in ataxia models and patients .\",\n \"Induction of autophagy through administration of Temsirolimus , a rapamycin ester , and lentiviral overexpression of BECN1 in SCA3 model mice increase autophagy and the clearance of mutant protein aggregates , and reduce the ataxic phenotype ( Menzies et al . , 2010; Nascimento-Ferreira et al . , 2013 ) .\",\n \"In a single patient trial , trehalose treatment of patient fibroblasts increased autophagy and alleviated cellular pathogenic features by improving mitochondrial morphology and reducing free radicals in the cell ( Casarejos et al . , 2014; Sarkar et al . , 2007 ) .\",\n \"Trehalose also showed success in trials involving models of SCA17 ( Chen et al . , 2015 ) .\",\n \"Lithium , another inducer of autophagy , improved symptoms in a SCA1 mouse model ( Watase et al . , 2007 ) , but did not slow or reduce symptoms in a treatment trial in SCA2 patients ( Sacca et al . , 2015 ) .\",\n \"An autophagy enhancer may be an appropriate treatment to test in the presented subjects , as autophagic flux is attenuated , but not completely abrogated , by the ATG5E122D/E122D mutation .\",\n \"This study’s finding of the pathogenic human E122D mutation in ATG5 , a gene encoding part of the autophagy-controlling core machinery , is important and novel , but consistent with reports of neurodegenerative disorders in other autophagy-related genes ( Frake et al . , 2015 ) .\",\n \"Our results suggest that other mutations in this and other ATG genes , which impair but do not completely abolish autophagy , may result in similar forms of ataxia , intellectual disability and developmental delay .\",\n \"This study exemplifies the utility of exome sequencing in the identification of rare disease-causing variants , and supports the role of impaired autophagy in neurodegenerative disease .\",\n \"In addition , we demonstrate the utility of a combined genetic , biochemical and cell biological analysis in multiple model systems to elucidate the underlying pathogenic mechanism of rare human diseases .\"\n ],\n [\n \"Study protocols including written informed consents have been approved by the University of Michigan Institutional Review Board and the Boğaziçi University Institutional Review Board for Research with Human Participants .\",\n \"Two Turkish brothers , ages 5 and 7 in 2004 , presented with ataxia and developmental delay , as previously described ( Yapici and Eraksoy , 2005 ) .\",\n \"Parents were initially reported to be unrelated , but recently suggested they might be remotely related .\",\n \"Both patients were delayed in walking , had truncal ataxia and dysmetria , nystagmus , and lower IQ ( 68 and 70 ) .\",\n \"MRI revealed cerebellar hypoplasia .\",\n \"Follow-up examinations showed no progression of symptoms .\",\n \"DNA was isolated from peripheral whole blood using the Qiagen ( Germantown , MD ) Gentra Puregene isolation kit .\",\n \"Linkage analysis was performed using the genotype data generated with Illumina HumanOmniExpress-24 chip for the mother and the four sibs .\",\n \"The Allegro module ( Gudbjartsson et al . , 2000 ) of easyLINKAGE software was used , assuming autosomal recessive inheritance and parents as third cousins .\",\n \"No deletion or duplications common to just the two affected brothers were detected using cnvPartition plug-in in Illumina Genome Studio v . 1 . 02 software .\",\n \"Exome sequencing was performed independently twice on one subject .\",\n \"Capture for whole exome sequencing was performed with NimbleGen SeqCap EZ Exome Library v1 . 0 kit ( Roche , Indianapolis , IN ) .\",\n \"Captured regions were sequenced with Illumina HiSeq2000 instruments .\",\n \"Variants were filtered to remove common variants based on 1000 Genomes , Exome Sequencing Project , and Exome Aggregation Consortium databases , variants outside of identified linkage regions , variants not expected to change protein coding , and variants not following a recessive model of inheritance ( Exome Aggregation Consortium ( ExAC ) , 2015; Genomes Project Consortium et al . , 2012; NHLBI Go Exome Sequencing Project , 2015 ) .\",\n \"PCR followed by Sanger sequencing was performed to validate the variant identified through exome sequencing and test for segregation within the family .\",\n \"The variant of interest was further examined in two separate collections of a total of 500 Turkish samples , and found absent .\",\n \"Lymphoblastoid cell lines ( LCL ) of both subjects were generated from heparinized whole blood samples and cultured as described ( Doyle , 1990 ) .\",\n \"As they are made in house and cultured briefly , mycoplasma contamination risk is minimized .\",\n \"We used a baculovirus/insect cell expression system to examine formation of the human ATG12–ATG5 conjugate in a heterologous system described previously ( Qiu et al . , 2013 ) .\",\n \"Hi5 insect cells ( Invitrogen , Carlsbad , CA ) were infected with baculoviruses expressing human ATG7 , ATG10 , a GST-tagged version of ATG12 ( residues 53–140 , corresponding to the ubiquitin-like domain ) and a His-tagged version of either ATG5WT or ATG5E122D .\",\n \"Three days post infection , lysates were subjected to glutathione affinity chromatography , and the GST-ATG12–His-ATG5 conjugate was detected by SDS-PAGE followed by Coomassie Blue staining .\",\n \"To confirm that the baculoviruses produce protein , Hi5 cells were coinfected with baculoviruses expressing the His-tagged WT and mutant versions of ATG5 and the N-terminal domain of ATG16L1 ( residues 1–69 , as an MBP fusion ) .\",\n \"At three days post infection , lysates were subjected to nickel affinity purification .\",\n \"The ATG5-ATG16L1 complex formation was detected by SDS-PAGE and Coomassie Blue staining .\",\n \"The complex containing ATG5E122D and the N-terminal domain of ATG16L1 ( residues 1–69 ) was expressed in Hi5 insect cells , and purified by nickel affinity , ion exchange , and size exclusion chromatography into a final buffer of 20 mM Tris , pH 8 . 5 , 50 mM NaCl , 10 mM DTT .\",\n \"The complex was concentrated to 18 . 5 mg/ml , aliquoted , flash-frozen and stored at -80°C until further use .\",\n \"Crystals were grown by the hanging drop vapor diffusion method by mixing purified protein 1:1 with reservoir solutions of 37 . 5 mM MES , pH 5 . 2–5 . 8 , 0 . 2 M sodium tartrate , and 11–13% polyethylene glycol 3350 .\",\n \"Final crystals were obtained by micro-seeding with reservoir solution of 40 mM MES , pH 5 . 5 , 0 . 2 M sodium tartrate , 8 . 5% PEG3350 , 10 mM DTT .\",\n \"Crystals were cryoprotected in reservoir solution supplemented with 25% xylitol , and flash frozen in liquid nitrogen prior to data collection .\",\n \"Diffraction data were processed with XDS .\",\n \"The structure was determined by molecular replacement using Phaser ( McCoy et al . , 2007 ) with the structure of the WT ATG5-ATG16L1 ( 1–69 ) ( PDB: 4TQ0 ) complex as a search model ( Kim et al . , 2015a ) .\",\n \"Model construction and rebuilding were performed using Coot ( Emsley et al . , 2010 ) .\",\n \"The structure was refined using Phenix ( Adams et al . , 2010 ) .\",\n \"Diffraction data and refinement statistics are provided in Table 1 .\",\n \"Cells or tissues were lysed in cell lysis buffer ( 20 mM Tris-HCl , pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 2 . 5 mM sodium pyrophosphate , 1 mM beta-glycerophosphate , 1 mM Na3VO4 , 1% Triton X-100 ) or RIPA buffer ( 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 1% sodium deoxycholate , 1% NP-40 , 0 . 1% SDS ) containing protease inhibitor cocktail ( Roche ) .\",\n \"After being clarified with centrifugation , lysates were boiled in SDS sample buffer , separated by SDS-PAGE , transferred to polyvinylidene difluoride membranes and probed with the indicated antibodies .\",\n \"ATG5 ( 12994 ) , LC3 ( 3868 ) and SQSTM1/p62 ( 5114 ) antibodies were purchased from Cell Signaling Technology .\",\n \"Hemagglutinin ( HA , 3F10 ) antibody was from Roche .\",\n \"Actin ( JLA20 ) and tubulin ( T5168 ) antibodies were from Developmental Studies Hybridoma Bank and Sigma , respectively .\",\n \"Ref ( 2 ) P antibody was previously described ( Pircs et al . , 2012 ) .\",\n \"Wild-type human ATG5-coding sequence was from Addgene #24922 ( deposited by Dr . Toren Finkel ) ( Lee et al . , 2008 ) .\",\n \"The E122D mutation was introduced into ATG5 by PCR-based site-directed mutagenesis .\",\n \"ATG5WT and ATG5E122D were cloned into the plasmid pLU-CMV-Flag .\",\n \"The HA-ATG12-expressing plasmid was from Addgene #22950 ( deposited by Dr . Noboru Mizushima ) ( Mizushima et al . , 1998b ) .\",\n \"HEK293 cells ( the 293 A substrain from Invitrogen , tested negative for mycoplasma by PCR ) were cultured in Dulbecco’s modified Eagle’s medium ( DMEM , Invitrogen ) containing 10% fetal bovine serum ( FBS ) and penicillin/streptomycin at 37°C in 5% CO2 .\",\n \"For transient expression of proteins , HEK293 cells were transfected with purified plasmid constructs and polyethylenimine ( PEI , Sigma ) as previously described ( Horbinski et al . , 2001 ) .\",\n \"Cells were harvested 24 hr after transfection for immunoblot analyses .\",\n \"Saccharomyces cerevisiae strain WLY176 was used to generate an ATG5 knockout strain ( atg5∆ ) as previously described ( Gueldener et al . , 2002 ) .\",\n \"The MKO strain YCY137 ( SEY6210 atg1∆ , 2∆ , 4∆ , 5∆ , 6∆ , 7∆ , 8∆ , 9∆ , 10∆ , 11∆ , 12∆ , 13∆ , 14∆ , 16∆ , 17∆ , 18∆ , 19∆ , 20∆ , 21∆ , 23∆ , 24∆ , 27∆ , 29∆ ) ( Cao et al . , 2008 ) , was used for in vivo reconstitution of Atg8 conjugation .\",\n \"Site-directed mutagenesis was performed to generate ATG5 amplicons with the E141D mutation as previously described ( Liu and Naismith , 2008 ) .\",\n \"A pRS406 empty plasmid was digested with Spel and SalI , and then ligated with a DNA fragment encoding either wild-type or mutant Atg5-PA .\",\n \"atg5∆ was transformed with an empty pRS406 vector , or plasmids encoding Atg5-PA WT or Atg5-PA E141D .\",\n \"Wild-type WLY176 colonies were transformed with empty pRS406 vector as a control .\",\n \"Colonies were grown on SMD-URA medium and starved in nitrogen-deficient medium .\",\n \"Pho8Δ60 and western blot analyses were performed as described previously ( Noda and Klionsky , 2008; Shintani and Klionsky , 2004 ) .\",\n \"Quantification was performed using ImageJ software .\",\n \"The pATG8∆R-ATG7-ATG10 ( 414 ) , pATG5 ( WT ) -HA-ATG12 ( 416 ) and pATG5 ( WT ) -HA-ATG12-ATG16 ( 416 ) plasmids were described previously ( Cao et al . , 2008 ) .\",\n \"The pATG5 ( E141D ) -HA-ATG12 ( 416 ) and pATG5 ( E141D ) -HA-ATG12-ATG16 ( 416 ) plasmids were made by site-directed mutagenesis based on the wild-type constructs .\",\n \"Atg5-null Drosophila flies ( Atg55cc5 ) were generated by CRISPR-Cas9-mediated genome editing , using a double gRNA approach , both targeting the same gene , as described ( Kondo and Ueda , 2013 ) .\",\n \"The Atg55cc5 mutant was recovered by screening viable candidate lines for accumulation of the specific autophagy cargo Ref ( 2 ) P using western blots , followed by PCR and sequencing .\",\n \"Atg55cc5 mutants have a deletion in X:7 , 322 , 242–7 , 323 , 717 residues ( Drosophila melanogaster R6 . 06 ) , which deletes five out of six exons of the Atg5 gene , eliminating more than 85% of protein-coding sequences including the translation start site ( Figure 6A ) .\",\n \"The PhiC31 integrase-mediated site-specific transformation method was used to express human ATG5WT and ATG5E122D from an identical genomic locus ( Bateman et al . , 2006; Bischof et al . , 2007; Venken et al . , 2006 ) .\",\n \"In brief , flag-tagged ATG5WT and ATG5E122D were cloned into a pUAST-attB vector ( Bischof et al . , 2007 ) and fully sequenced .\",\n \"pUAST-attB-ATG5WT and pUAST-attB-ATG5E122D were microinjected into y1 M{vas-int . Dm}ZH-2A w*; M{3xP3-RFP . attP}ZH-51D flies and stable transformants were isolated by the presence of the mini-white+ marker ( Figure 6B ) .\",\n \"The UAS-ATG5WT or UAS-ATG5E122D transgenes were crossed with a double balancer strain ( Bl/CyO; TM2/TM6B ) and then with +/CyO; Tub-Gal4/TM2 to be constructed as stable Tub>ATG5 lines ( UAS-ATG5/UAS-ATG5; Tub-Gal4/TM6B ) .\",\n \"The Tub>ATG5 male flies were crossed with Atg55cc5/FM7 female flies to generate Atg5-null flies expressing human ATG5 transgenes .\",\n \"Climbing assays and TUNEL staining were performed as previously described ( Kim et al . , 2015b ) .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health .","Defects in the autophagy pathway have been observed in neurodegenerative disorders; however , no genetically-inherited pathogenic mutations in any of the core autophagy-related ( ATG ) genes have been reported in human patients to date .","We identified a homozygous missense mutation , changing a conserved amino acid , in ATG5 in two siblings with congenital ataxia , mental retardation , and developmental delay .","The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5 .","The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy .","Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder , in contrast to flies expressing the wild-type human protein .","Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health ."],"string":"[\n \"Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health .\",\n \"Defects in the autophagy pathway have been observed in neurodegenerative disorders; however , no genetically-inherited pathogenic mutations in any of the core autophagy-related ( ATG ) genes have been reported in human patients to date .\",\n \"We identified a homozygous missense mutation , changing a conserved amino acid , in ATG5 in two siblings with congenital ataxia , mental retardation , and developmental delay .\",\n \"The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5 .\",\n \"The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy .\",\n \"Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder , in contrast to flies expressing the wild-type human protein .\",\n \"Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Ataxia is a rare disease that affects balance and co-ordination , leading to difficulties in walking and other movements .","The disease mostly affects adults , but some children are born with it and they often have additional cognitive and developmental problems .","Mutations in at least 60 genes are known to be able to cause ataxia , but it is thought that there are still more to be found .","Kim , Sandford et al . studied two siblings with the childhood form of ataxia and found that they both had a mutation in a gene called ATG5 .","The protein produced by the mutant ATG5 gene was less able to interact with another protein called ATG12 .","Furthermore , the cells of both children had defects in a process called autophagy – which destroys old and faulty proteins to prevent them accumulating and causing damage to the cell .","Next , Kim , Sandford et al . examined the effect of this mutation in baker’s yeast cells .","Cells with a mutation in the yeast equivalent of human ATG5 had lower levels of autophagy than normal cells .","Further experiments used fruit flies that lacked fly Atg5 , which were unable to fly or walk properly .","Inserting the normal form of human ATG5 into the flies restored normal movement , but the mutant form of the gene had less of an effect .","These findings suggest that a mutation in ATG5 can be responsible for the symptoms of childhood ataxia .","Kim , Sandford et al . think that other people with severe ataxia may have mutations in genes involved in autophagy .","Therefore , the next step is to study autophagy in cells from many other ataxia patients ."],"string":"[\n \"Ataxia is a rare disease that affects balance and co-ordination , leading to difficulties in walking and other movements .\",\n \"The disease mostly affects adults , but some children are born with it and they often have additional cognitive and developmental problems .\",\n \"Mutations in at least 60 genes are known to be able to cause ataxia , but it is thought that there are still more to be found .\",\n \"Kim , Sandford et al . studied two siblings with the childhood form of ataxia and found that they both had a mutation in a gene called ATG5 .\",\n \"The protein produced by the mutant ATG5 gene was less able to interact with another protein called ATG12 .\",\n \"Furthermore , the cells of both children had defects in a process called autophagy – which destroys old and faulty proteins to prevent them accumulating and causing damage to the cell .\",\n \"Next , Kim , Sandford et al . examined the effect of this mutation in baker’s yeast cells .\",\n \"Cells with a mutation in the yeast equivalent of human ATG5 had lower levels of autophagy than normal cells .\",\n \"Further experiments used fruit flies that lacked fly Atg5 , which were unable to fly or walk properly .\",\n \"Inserting the normal form of human ATG5 into the flies restored normal movement , but the mutant form of the gene had less of an effect .\",\n \"These findings suggest that a mutation in ATG5 can be responsible for the symptoms of childhood ataxia .\",\n \"Kim , Sandford et al . think that other people with severe ataxia may have mutations in genes involved in autophagy .\",\n \"Therefore , the next step is to study autophagy in cells from many other ataxia patients .\"\n]","isConversational":false},"year":{"kind":"string","value":"2016"}}},{"rowIdx":295,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"The auditory representation of speech sounds in human motor cortex"},"id":{"kind":"string","value":"elife-12577-v2"},"sections":{"kind":"list like","value":[["Our motor and sensory cortices are traditionally thought to be functionally separate systems .","However , an accumulating number of studies has revealed their roles in action and perception to be highly integrated ( Pulvermüller and Fadiga , 2010 ) .","For example , a number of studies have demonstrated that both sensory and motor cortices are engaged during perception ( Gallese et al . , 1996; Wilson et al . , 2004; Tkach et al . , 2007; Cogan et al . , 2014 ) .","In humans , this phenomenon has been observed in the context of speech , where listening to speech sounds evokes robust neural activity in the motor cortex ( Wilson et al . , 2004; Pulvermüller et al . , 2006; Edwards et al . , 2010; Cogan et al . , 2014 ) .","This observation has re-ignited an intense scientific debate over the role of the motor system in speech perception over the past decade ( Lotto et al . , 2009; Scott et al . , 2009; Pulvermüller and Fadiga , 2010 ) .","One interpretation of the observed motor activity during speech perception is that “the objects of speech perception are the intended phonetic gestures of the speaker”- as posited by Liberman’s motor theory of speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) .","The motor theory is a venerable and well-differentiated exemplar of a set of speech perception theories that we could call 'production-referencing' theories .","Unlike motor theory , more modern production referencing theories do not assume that sensorimotor circuits are necessarily referenced in order for speech to be recognized , but they allow for motor involvement in perception in certain phonetic modes .","For example , Lindblom , 1996 suggested that a direct link between spectrotemporal analysis and word recognition is the normal mode of speech perception ( the 'what' mode of perception ) , but in some cases listeners do use a route through sensorimotor circuits ( the 'how' mode of perception ) if , for example , the listener is attempting to imitate a new sound ( Lindblom , 1996 ) .","While demonstrations of evoked motor cortex activity by speech sounds strengthen production-referencing theories , it remains unclear what information is actually represented by such activity .","Determining what phonetic properties are encoded in the motor cortex has significant implications for elucidating the role it may play in speech perception .","To address this , we recorded direct neural activity from the peri-Sylvian speech cortex in nine human participants undergoing clinical monitoring for epilepsy surgery .","This includes but is not limited to two relevant areas comprising the supra-Sylvian ventral half of the lateral sensorimotor cortex ( vSMC ) for the motor control of articulation ( Penfield and Boldrey , 1937 ) and the infra-Sylvian superior temporal gyrus ( STG ) for the auditory processing of speech sounds ( Ojemann et al . , 1989; Boatman et al . , 1995 ) .","Since cortical processing of speech sounds is spatially discrete and temporally fast ( Formisano et al . , 2008; Chang et al . , 2011; Steinschneider et al . , 2011 ) , we used customized high-density electrode grids ( a four-fold increase over conventional recordings ) ( Bouchard et al . , 2013; Mesgarani et al . , 2014 ) .","Importantly , these recordings have simultaneous high spatial and temporal resolution in order to study the detailed speech representations in the vSMC ( Crone et al . , 1998; Edwards et al . , 2009 ) .","With this approach , we seek to address unanswered questions about the representation of speech sounds in motor cortex , including how the spatiotemporal patterns compare when speaking and listening and whether auditory representations in motor cortex are organized along articulatory or acoustic dimensions ."],["Participants first listened passively to consonant-vowel ( CV ) syllables ( 8 consonants followed by the /a/ vowel ) .","In a separate trial block , they spoke aloud these same CV syllables .","We measured the average evoked cortical activity during these listening and speaking CV tasks .","We focused our analysis on high gamma ( 70–150 Hz ) cortical surface local field potentials , which strongly correlate with extracellular multi-unit neuronal spiking ( Steinschneider et al . , 2008; Ray and Maunsell , 2011 ) .","We aligned neural responses to the onset of speech acoustics ( t = 0 ) in listening and speaking tasks to provide a common reference point across speech sounds .","We first determined which peri-Sylvian cortical areas were activated during passive listening to speech sounds .","Figure 1a and b shows the locations of cortical areas that demonstrated cortical evoked responses in a single representative subject during listening and speaking respectively .","During listening , evoked responses spanned middle and posterior STG as expected , with weaker responses in middle temporal gyrus ( MTG ) ( Figure 1a ) .","In the vSMC , ( composed of the pre- and post- central gyri ) we found electrodes in the superior-most and inferior-most aspects ( Figure 1a , Figure 1—figure supplement 1 , 2 ) that demonstrated reliable and robust single-trial responses to speech sounds during passive listening ( Figure 1b ) .","Neural responses were also found at a few sites scattered across supramarginal , inferior- , and middle- frontal gyri—though these were not consistent across subjects ( Figure 1—figure supplement 1 ) .","By performing spatial clustering analysis on the electrode positions in each subject , we found that 3/5 subjects showed significant clustering of regions responsive to auditory stimuli ( Hartigan’s Dip statistic , p<0 . 05 ( see Materials and methods ) ; Figure 1—figure supplement 1 ) .","Out of these 3 subjects , k-means clustering revealed two subjects with k=2 electrode clusters ( subjects 1 and 4 , clusters in inferior and superior vSMC ) , and one subject with k=5 clusters .","When participants spoke the same CV syllables , in contrast , articulatory movement-related cortical activity was well distributed throughout vSMC ( Figure 1c ) , with auditory feedback cortical activity seen in the STG . 10 . 7554/eLife . 12577 . 003Figure 1 . Speech sounds evoke responses in the human motor cortex .","( a ) Magnetic resonance image surface reconstruction of one representative subject’s cerebrum ( subject 1: S1 ) .","Individual electrodes are plotted as dots , and the average cortical response magnitude ( z-scored high gamma activity ) when listening to CV syllables is signified by the color opacity .","CS denotes the central sulcus; SF denotes the Sylvian fissure .","( b ) Acoustic waveform , spectrogram , single-trial cortical activity ( raster ) , and mean cortical activity ( high gamma z-score , with standard error ) from two vSMC sites and one STG site when a subject is listening to /da/ .","Time points significantly above a pre-stimulus silence period ( p<0 . 01 , bootstrap resampled , FDR corrected , alpha < 0 . 005 ) are marked along the horizontal axis .","The vertical dashed line indicates the onset of the syllable acoustics ( t=0 ) .","( c ) Same subject as in","( a ) ; distributed vSMC cortical activity when speaking CV syllables ( mean high gamma z-score ) .","( d ) Total number of significantly active sites in all subjects during listening , speaking , and both conditions ( p<0 . 01 , t-test , responses compared to silence and speech ) .","Electrode sites are broken down by their anatomical locations .","S denotes superior vSMC sites; I denotes inferior vSMC sites . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00310 . 7554/eLife . 12577 . 004Figure 1—figure supplement 1 . Average cortical responses to speaking and listening in all subjects ( S2-S5 ) .","( a ) Average Z-scored responses in active electrodes when listening to ( left ) or speaking ( right ) CV syllables in all subjects , as shown in Figure 1a and c .","( b ) Results from spatial clustering of significantly active electrodes in each subject .","S1 , S4 , and S5 showed spatially segregated clusters ( see Materials and methods ) and were included in k-means clustering analysis .","The silhouette index shows that the best number of clusters for S1 ( from Figure 1 ) and S4 was k=2 , whereas the best number of clusters for S5 was k=5 . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00410 . 7554/eLife . 12577 . 005Figure 1—figure supplement 2 . Neural responses while listening to CV syllables in 4 additional subjects not included in MDS analyses ( S6 - S9 ) .","Responses are plotted on each subject’s brain surface and are shown for each electrode as the average Z-scored high gamma activity across all syllables , aligned to the acoustic onset of the stimulus ( indicated by dashed line at t = 0 ) .","As in Figure 1 and Figure 1—figure supplement 1 , strong responses are seen during listening in inferior and superior regions of the vSMC ( primarily precentral gyrus ) in addition to the superior temporal gyrus ( STG ) .","For each subject , SF indicates the location of the Sylvian fissure , and CS indicates the central sulcus . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 005 Across all participants , we identified 115 electrodes that demonstrated significant neural activity in vSMC during listening ( p<0 . 01 , t-test , compared to pre-stimulus silent rest period; Figure 1d ) .","When speaking , in contrast , a total of 362 electrodes in vSMC were found to be significantly active ( Figure 1d , p<0 . 01 , t-test , compared to pre-stimulus silent rest period ) .","We compared the relative proportions of electrodes that were found in different supra-Sylvian anatomical regions .","Critically , only a subset of sites in vSMC ( 98 out of 362 , ~27% ) was active during both listening and speaking ( Figure 1d ) .","These sites were primarily localized to the pre-central gyrus , whereas speaking evoked activity across both pre- and post-central gyri sites .","Neural responses in the vSMC during listening were found in the superior ( S in Figure 1d ) pre-central gyrus and inferior , anterior aspect of the sub-central gyrus of the vSMC ( I in Figure 1d ) .","We next compared the patterns of cortical activity to specific speech sounds during listening and speaking .","During speaking , specific articulator representations have been identified in the somatotopically-organized vSMC ( Bouchard et al . , 2013 ) .","For example , the plosive consonants /b/ , /d/ , and /g/ are produced by the closure of the vocal tract at the lips , front tongue , and back tongue , respectively ( Figure 2a , b , see Figure 2—figure supplement 1 for all syllable tokens ) ( Ladefoged and Johnson , 2010 ) .","The cortical representations for these articulators are laid out along a superior-to-inferior ( medial-to-lateral ) sequence in the vSMC ( Penfield and Boldrey , 1937 ) .","We first examined average cortical activity at single electrode sites distributed along the vSMC axis for articulating individual speech sounds .","Figure 2c shows single electrode activity from a single representative subject ( the same from Figure 1 ) for speaking ( blue lines ) and listening ( red lines ) for three CV syllables , which have different place of articulation ( /ba/ , /da/ , and /ga/ ) .","The exact location of these electrodes on the vSMC is shown in Figure 2d .","The production of labial consonants ( /b/ ) is associated with activity in lip cortical representations as evidenced by strong responses to the bilabial /ba/ ( Figure 2c , electrodes 5–6 , blue lines ) .","These are located superior to the tongue representations associated with the /d/ and /g/ consonants , as shown previously ( Bouchard et al . , 2013 ) .","Those tongue sites were sub-specified by ‘coronal’ ( i . e . anterior-based ) tongue position for /d/ ( electrodes 8–10 , blue lines ) superiorly , and ‘dorsal’ ( i . e . posterior-based ) tongue position for /g/ inferiorly ( electrode 13 , blue line ) .","Other sites ( electrodes 1–4 , 11–12 , blue lines ) showed the same neural activity across all three syllables . 10 . 7554/eLife . 12577 . 006Figure 2 . Site-by-site differences in vSMC neural activity when speaking and listening to CV syllables .","( a ) Top , vocal tract schematics for three syllables ( /ba/ , /da/ , /ga/ ) produced by occlusion at the lips , tongue tip , and tongue body , respectively ( arrow ) .","( b ) Acoustic waveforms and spectrograms of spoken syllables .","( c ) Average neural activity at electrodes along the vSMC for speaking ( blue ) and listening ( red ) to the three syllables ( high gamma z-score ) .","Solid lines indicate activity was significantly different from pre-stimulus silence activity ( p<0 . 01 ) .","Transparent lines indicate activity was not different from pre-stimulus silence activity ( p>0 . 01 ) .","Vertical dashed line denotes the onset of the syllable acoustics ( t=0 ) .","( d ) Location of electrodes 1–13 in panel c , shown on whole brain and with inset detail .","CS = central sulcus , SF = Sylvian fissure . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00610 . 7554/eLife . 12577 . 007Figure 2—figure supplement 1 . Syllable token set .","( a ) Consonants of all syllable tokens .","Consonants were paired with the vowel /a/ , and are organized by place and manner of articulation .","( b ) Vocal tract schematics for three occlusions made with the lips , tongue tip , and tongue body , respectively ( arrow ) .","( c ) Acoustic waveform and spectrogram of the syllable tokens . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 007 We next examined those same vSMC electrodes during listening , and found that the majority of those cortical vSMC electrodes were not active ( p>0 . 01 , t-test compared to silence , Figure 2c transparent red lines ) .","The few that were active ( electrodes 1 , 2 , 4 , 11–12 , solid red lines ) were similar for all three CV syllables , with activity increasing approximately 100ms after the acoustic onset .","Across the entire population of vSMC electrodes that were active during listening , onset latencies were generally shorter than those in STG sites , with significant increases in both inferior vSMC ( p<0 . 001 ) and superior vSMC ( p<0 . 05 ) compared to STG ( Figure 3a , Wilcoxon rank sum test , see Figure 3c for average responses to all syllables ) .","The latency to the response peak was also significantly higher in superior vSMC compared to STG ( Figure 3b , p<0 . 01 , Wilcoxon rank sum test ) .","A cross-correlation analysis between these vSMC electrodes and STG electrodes revealed a diverse array of relationships between these populations ( Figure 3d–f ) , including STG electrode activity leading vSMC electrode activity and vice versa .","In contrast to speaking , we did not observe somatotopic organization of cortical responses when listening to speech .","Therefore , the pattern of raw evoked responses during listening shows critical differences from those during speaking . 10 . 7554/eLife . 12577 . 008Figure 3 . Dynamics of responses during CV listening in STG , inferior vSMC , and superior vSMC .","( a ) STG onset latencies were significantly lower than both inferior vSMC ( p<0 . 001 , Z = −4 . 03 ) and superior vSMC ( p<0 . 05 , Z = −2 . 28 ) .","( b ) STG peak latencies were significantly lower than superior vSMC ( p<0 . 01 , Z = −2 . 93 ) , but not significantly different from peak latencies in inferior vSMC ( p>0 . 1 ) .","In","( a ) and","( b ) , red bar indicates the median , boxes indicate 25th and 75th percentile , and error bars indicate the range .","Response latencies were pooled across all subjects .","All p-values in","( a ) and","( b ) are from the Wilcoxon rank sum test .","( c ) Average evoked responses to all syllable tokens across sites in superior vSMC ( n=32 ) , inferior vSMC ( n=37 ) , and STG .","Responses were aligned to the syllable acoustic onset ( t=0 ) .","A random subset of STG responses ( n=52 out of the 273 that were used in the latency analysis in","( a ) and","( b ) ) are shown here for ease of viewing .","( d ) Example cross-correlations between three vSMC electrodes and all STG electrodes in one patient , for a maximum lag of ± 0 . 75 s .","More power in the negative lags indicates a faster response in the STG compared to the vSMC electrode , and more power in the positive lags indicates a faster response in vSMC compared to STG .","We observe vSMC electrodes that tend to respond later than STG ( e248 , left panel ) , vSMC electrodes that tend to respond before STG ( e136 , middle panel ) , and vSMC electrodes that respond at similar times to some STG electrodes ( e169 , right panel ) .","( e ) Average evoked responses during CV listening for all STG electrodes from this patient and the three vSMC electrodes shown in panel","( d ) .","Responses were aligned to the syllable acoustic onset ( t=0 ) , as in panel","( c ) .","( f ) Percentage of sites with STG leading , coactive , or vSMC leading as expressed by the asymmetry index ( see Materials and methods ) .","Both inferior and superior vSMC show leading and lagging responses compared to STG , as well as populations of coactive pairs . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 008 We next evaluated quantitatively whether the structure of distributed vSMC neural activity during listening was more similar to that of vSMC during speaking or the STG during listening .","In previous studies , we demonstrated that the structure of evoked responses are primarily organized by different feature sensitivities: place of articulation in the vSMC ( Bouchard et al . , 2013 ) , and manner of articulation in the STG ( Mesgarani et al . , 2014 ) .","We visualized the similarity of population activity evoked by different consonants using unsupervised multidimensional scaling ( MDS ) , where the 2-dimensional Euclidean distances between stimuli correspond to the similarity of their neural responses .","Visual inspection of MDS plots shows that , during speaking , evoked activity in vSMC clustered into place of articulation features ( Figure 4a ) : labials ( /b/ , /p/ ) , alveolars ( /s/ , /sh/ , /t/ , /d/ ) , and velars ( /g/ , /k/ ) ( Figure 4b ) .","In contrast , neural responses during listening did not cluster into the same features ( Figure 4c ) .","To quantify the degree to which the evoked activity clustered into place of articulation features , we used unsupervised K-means clustering to assign the neural responses to clusters ( k=3 ) , and the adjusted Rand Index ( RIadj ) ( Rand , 1971; Hubert and Arabie , 1985 ) to measure the degree to which the neural clustering agreed with linguistically defined place of articulation consonant clusters .","The RIadj quantifies the degree of agreement between two clustering patterns , where RIadj = 1 denotes identical clustering patterns and RIadj = 0 denotes independent clustering patterns .","We found that while evoked activity during speaking clustered by place of articulation features , activity during listening did not ( Figure 4d; see Figure 4—figure supplement 1 for moving time window analysis ) .","Even when the vSMC electrode subset was restricted to short-latency vSMC electrodes leading STG activity ( as evidenced by a positive asymmetry index in Figure 3f ) , activity during listening did not cluster according to place of articulation features ( Figure 4—figure supplement 2 ) .","Thus , responses in motor areas during speech perception do not show a spatially distributed representation of speech motor articulator features . 10 . 7554/eLife . 12577 . 009Figure 4 . Organization of motor cortex activity patterns .","( a ) Consonants of all syllable tokens organized by place and manner of articulation .","Where consonants appear in pairs , the right is a voiced consonant , and the left is a voiceless consonant .","( b ) Relational organization of vSMC patterns ( similarity ) using multidimensional scaling ( MDS ) during speaking .","Neural pattern similarity is proportional to the Euclidean distance ( that is , similar response patterns are grouped closely together , whereas dissimilar patterns are positioned far apart ) .","Tokens are colored by the main place of articulation of the consonants ( labial , velar , or alveolar ) .","( c ) Similarity of vSMC response patterns during listening .","Same coloring by place of articulation .","( d ) Organization by motor articulators .","K-means clustering was used to assign mean neural responses to 3 groups ( labial , alveolar , velar ) for both listening and speaking neural organizations ( b , c ) .","The similarity of the grouping to known major articulators was measured by the adjusted Rand Index .","An index of 1 indicates neural responses group by place of articulation features .","***p<0 . 001 , Wilcoxon rank-sum","( e ) Organization of mean STG responses using MDS when listening .","In contrast to c and d , tokens are now colored by their main acoustic feature ( fricative , voiced plosive , or voiceless plosive ) .","( f ) Organization of mean vSMC responses using MDS when listening colored by their main acoustic feature .","( Identical to C , but recolored here by acoustic features ) .","( g ) Organization by manner of articulation acoustic features ( fricative , voiced plosive , voiceless plosive ) for both STG and vSMC organizations when listening ( e ,","f ) .","The similarity of the grouping to known acoustic feature groupings was measured by the adjusted Rand Index .","***p<0 . 001 , Wilcoxon rank sum .","( h ) During listening , responses in vSMC show significantly greater organization by acoustic manner features compared to place features as assessed by the adjusted Rand Index , indicating an acoustic rather than articulatory representation ( ***p<0 . 001 , Wilcoxon rank-sum ) .","Bars in this panel are the same as the red bars in","( d ) and","( g ) .","In","( d ) ,","( g ) , and","( h ) , bars indicate mean ± standard deviation , DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00910 . 7554/eLife . 12577 . 010Figure 4—figure supplement 1 . Clustering trajectory analysis of neural responses to syllables . The clustering trajectory across time was derived from vSMC activity ( n=98 electrodes ) when speaking ( blue ) and listening ( red ) using the ΔRIadj metric .","An index of 1 denotes the neural responses organized by acoustic features , and an index of -1 denotes an organization by place of articulation features .","Time points indicated by the blue and red windows are significantly organized according to place or acoustics , respectively ( FDR-corrected p<0 . 05 , permutation test comparing random clustering at each time point to clustering by acoustics or place ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01010 . 7554/eLife . 12577 . 011Figure 4—figure supplement 2 . Analysis of short latency responses in vSMC . We restricted the MDS analysis to vSMC responses with a positive asymmetry index ( see Figure 3d and f , n=50 electrodes ) and found significant organization by acoustic manner features during listening in these sites .","( a ) Similarity of responses to syllables during listening in the subset of vSMC electrodes leading activity in STG .","Tokens are colored according to place of articulation features ( left ) or acoustic manner features ( right ) as in Figure 4c and f .","( b ) During listening , responses of short latency sites in the vSMC still show significantly greater organization by acoustic manner features compared to place features as assessed by the adjusted Rand Index , indicating an acoustic rather than articulatory representation ( ***p<0 . 001 , Wilcoxon rank-sum ) .","Compare with Figure 4h .","Bars indicate mean ± standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01110 . 7554/eLife . 12577 . 012Figure 4—figure supplement 3 . Organization of syllable tokens and auditory cortical activity patterns .","( a ) Organization of the acoustic spectrograms of the CV tokens using MDS .","( b ) Organization of mean STG responses using MDS when speaking .","Tokens in","( a ) and","( b ) are colored by their main acoustic feature .","( c ) Organization of vSMC responses by place of articulation using all speech-responsive electrodes ( n=362 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 012 Finding no evidence that major articulator features are either locally or spatially distributed in the vSMC in response to speech sounds , we next compared vSMC responses to population responses in the STG .","STG has an acoustic sensory representation of speech that best discriminates speech sounds by manner of articulation features with salient acoustic differences ( Mesgarani et al . , 2014 ) .","Using multidimensional scaling , STG spatial patterns during listening showed clustering according to three high-order acoustic features ( Figure 4e ) : voiced plosives ( /b/ , /d/ , /g/ ) , unvoiced plosives ( /p/ , /t/ , /k/ ) , and fricatives ( /s/ , /sh/ ) ( Ladefoged and Johnson , 2010 ) .","This is consistent with the relational organization derived by analysis of structure in the stimulus acoustics ( Figure 4—figure supplement 3a ) , and the structure of STG during speaking ( Figure 4—figure supplement 3b ) .","With the same analyses , we observed that activity in motor cortex clustered into the same three acoustic features ( Figure 4f , note this panel is identical to Figure 4c simply re-colored ) .","Unsupervised K-means clustering analysis confirmed that vSMC activity , during listening , organized into these linguistically defined acoustic feature groups , but was significantly weaker than the organization of STG ( p<0 . 001 , Wilcoxon rank-sum , Figure 4g ) .","Importantly , however , clustering by acoustic manner features was significantly stronger than clustering by place features in vSMC electrodes during listening ( p<0 . 001 , Wilcoxon rank-sum , Figure 4h ) .","This organization suggests that motor cortex activity during speech perception reflects an acoustic sensory representation of speech in the vSMC that mirrors acoustic representations of speech in auditory cortex .","To further define the acoustic selectivity and tuning of vSMC motor electrodes , participants listened to natural , continuous speech samples from a corpus with a range of American English speakers ( Garofolo et al . , 1993 ) .","We fit spectrotemporal receptive field ( STRF ) models for each vSMC electrode using normalized reverse correlation ( see Materials and methods ) , which describes the spectrotemporal properties of speech acoustics that predict the activity of a single site in motor cortex .","To compute the STRF , we calculate the correlation between the neural response at an electrode and the stimulus spectrogram at multiple time lags .","The result is then normalized by the auto-correlation in the stimulus .","This results in a linear filter for each electrode ( the STRF ) , which , when convolved with the stimulus spectrogram , produces a predicted neural response to that stimulus .","The prediction performance of each STRF was determined by calculating the correlation between the activity predicted by the STRF and the actual response on held out data .","A fraction of vSMC sites ( 16/98 sites total ) were reasonably well-predicted with a linear STRF ( r>=0 . 10 and p<0 . 01 , permutation test ) ( Theunissen et al . , 2001 ) .","STRFs with significant correlation coefficients were localized to superior and inferior vSMC ( primarily precentral gyrus ) in addition to STG ( Figure 5a ) .","Still , the prediction performance of STRFs in vSMC was generally lower than that of the STG ( Figure 5b ) .","Furthermore , the majority of STRFs in both regions showed strong low frequency tuning ( 100–200 Hz ) properties related to voicing ( Figure 5c ) , though some also showed high frequency tuning consistent with selectivity for fricatives and stop consonants by visual inspection ( Mesgarani et al . , 2014 ) .","We also estimated the mean cortical response at each motor site to every phoneme in English and found a diverse set of responses ( Figure 5—figure supplement 1a ) that were notably weaker in magnitude compared to STG responses ( Figure 5—figure supplement 1b ) .","Weak selectivity to phonetic features measured by the Phoneme Selectivity Index ( PSI ) was also observed ( Figure 5—figure supplement 1c ) ( Mesgarani et al . , 2014 ) .","These findings reveal that individual sites in motor cortex reflect sensory responses to definable spectrotemporal features speech acoustics , including voicing attributes .","Presumably , this tuning gives rise to the acoustic organization found in the previous analysis of distributed spatial patterns of neural activity . 10 . 7554/eLife . 12577 . 013Figure 5 . Acoustic spectrotemporal tuning in vSMC .","( a ) All STRF correlations and locations are plotted with opacity signifying the strength of the correlation .","CS denotes the central sulcus; SF denotes the Sylvian fissure .","( b ) Distribution of STRF prediction correlations for significantly active vSMC and STG sites .","Cut-off at r = 0 . 1 is shown as a dashed line .","( c ) Individual STRFs from all subjects ( S1-S5 , STRF correlation>0 . 1 ) plotted as a function of distance from the central sulcus and Sylvian fissure , with opacity signifying the strength of the STRF correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01310 . 7554/eLife . 12577 . 014Figure 5—figure supplement 1 . Summary and comparison of vSMC and STG responses to syllables .","( a ) Average responses at sample electrodes to all English phonemes and their PSI vectors .","( b ) Peak high-gamma z-score distributions for significantly active vSMC and STG sites when listening ( p<0 . 01 , comparing silence to stimulus activity ) .","( c ) Average PSI distributions for significantly active vSMC and STG sites . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 014"],["Our principal objective was to determine the vSMC motor cortex representation of auditory speech sounds .","We used high-resolution cortical recordings and a wide array of speech sounds to determine how the vSMC structure of speech sounds compared to the structure of motor commands in vSMC and sensory processing in STG .","We found evidence for both spatially local and distributed activity correlated to speech acoustics , which suggests an auditory representation of speech in motor cortex .","The proposal that the motor cortex critically integrates observations with motor commands largely stems from the discovery of mirror neurons ( in area F5 of macaques ) that fire both when a monkey produced an action and observed a similar action ( di Pellegrino et al . , 1992; Rizzolatti and Craighero , 2004; Pulvermüller and Fadiga , 2010 ) .","This 'integrative' view is reminiscent of linguistic production-referencing theories , including the motor theory of speech perception , which propose that motor circuits are involved in speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) .","In line with these theories , human neuroimaging studies have showed mirror activity in ventral premotor cortex during listening ( Wilson et al . , 2004; Pulvermüller et al . , 2006; Edwards et al . , 2010 ) , and modulated premotor activity in phoneme categorization tasks ( Alho et al . , 2012; Chevillet et al . , 2013 ) .","Our results extend these findings by detailing the representational selectivity and encoding of vSMC in perception .","Consistent with previous findings , we demonstrated local ‘audiomotor’ responses to speech sounds in vSMC .","When the responses were further examined for phonetic structure , we found major motor articulatory place features , such as labial , alveolar , and velar , were not represented with single site activity or distributed spatial activity .","This observation is in direct contrast with structural predictions made by the original motor theory of speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) , while confirming that motor cortex plays a role in perception ( Lindblom , 1996; Hickok and Poeppel , 2007 ) .","We localized activity during speech perception to regions of the vSMC that have been implicated in phonation and laryngeal control ( Penfield and Boldrey , 1937; Brown et al . , 2008 ) .","When listening to speech , we observed these regions reflected acoustic sensory properties of speech , with individual sites tuned for spectrotemporal acoustic properties .","The tuning properties of responsive sites in vSMC are similar to properties observed in STG during listening ( Mesgarani et al . , 2014 ) and appear to give rise to an acoustic sensory organization of speech sounds ( rather than purely motor organization ) in motor cortex during listening .","There is an emerging consensus that frontal and motor regions are recruited during effortful listening ( Du et al . , 2014 ) .","For example , previous studies have demonstrated that frontal areas come online to process degraded speech for the attentional enhancement of auditory processing ( Wild et al . , 2012 ) .","Our results may complement this interpretation in that the audiomotor cortex enhancement is specific to an auditory representation , without transforming information to a motor articulatory representation .","That being said , the auditory encoding that we observed in the motor cortex did not appear to be as strong as that as that observed in the STG , and exhibited comparatively weaker activity and weaker phoneme selectivity ( Figure 5—figure supplement 1b and c , and see ( Mesgarani et al . , 2014 ) .","In addition to having implications for perceptual models , we speculate that these results have strong implications for speech production , as auditory feedback is potentially processed directly in the vSMC in addition to the canonical auditory cortex .","Speech production models currently propose a complex role for sensory feedback , where pathways exist for the activation of auditory cortex from vSMC activation ( the forward prediction of production consequences ) , and the activation of vSMC from auditory and somatosensory input ( the error correction signal ) ( Guenther et al . , 2006; Houde and Nagarajan , 2011 ) .","In the current study , it appears that the motor cortex contains both sensory and motor representations , where the sensory representations are active during passive listening , whereas motor representations dominate during speech production .","Analysis of the time course of vSMC and STG responses revealed a heterogeneous population of both short- and longer-latencies in the inferior and superior vSMC that are generally slower than the STG ( Figure 3a–c ) .","Early responses in vSMC may reflect bidirectional connections from STG ( Zatorre et al . , 2007 ) , primary auditory cortex ( Nelson et al . , 2013; Schneider et al . , 2014 ) or auditory thalamus ( Henschke et al . , 2014 ) , whereas later responses might reflect indirect connectivity in areas downstream from the STG ( Rauschecker and Scott , 2009 ) .","Indeed , our cross-correlation analysis revealed bidirectional dynamical relationships between vSMC and STG responses , in which STG responses led vSMC responses and vice versa ( Figure 3d–f ) .","Still , this analysis was independent of the diverse tuning properties in the vSMC and STG electrode sets , so longer latency responses likely reflect the later responses to vowels relative to consonants .","Even so , we found a wide variety of tuning and dynamical profiles in the vSMC electrodes that responded during listening .","Given these proposed functional connections , activity in vSMC from speech sounds may be a consequence of sounds activating the sensory feedback circuit ( Hickok et al . , 2011 ) .","Alternatively , evoked responses in the motor cortex during passive listening may directly reflect auditory inputs arising from aggregated activity picked up by the electrode .","We believe the latter scenario to be less likely , however , given that auditory responses were observed in dorsal vSMC on electrode contacts several centimeters away from auditory inputs in the STG .","In addition , the spatial spread of neural signals in the high gamma range is substantially smaller than this difference – high gamma signal correlations at <2 mm spacing are only around r=0 . 5 , and at distances of 1 cm reach a noise floor ( Chang , 2015 ) ; Muller et al , unpublished findings ) .","Given the observed acoustic rather than place selectivity observed during listening in the vSMC , our results suggest that motor theories of speech perception may need to be revised to incorporate a novel sensorimotor representation of sound in the vSMC ."],["Nine human participants were implanted with high-density multi-electrode cortical surface arrays as part of their clinical evaluation for epilepsy surgery .","The array contained 256 electrodes with 4 mm pitch .","Arrays were implanted on the lateral left hemispheres over the peri-Sylvian cortex , but exact placement was determined entirely by clinical indications ( Figure 1—figure supplement 1 and Figure 1—figure supplement 2 ) .","Using anatomic image fusion software from BrainLab ( Munich , Germany ) , electrode positions were extracted from the computed tomography ( CT ) scan , co-registered with the patient’s MRI and then superimposed on the participant’s 3D MRI surface reconstruction image .","All participants were left hemisphere language dominant , as assessed by the Wada test .","Participants had self-reported normal hearing .","The study protocol was approved by the UC San Francisco Committee on Human Research , and all participants provided written informed consent .","Participants completed three separate tasks that were designed to sample a range of phonetic features .","First , participants listened to eight consonant-vowel ( CV ) syllables ( /ba/ , /da/ , /ga/ , /pa/ , /ta/ , /ka/ , /∫a/ , /sa/ ) produced by a male speaker unknown to the participant .","Stimuli were presented randomly , with 4–21 repetitions of each CV syllable for 5 out of the 9 subjects included in all subsequent analyses , and one repetition of each CV syllable for 4 subjects shown only in Figure 1—figure supplement 2 .","To remain alert , participants were asked to identify the syllable they heard by selecting from a multiple-choice question on a computer with their ipsilateral ( left ) hand .","In the second task , participants spoke aloud the same CV syllables prompted by a visual cue on the laptop computer display .","In the third task , participants passively listened to natural speech samples from a phonemically transcribed continuous speech corpus ( TIMIT ) .","We chose 499 unique sentences from 400 different male and female speakers .","Each sentence was repeated two times .","For the phoneme selectivity analysis , we chose a subset of TIMIT phonemes that occurred more than 30 times .","This resulted in an analysis of 33 phonemes .","For spectrotemporal receptive field analysis ( see below ) , data from all sentences were used .","Electrocorticographic ( ECoG ) signals were recorded with a multichannel PZ2 amplifier connected to an RZ2 digital signal acquisition system ( Tucker-Davis Technologies , Alachua , FL , USA ) sampling at 3 , 052 Hz .","The produced speech was recorded with a microphone , digitized , and simultaneously recorded .","The speech sound signals were presented monaurally from loudspeakers at a comfortable level , digitized , and also simultaneously recorded with the ECoG signals .","Line noise ( 60 Hz and harmonics at 120 and 180 Hz ) was next removed from the signal with a notch filter .","Each time series was visually and quantitatively inspected for excessive noise , and was subsequently removed from further analyses if its periodogram deviated more than two standard deviations away from the average periodogram of all other time series .","The remaining time series were then common-average referenced ( CAR ) and used for analyses .","The CAR was taken across 16 channel banks in order to remove non-neural electrical noise from shared inputs to the PZ2 .","We find that this method of CAR significantly reduces movement-related and other non-neural artifacts while not adversely affecting our signals of interest .","The analytic amplitude of each time series was extracted using eight bandpass filters ( Gaussian filters , logarithmically increasing center frequencies ( 70–150 Hz ) with semi-logarithmically increasing bandwidths ) with the Hilbert transform .","The high-gamma power was calculated by averaging the analytic amplitude across these eight bands , and downsampling the signal to 100 Hz .","The signal was finally z-scored relative to the mean and standard deviation of baseline rest data for each channel .","Supra-Sylvian cortical sites with robust evoked responses to both speech sounds and speech production were selected for this analysis .","To identify if a site was responsive to speech sounds , we implemented a bootstrap t-test comparing a site's responses randomly sampled over time during speech sound presentations to responses randomly sampled over time during pre-stimulus silent intervals ( p<0 . 01 ) .","This resulted in 10 , 22 , 29 , 27 , and 27 sites for the five participants ( n=115 ) .","Next we implemented a bootstrap t-test comparing neural responses during speech production and pre-stimulus silence ( p<0 . 01 ) , resulting in 25 , 74 , 87 , 92 , and 84 sites ( n=362 ) .","Finally , we took the intersection of these two groups to arrive at our final supra-Sylvian sites set of 8 , 16 , 28 , 22 , and 24 sites active during listening and speaking ( n=98 ) .","To analyze the responses of the auditory cortex , we restricted the infra-Sylvian cortical sites to those that were reliably evoked by speech sounds ( p<0 . 01 , t-test between silence and speech sounds neural responses ) .","This resulted in 73 , 61 , 40 , 77 , and 89 infra-Sylvian temporal cortical sites ( n=340 ) responsive to speech sounds .","To investigate the degree of spatial clustering in the vSMC electrodes responsive during listening , we used the Dip-means method ( Kalogeratos and Likas , 2012 ) , which allows us to test whether data shows any form of clustering .","Importantly , unlike the silhouette index , this allows us to distinguish between k=1 and k>1 clusters .","For each subject , the pairwise distances between the spatial locations of all electrodes in a single subject were computed .","Using each electrode in turn as a 'viewer' ( Kalogeratos and Likas , 2012 ) , we tested to see whether the distribution of distances to that electrode significantly deviated from unimodality ( Hartigan and Hartigan , 1985 ) .","If one or more electrodes showed a signficantly non-unimodal pairwise distance histogram , then the data were considered to be clustered .","Following this procedure , k-means clustering was performed with k=2 through k=6 clusters , and the silhouette index was used to determine the best number of clusters for a given subject .","The silhouette index for a given data point is defined ass ( i ) =b ( i ) −aimax{a ( i ) , b ( i ) } where b ( i ) is the lowest average distance of i to any other cluster of which i is not a member , and a ( i ) is the average distance between i and any other data point assigned to the same cluster .","The silhouette index ranges from −1 to 1 , with higher positive values indicating good clustering .","For the speaking and listening CV syllable tasks , the start of the syllable acoustics was used to align the responses of each electrode site .","For the phoneme responses , the TIMIT phonetic transcriptions were used to align responses to the phoneme onset .","Once responses were aligned to a stimulus , the average activity for each site to each stimulus was measured by taking the mean response over different trials of the same stimuli .","The maximum of the mean responses to different stimuli were then used to measure the peak-high gamma distributions between different tasks and sites .","We measured the onset latencies for responses to listening in STG and vSMC by calculating the average z-scored high gamma activity across all CV syllables , and then calculating the first time at which activity was significantly higher than the 500-ms pre-stimulus silent rest period ( one-tailed Wilcoxon rank sum test , p<0 . 001 ) .","We also calculated the peak latency as the time at which the average z-scored response reached its maximum value .","Differences in onset and peak latencies were compared across STG , inferior , and superior vSMC using the a two-tailed Wilcoxon rank sum test at a significance level of p<0 . 05 ( uncorrected ) .","To measure the timing/dynamics between pairs of vSMC and STG sites during CV syllable listening , we performed a cross-correlation analysis between pairs of electrodes in these two regions .","The cross-correlation measures the similarity of two time series at different time lags by taking pairs of electrode responses and calculating the correlation between one response and a time-shifted version of the second response .","If the peak in the cross-correlation between an STG electrode and a vSMC electrode occurs at a negative lag , this indicates that the STG response leads ( occurs earlier than ) the vSMC response and that STG activity in the past is predictive of future activity in the vSMC .","In contrast , if the peak in the cross-correlation between an STG electrode and a vSMC electrode occurs at a positive lag , this indicates that the vSMC response leads ( occurs earlier than ) the STG response .","The cross-correlation at time lag τ is calculated between the response at an STG electrode ( denoted","x ) and the response at a vSMC electrode","( y ) as follows:x*y[τ] = def∑t=-0 . 5 st=1 sx*[t]y[t+τ] Where the maximum lag τ was chosen to be 0 . 75 s .","Cross-correlations were normalized by 1M−|τ| ( where M is the total number of time points in the response ) to obtain an unbiased estimate at each time lag τ .","The cross-correlation between vSMC and STG electrodes was calculated separately for each CV syllable trial , and then averaged across trials ( see examples in Figure 3d ) .","To determine the incidence of relationships within our electrode population where STG leads vSMC , vSMC leads STG , or both are coactive , we calculated an asymmetry index .","This index ranges from −1 to 1 and describes the relative power in the positive versus negative lags for each vSMC electrode .","It is calculated for each vSMC electrode by taking the sum of the positive cross-correlations in the negative lags and the sum of the positive cross-correlations in the positive lags , and then computing the ratio:asymmetry index = Ppos−PnegPpos+Pneg For a given vSMC electrode , an asymmetry index of −1 indicates that the cross-correlations lie fully in the negative lags ( indicating that STG responses lead the vSMC response in that electrode ) .","In contrast , a value of 1 indicates that the cross-correlations are in the positive lags only , indicating that the vSMC electrode leads all STG electrodes .","To examine the relational organization of the neural responses to syllables , we applied unsupervised multidimensional scaling ( MDS ) to the distance matrix of the mean neural responses at the sites of interest described in Materials and methods: Electrode selection .","For analysis of speaking and listening responses , the vSMC sites used were those identified as significantly active during both speech production and speech perception ( n=98 , Figure 4b , c , f ) .","However , clustering results for speaking were similar when all vSMC sites identified as significantly active during speech production were included ( n=362 , Figure 4—figure supplement 3c ) .","The STG sites used were those identified as significantly active during speech perception ( n=340 , Figure 4e , Figure 4—figure supplement 3b ) .","Syllables placed closer together in MDS space elicited similar neural response patterns , and those further apart from one another elicited more dissimilar patterns .","To calculate the distance between a pair of mean neural responses , a mean neural response to one syllable was linearly correlated to another , and the resulting correlation coefficient was subtracted from 1 .","We used unsupervised K-means clustering to examine the grouping of the mean neural activity to syllables of the electrodes of interest described in Methods: Electrode selection .","We clustered the mean activity into 3 distinct clusters .","This number of clusters was chosen because there are 3 major place of articulations and manner of articulations in the syllable stimuli set ( Figure 4a ) that have been shown to play a major role in the neural organization of motor cortex during speech production and auditory cortex during speech perception .","After clustering the neural responses into three distinct groups , we measured the similarity of the grouping to the linguistically defined grouping of consonants by place of articulation and acoustic features ( Figure 4a and Figure 2—figure supplement 1 ) using the adjusted Rand Index ( RIadj ) .","The RIadj is frequently used in statistics for cluster validation .","It measures the amount of agreement between two clustering schemes: one by a given clustering process ( e . g . K-means ) , and the other by some external criteria , or gold-standard ( e . g . place of articulation linguistic features ) .","The RIadj takes an intuitive approach to measuring cluster similarity by counting the number of pairs of objects classified in the same cluster under both clustering schemes , and controlling for chance ( hence , 'adjusted' RI ) .","It has an expected value of 0 for independent clusterings , and a maximum value of 1 for identical clustering .","It is defined as the following: Let S be a set of n objects , S = ( o1 , o2 , … , on ) .","Partitioning the objects in two different ways such that U = ( U1 , … , Ur ) is a partition of S into r subsets , and V = ( V1 , … , Vt ) is a partition of S into t subsets , let: a = number of pair of objects that are in the same set in U and in the same set in V , b = number of pair of objects that are in the same set in U and in different sets in V , c = number of pair of objects that are in different sets in U and in the same set in V , d = number of pair of objects that are in different sets in U and in different sets in V . Without adjusting for chance , the RI is simply:RI = a+da+b+c+d=a+dn2 .","Taking into account chance pairings , RIadj becomes:RIadj = n2 ( a+d ) - ( a+b ) ( a+c ) + ( c+d ) ( b+d ) n22- ( a+b ) ( a+c ) + ( c+d ) ( b+d ) .","To localize an unbiased time window for analysis , the ΔRIadj metric was derived for all time windows by subtracting the RIadj measured with the place of articulation features gold-standard from the RIadj measured with the acoustic feature gold-standard ( Figure 4—figure supplement 1 ) .","An ΔRIadj = 1 denotes organization by acoustic features , and an ΔRIadj = −1 denotes organization by place features .","The significance of the ΔRIadj was computed by calculating the RIadj for a randomized labeling of neural responses compared to either acoustic feature or place feature clustering , taking the difference ( ∆RIadj ) , and repeating this procedure 1000 times with different randomized labelings to create a null distribution of ∆RIadj values .","The p-value was calculated as the number of times this random ∆RIadj exceeded the observed ∆RIadj , and was thresholded at an FDR-corrected p<0 . 05 using the Benjamini-Hochberg procedure ( Benjamini and Hochberg , 1995 ) .","To characterize the phoneme selectivity of each electrode site , we implemented the PSI calculation described by Mesgarani et al . , 2014 .","In short , for a single site , we summed the number of responses that were statistically different ( Wilcoxon rank-sum test , p<0 . 01 , corrected for multiple comparisons ) from the response to a particular phoneme .","This resulted in a PSI that ranges from 0 to 32 , where a PSI = 32 is an extremely selective electrode and a PSI = 0 is not selective .","A PSI describes an electrode’s selectivity to one phoneme , and a vector of PSIs describes an electrode’s selectivity profile to all phonemes .","The spectrotemporal representation of speech sounds was first estimated using a cochlear frequency model , consisting of a bank of logarithmically spaced constant Q asymmetric filters .","The filter bank output was subjected to nonlinear compression , followed by a first order derivative along the spectral axis modeling a lateral inhibitory network , and an envelope estimation operation ( Wang and Shamma , 1994 ) .","This resulted in a two dimensional spectrotemporal representation ( spectrogram ) of speech sounds simulating the pattern of activity on the auditory nerve .","We then estimated the spectrotemporal receptive fields ( STRFs ) of the sites from passive listening to TIMIT using normalized reverse correlation ( Aertsen and Johannesma , 1981; Klein et al . , 2000; Theunissen et al . , 2001; Woolley et al . , 2006 ) between spectrotemporal representation of the sentences and the evoked neural activity ( STRFLab software package: http://strflab . berkeley . edu , DirectFit routine ) .","The STRF is a linear filter that describes which combinations of spectrotemporal features will elicit a neural response in a given electrode .","The relationship between the STRF , H , stimulus spectrogram , S ( as estimated above ) , and the predicted response , r^ ( t ) , of an electrode are given by the following equation:r^ ( t ) =∑i=0M-1 ∑τ=0N-1 H ( τ , f ) S ( t -τ , f ) where N is the number of delays of length τ after which the STRF will be estimated ( reflecting memory for the stimulus ) , and M is the number of frequency bands in the spectrogram .","To estimate the STRF , we minimize the mean squared error between the predicted and observed responses .","To prevent overfitting , we used an L2 regularization procedure in which a ridge hyperparameter and sparseness hyperparameter were calculated for each electrode's STRF ( details in [Woolley et al . , 2006] ) .","The ridge hyperparameter acts as a smoothing factor on the STRF , whereas the sparseness hyperparameter controls the number of non-zero weights in the STRF .","These hyperparameters were optimized with a systematic hyperparameter grid search maximizing for mutual information ( bits/s ) .","With the optimized hyperparameters , we calculated the final STRF and correlation between the predicted and actual neural response using cross-validation .","To do this , a STRF was derived using 9/10 of the stimuli-response pairs , and the Pearson correlation coefficient ( indicating the STRF goodness-of-fit ) was measured by predicting the remaining one-tenth responses .","This was repeated 10 times with 10 non-overlapping stimuli-response pair sets .","The final STRF and correlation number were derived by averaging the 10 STRFs and correlation coefficients .","To assess statistical differences , we used independent sample t-tests when the data were found not to deviate significantly from normality ( KS test ) .","When data were not normally distributed , we used the nonparametric Wilcoxon rank sum test .","In some cases , a bootstrap t-test was used ."]],"string":"[\n [\n \"Our motor and sensory cortices are traditionally thought to be functionally separate systems .\",\n \"However , an accumulating number of studies has revealed their roles in action and perception to be highly integrated ( Pulvermüller and Fadiga , 2010 ) .\",\n \"For example , a number of studies have demonstrated that both sensory and motor cortices are engaged during perception ( Gallese et al . , 1996; Wilson et al . , 2004; Tkach et al . , 2007; Cogan et al . , 2014 ) .\",\n \"In humans , this phenomenon has been observed in the context of speech , where listening to speech sounds evokes robust neural activity in the motor cortex ( Wilson et al . , 2004; Pulvermüller et al . , 2006; Edwards et al . , 2010; Cogan et al . , 2014 ) .\",\n \"This observation has re-ignited an intense scientific debate over the role of the motor system in speech perception over the past decade ( Lotto et al . , 2009; Scott et al . , 2009; Pulvermüller and Fadiga , 2010 ) .\",\n \"One interpretation of the observed motor activity during speech perception is that “the objects of speech perception are the intended phonetic gestures of the speaker”- as posited by Liberman’s motor theory of speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) .\",\n \"The motor theory is a venerable and well-differentiated exemplar of a set of speech perception theories that we could call 'production-referencing' theories .\",\n \"Unlike motor theory , more modern production referencing theories do not assume that sensorimotor circuits are necessarily referenced in order for speech to be recognized , but they allow for motor involvement in perception in certain phonetic modes .\",\n \"For example , Lindblom , 1996 suggested that a direct link between spectrotemporal analysis and word recognition is the normal mode of speech perception ( the 'what' mode of perception ) , but in some cases listeners do use a route through sensorimotor circuits ( the 'how' mode of perception ) if , for example , the listener is attempting to imitate a new sound ( Lindblom , 1996 ) .\",\n \"While demonstrations of evoked motor cortex activity by speech sounds strengthen production-referencing theories , it remains unclear what information is actually represented by such activity .\",\n \"Determining what phonetic properties are encoded in the motor cortex has significant implications for elucidating the role it may play in speech perception .\",\n \"To address this , we recorded direct neural activity from the peri-Sylvian speech cortex in nine human participants undergoing clinical monitoring for epilepsy surgery .\",\n \"This includes but is not limited to two relevant areas comprising the supra-Sylvian ventral half of the lateral sensorimotor cortex ( vSMC ) for the motor control of articulation ( Penfield and Boldrey , 1937 ) and the infra-Sylvian superior temporal gyrus ( STG ) for the auditory processing of speech sounds ( Ojemann et al . , 1989; Boatman et al . , 1995 ) .\",\n \"Since cortical processing of speech sounds is spatially discrete and temporally fast ( Formisano et al . , 2008; Chang et al . , 2011; Steinschneider et al . , 2011 ) , we used customized high-density electrode grids ( a four-fold increase over conventional recordings ) ( Bouchard et al . , 2013; Mesgarani et al . , 2014 ) .\",\n \"Importantly , these recordings have simultaneous high spatial and temporal resolution in order to study the detailed speech representations in the vSMC ( Crone et al . , 1998; Edwards et al . , 2009 ) .\",\n \"With this approach , we seek to address unanswered questions about the representation of speech sounds in motor cortex , including how the spatiotemporal patterns compare when speaking and listening and whether auditory representations in motor cortex are organized along articulatory or acoustic dimensions .\"\n ],\n [\n \"Participants first listened passively to consonant-vowel ( CV ) syllables ( 8 consonants followed by the /a/ vowel ) .\",\n \"In a separate trial block , they spoke aloud these same CV syllables .\",\n \"We measured the average evoked cortical activity during these listening and speaking CV tasks .\",\n \"We focused our analysis on high gamma ( 70–150 Hz ) cortical surface local field potentials , which strongly correlate with extracellular multi-unit neuronal spiking ( Steinschneider et al . , 2008; Ray and Maunsell , 2011 ) .\",\n \"We aligned neural responses to the onset of speech acoustics ( t = 0 ) in listening and speaking tasks to provide a common reference point across speech sounds .\",\n \"We first determined which peri-Sylvian cortical areas were activated during passive listening to speech sounds .\",\n \"Figure 1a and b shows the locations of cortical areas that demonstrated cortical evoked responses in a single representative subject during listening and speaking respectively .\",\n \"During listening , evoked responses spanned middle and posterior STG as expected , with weaker responses in middle temporal gyrus ( MTG ) ( Figure 1a ) .\",\n \"In the vSMC , ( composed of the pre- and post- central gyri ) we found electrodes in the superior-most and inferior-most aspects ( Figure 1a , Figure 1—figure supplement 1 , 2 ) that demonstrated reliable and robust single-trial responses to speech sounds during passive listening ( Figure 1b ) .\",\n \"Neural responses were also found at a few sites scattered across supramarginal , inferior- , and middle- frontal gyri—though these were not consistent across subjects ( Figure 1—figure supplement 1 ) .\",\n \"By performing spatial clustering analysis on the electrode positions in each subject , we found that 3/5 subjects showed significant clustering of regions responsive to auditory stimuli ( Hartigan’s Dip statistic , p<0 . 05 ( see Materials and methods ) ; Figure 1—figure supplement 1 ) .\",\n \"Out of these 3 subjects , k-means clustering revealed two subjects with k=2 electrode clusters ( subjects 1 and 4 , clusters in inferior and superior vSMC ) , and one subject with k=5 clusters .\",\n \"When participants spoke the same CV syllables , in contrast , articulatory movement-related cortical activity was well distributed throughout vSMC ( Figure 1c ) , with auditory feedback cortical activity seen in the STG . 10 . 7554/eLife . 12577 . 003Figure 1 . Speech sounds evoke responses in the human motor cortex .\",\n \"( a ) Magnetic resonance image surface reconstruction of one representative subject’s cerebrum ( subject 1: S1 ) .\",\n \"Individual electrodes are plotted as dots , and the average cortical response magnitude ( z-scored high gamma activity ) when listening to CV syllables is signified by the color opacity .\",\n \"CS denotes the central sulcus; SF denotes the Sylvian fissure .\",\n \"( b ) Acoustic waveform , spectrogram , single-trial cortical activity ( raster ) , and mean cortical activity ( high gamma z-score , with standard error ) from two vSMC sites and one STG site when a subject is listening to /da/ .\",\n \"Time points significantly above a pre-stimulus silence period ( p<0 . 01 , bootstrap resampled , FDR corrected , alpha < 0 . 005 ) are marked along the horizontal axis .\",\n \"The vertical dashed line indicates the onset of the syllable acoustics ( t=0 ) .\",\n \"( c ) Same subject as in\",\n \"( a ) ; distributed vSMC cortical activity when speaking CV syllables ( mean high gamma z-score ) .\",\n \"( d ) Total number of significantly active sites in all subjects during listening , speaking , and both conditions ( p<0 . 01 , t-test , responses compared to silence and speech ) .\",\n \"Electrode sites are broken down by their anatomical locations .\",\n \"S denotes superior vSMC sites; I denotes inferior vSMC sites . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00310 . 7554/eLife . 12577 . 004Figure 1—figure supplement 1 . Average cortical responses to speaking and listening in all subjects ( S2-S5 ) .\",\n \"( a ) Average Z-scored responses in active electrodes when listening to ( left ) or speaking ( right ) CV syllables in all subjects , as shown in Figure 1a and c .\",\n \"( b ) Results from spatial clustering of significantly active electrodes in each subject .\",\n \"S1 , S4 , and S5 showed spatially segregated clusters ( see Materials and methods ) and were included in k-means clustering analysis .\",\n \"The silhouette index shows that the best number of clusters for S1 ( from Figure 1 ) and S4 was k=2 , whereas the best number of clusters for S5 was k=5 . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00410 . 7554/eLife . 12577 . 005Figure 1—figure supplement 2 . Neural responses while listening to CV syllables in 4 additional subjects not included in MDS analyses ( S6 - S9 ) .\",\n \"Responses are plotted on each subject’s brain surface and are shown for each electrode as the average Z-scored high gamma activity across all syllables , aligned to the acoustic onset of the stimulus ( indicated by dashed line at t = 0 ) .\",\n \"As in Figure 1 and Figure 1—figure supplement 1 , strong responses are seen during listening in inferior and superior regions of the vSMC ( primarily precentral gyrus ) in addition to the superior temporal gyrus ( STG ) .\",\n \"For each subject , SF indicates the location of the Sylvian fissure , and CS indicates the central sulcus . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 005 Across all participants , we identified 115 electrodes that demonstrated significant neural activity in vSMC during listening ( p<0 . 01 , t-test , compared to pre-stimulus silent rest period; Figure 1d ) .\",\n \"When speaking , in contrast , a total of 362 electrodes in vSMC were found to be significantly active ( Figure 1d , p<0 . 01 , t-test , compared to pre-stimulus silent rest period ) .\",\n \"We compared the relative proportions of electrodes that were found in different supra-Sylvian anatomical regions .\",\n \"Critically , only a subset of sites in vSMC ( 98 out of 362 , ~27% ) was active during both listening and speaking ( Figure 1d ) .\",\n \"These sites were primarily localized to the pre-central gyrus , whereas speaking evoked activity across both pre- and post-central gyri sites .\",\n \"Neural responses in the vSMC during listening were found in the superior ( S in Figure 1d ) pre-central gyrus and inferior , anterior aspect of the sub-central gyrus of the vSMC ( I in Figure 1d ) .\",\n \"We next compared the patterns of cortical activity to specific speech sounds during listening and speaking .\",\n \"During speaking , specific articulator representations have been identified in the somatotopically-organized vSMC ( Bouchard et al . , 2013 ) .\",\n \"For example , the plosive consonants /b/ , /d/ , and /g/ are produced by the closure of the vocal tract at the lips , front tongue , and back tongue , respectively ( Figure 2a , b , see Figure 2—figure supplement 1 for all syllable tokens ) ( Ladefoged and Johnson , 2010 ) .\",\n \"The cortical representations for these articulators are laid out along a superior-to-inferior ( medial-to-lateral ) sequence in the vSMC ( Penfield and Boldrey , 1937 ) .\",\n \"We first examined average cortical activity at single electrode sites distributed along the vSMC axis for articulating individual speech sounds .\",\n \"Figure 2c shows single electrode activity from a single representative subject ( the same from Figure 1 ) for speaking ( blue lines ) and listening ( red lines ) for three CV syllables , which have different place of articulation ( /ba/ , /da/ , and /ga/ ) .\",\n \"The exact location of these electrodes on the vSMC is shown in Figure 2d .\",\n \"The production of labial consonants ( /b/ ) is associated with activity in lip cortical representations as evidenced by strong responses to the bilabial /ba/ ( Figure 2c , electrodes 5–6 , blue lines ) .\",\n \"These are located superior to the tongue representations associated with the /d/ and /g/ consonants , as shown previously ( Bouchard et al . , 2013 ) .\",\n \"Those tongue sites were sub-specified by ‘coronal’ ( i . e . anterior-based ) tongue position for /d/ ( electrodes 8–10 , blue lines ) superiorly , and ‘dorsal’ ( i . e . posterior-based ) tongue position for /g/ inferiorly ( electrode 13 , blue line ) .\",\n \"Other sites ( electrodes 1–4 , 11–12 , blue lines ) showed the same neural activity across all three syllables . 10 . 7554/eLife . 12577 . 006Figure 2 . Site-by-site differences in vSMC neural activity when speaking and listening to CV syllables .\",\n \"( a ) Top , vocal tract schematics for three syllables ( /ba/ , /da/ , /ga/ ) produced by occlusion at the lips , tongue tip , and tongue body , respectively ( arrow ) .\",\n \"( b ) Acoustic waveforms and spectrograms of spoken syllables .\",\n \"( c ) Average neural activity at electrodes along the vSMC for speaking ( blue ) and listening ( red ) to the three syllables ( high gamma z-score ) .\",\n \"Solid lines indicate activity was significantly different from pre-stimulus silence activity ( p<0 . 01 ) .\",\n \"Transparent lines indicate activity was not different from pre-stimulus silence activity ( p>0 . 01 ) .\",\n \"Vertical dashed line denotes the onset of the syllable acoustics ( t=0 ) .\",\n \"( d ) Location of electrodes 1–13 in panel c , shown on whole brain and with inset detail .\",\n \"CS = central sulcus , SF = Sylvian fissure . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00610 . 7554/eLife . 12577 . 007Figure 2—figure supplement 1 . Syllable token set .\",\n \"( a ) Consonants of all syllable tokens .\",\n \"Consonants were paired with the vowel /a/ , and are organized by place and manner of articulation .\",\n \"( b ) Vocal tract schematics for three occlusions made with the lips , tongue tip , and tongue body , respectively ( arrow ) .\",\n \"( c ) Acoustic waveform and spectrogram of the syllable tokens . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 007 We next examined those same vSMC electrodes during listening , and found that the majority of those cortical vSMC electrodes were not active ( p>0 . 01 , t-test compared to silence , Figure 2c transparent red lines ) .\",\n \"The few that were active ( electrodes 1 , 2 , 4 , 11–12 , solid red lines ) were similar for all three CV syllables , with activity increasing approximately 100ms after the acoustic onset .\",\n \"Across the entire population of vSMC electrodes that were active during listening , onset latencies were generally shorter than those in STG sites , with significant increases in both inferior vSMC ( p<0 . 001 ) and superior vSMC ( p<0 . 05 ) compared to STG ( Figure 3a , Wilcoxon rank sum test , see Figure 3c for average responses to all syllables ) .\",\n \"The latency to the response peak was also significantly higher in superior vSMC compared to STG ( Figure 3b , p<0 . 01 , Wilcoxon rank sum test ) .\",\n \"A cross-correlation analysis between these vSMC electrodes and STG electrodes revealed a diverse array of relationships between these populations ( Figure 3d–f ) , including STG electrode activity leading vSMC electrode activity and vice versa .\",\n \"In contrast to speaking , we did not observe somatotopic organization of cortical responses when listening to speech .\",\n \"Therefore , the pattern of raw evoked responses during listening shows critical differences from those during speaking . 10 . 7554/eLife . 12577 . 008Figure 3 . Dynamics of responses during CV listening in STG , inferior vSMC , and superior vSMC .\",\n \"( a ) STG onset latencies were significantly lower than both inferior vSMC ( p<0 . 001 , Z = −4 . 03 ) and superior vSMC ( p<0 . 05 , Z = −2 . 28 ) .\",\n \"( b ) STG peak latencies were significantly lower than superior vSMC ( p<0 . 01 , Z = −2 . 93 ) , but not significantly different from peak latencies in inferior vSMC ( p>0 . 1 ) .\",\n \"In\",\n \"( a ) and\",\n \"( b ) , red bar indicates the median , boxes indicate 25th and 75th percentile , and error bars indicate the range .\",\n \"Response latencies were pooled across all subjects .\",\n \"All p-values in\",\n \"( a ) and\",\n \"( b ) are from the Wilcoxon rank sum test .\",\n \"( c ) Average evoked responses to all syllable tokens across sites in superior vSMC ( n=32 ) , inferior vSMC ( n=37 ) , and STG .\",\n \"Responses were aligned to the syllable acoustic onset ( t=0 ) .\",\n \"A random subset of STG responses ( n=52 out of the 273 that were used in the latency analysis in\",\n \"( a ) and\",\n \"( b ) ) are shown here for ease of viewing .\",\n \"( d ) Example cross-correlations between three vSMC electrodes and all STG electrodes in one patient , for a maximum lag of ± 0 . 75 s .\",\n \"More power in the negative lags indicates a faster response in the STG compared to the vSMC electrode , and more power in the positive lags indicates a faster response in vSMC compared to STG .\",\n \"We observe vSMC electrodes that tend to respond later than STG ( e248 , left panel ) , vSMC electrodes that tend to respond before STG ( e136 , middle panel ) , and vSMC electrodes that respond at similar times to some STG electrodes ( e169 , right panel ) .\",\n \"( e ) Average evoked responses during CV listening for all STG electrodes from this patient and the three vSMC electrodes shown in panel\",\n \"( d ) .\",\n \"Responses were aligned to the syllable acoustic onset ( t=0 ) , as in panel\",\n \"( c ) .\",\n \"( f ) Percentage of sites with STG leading , coactive , or vSMC leading as expressed by the asymmetry index ( see Materials and methods ) .\",\n \"Both inferior and superior vSMC show leading and lagging responses compared to STG , as well as populations of coactive pairs . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 008 We next evaluated quantitatively whether the structure of distributed vSMC neural activity during listening was more similar to that of vSMC during speaking or the STG during listening .\",\n \"In previous studies , we demonstrated that the structure of evoked responses are primarily organized by different feature sensitivities: place of articulation in the vSMC ( Bouchard et al . , 2013 ) , and manner of articulation in the STG ( Mesgarani et al . , 2014 ) .\",\n \"We visualized the similarity of population activity evoked by different consonants using unsupervised multidimensional scaling ( MDS ) , where the 2-dimensional Euclidean distances between stimuli correspond to the similarity of their neural responses .\",\n \"Visual inspection of MDS plots shows that , during speaking , evoked activity in vSMC clustered into place of articulation features ( Figure 4a ) : labials ( /b/ , /p/ ) , alveolars ( /s/ , /sh/ , /t/ , /d/ ) , and velars ( /g/ , /k/ ) ( Figure 4b ) .\",\n \"In contrast , neural responses during listening did not cluster into the same features ( Figure 4c ) .\",\n \"To quantify the degree to which the evoked activity clustered into place of articulation features , we used unsupervised K-means clustering to assign the neural responses to clusters ( k=3 ) , and the adjusted Rand Index ( RIadj ) ( Rand , 1971; Hubert and Arabie , 1985 ) to measure the degree to which the neural clustering agreed with linguistically defined place of articulation consonant clusters .\",\n \"The RIadj quantifies the degree of agreement between two clustering patterns , where RIadj = 1 denotes identical clustering patterns and RIadj = 0 denotes independent clustering patterns .\",\n \"We found that while evoked activity during speaking clustered by place of articulation features , activity during listening did not ( Figure 4d; see Figure 4—figure supplement 1 for moving time window analysis ) .\",\n \"Even when the vSMC electrode subset was restricted to short-latency vSMC electrodes leading STG activity ( as evidenced by a positive asymmetry index in Figure 3f ) , activity during listening did not cluster according to place of articulation features ( Figure 4—figure supplement 2 ) .\",\n \"Thus , responses in motor areas during speech perception do not show a spatially distributed representation of speech motor articulator features . 10 . 7554/eLife . 12577 . 009Figure 4 . Organization of motor cortex activity patterns .\",\n \"( a ) Consonants of all syllable tokens organized by place and manner of articulation .\",\n \"Where consonants appear in pairs , the right is a voiced consonant , and the left is a voiceless consonant .\",\n \"( b ) Relational organization of vSMC patterns ( similarity ) using multidimensional scaling ( MDS ) during speaking .\",\n \"Neural pattern similarity is proportional to the Euclidean distance ( that is , similar response patterns are grouped closely together , whereas dissimilar patterns are positioned far apart ) .\",\n \"Tokens are colored by the main place of articulation of the consonants ( labial , velar , or alveolar ) .\",\n \"( c ) Similarity of vSMC response patterns during listening .\",\n \"Same coloring by place of articulation .\",\n \"( d ) Organization by motor articulators .\",\n \"K-means clustering was used to assign mean neural responses to 3 groups ( labial , alveolar , velar ) for both listening and speaking neural organizations ( b , c ) .\",\n \"The similarity of the grouping to known major articulators was measured by the adjusted Rand Index .\",\n \"An index of 1 indicates neural responses group by place of articulation features .\",\n \"***p<0 . 001 , Wilcoxon rank-sum\",\n \"( e ) Organization of mean STG responses using MDS when listening .\",\n \"In contrast to c and d , tokens are now colored by their main acoustic feature ( fricative , voiced plosive , or voiceless plosive ) .\",\n \"( f ) Organization of mean vSMC responses using MDS when listening colored by their main acoustic feature .\",\n \"( Identical to C , but recolored here by acoustic features ) .\",\n \"( g ) Organization by manner of articulation acoustic features ( fricative , voiced plosive , voiceless plosive ) for both STG and vSMC organizations when listening ( e ,\",\n \"f ) .\",\n \"The similarity of the grouping to known acoustic feature groupings was measured by the adjusted Rand Index .\",\n \"***p<0 . 001 , Wilcoxon rank sum .\",\n \"( h ) During listening , responses in vSMC show significantly greater organization by acoustic manner features compared to place features as assessed by the adjusted Rand Index , indicating an acoustic rather than articulatory representation ( ***p<0 . 001 , Wilcoxon rank-sum ) .\",\n \"Bars in this panel are the same as the red bars in\",\n \"( d ) and\",\n \"( g ) .\",\n \"In\",\n \"( d ) ,\",\n \"( g ) , and\",\n \"( h ) , bars indicate mean ± standard deviation , DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 00910 . 7554/eLife . 12577 . 010Figure 4—figure supplement 1 . Clustering trajectory analysis of neural responses to syllables . The clustering trajectory across time was derived from vSMC activity ( n=98 electrodes ) when speaking ( blue ) and listening ( red ) using the ΔRIadj metric .\",\n \"An index of 1 denotes the neural responses organized by acoustic features , and an index of -1 denotes an organization by place of articulation features .\",\n \"Time points indicated by the blue and red windows are significantly organized according to place or acoustics , respectively ( FDR-corrected p<0 . 05 , permutation test comparing random clustering at each time point to clustering by acoustics or place ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01010 . 7554/eLife . 12577 . 011Figure 4—figure supplement 2 . Analysis of short latency responses in vSMC . We restricted the MDS analysis to vSMC responses with a positive asymmetry index ( see Figure 3d and f , n=50 electrodes ) and found significant organization by acoustic manner features during listening in these sites .\",\n \"( a ) Similarity of responses to syllables during listening in the subset of vSMC electrodes leading activity in STG .\",\n \"Tokens are colored according to place of articulation features ( left ) or acoustic manner features ( right ) as in Figure 4c and f .\",\n \"( b ) During listening , responses of short latency sites in the vSMC still show significantly greater organization by acoustic manner features compared to place features as assessed by the adjusted Rand Index , indicating an acoustic rather than articulatory representation ( ***p<0 . 001 , Wilcoxon rank-sum ) .\",\n \"Compare with Figure 4h .\",\n \"Bars indicate mean ± standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01110 . 7554/eLife . 12577 . 012Figure 4—figure supplement 3 . Organization of syllable tokens and auditory cortical activity patterns .\",\n \"( a ) Organization of the acoustic spectrograms of the CV tokens using MDS .\",\n \"( b ) Organization of mean STG responses using MDS when speaking .\",\n \"Tokens in\",\n \"( a ) and\",\n \"( b ) are colored by their main acoustic feature .\",\n \"( c ) Organization of vSMC responses by place of articulation using all speech-responsive electrodes ( n=362 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 012 Finding no evidence that major articulator features are either locally or spatially distributed in the vSMC in response to speech sounds , we next compared vSMC responses to population responses in the STG .\",\n \"STG has an acoustic sensory representation of speech that best discriminates speech sounds by manner of articulation features with salient acoustic differences ( Mesgarani et al . , 2014 ) .\",\n \"Using multidimensional scaling , STG spatial patterns during listening showed clustering according to three high-order acoustic features ( Figure 4e ) : voiced plosives ( /b/ , /d/ , /g/ ) , unvoiced plosives ( /p/ , /t/ , /k/ ) , and fricatives ( /s/ , /sh/ ) ( Ladefoged and Johnson , 2010 ) .\",\n \"This is consistent with the relational organization derived by analysis of structure in the stimulus acoustics ( Figure 4—figure supplement 3a ) , and the structure of STG during speaking ( Figure 4—figure supplement 3b ) .\",\n \"With the same analyses , we observed that activity in motor cortex clustered into the same three acoustic features ( Figure 4f , note this panel is identical to Figure 4c simply re-colored ) .\",\n \"Unsupervised K-means clustering analysis confirmed that vSMC activity , during listening , organized into these linguistically defined acoustic feature groups , but was significantly weaker than the organization of STG ( p<0 . 001 , Wilcoxon rank-sum , Figure 4g ) .\",\n \"Importantly , however , clustering by acoustic manner features was significantly stronger than clustering by place features in vSMC electrodes during listening ( p<0 . 001 , Wilcoxon rank-sum , Figure 4h ) .\",\n \"This organization suggests that motor cortex activity during speech perception reflects an acoustic sensory representation of speech in the vSMC that mirrors acoustic representations of speech in auditory cortex .\",\n \"To further define the acoustic selectivity and tuning of vSMC motor electrodes , participants listened to natural , continuous speech samples from a corpus with a range of American English speakers ( Garofolo et al . , 1993 ) .\",\n \"We fit spectrotemporal receptive field ( STRF ) models for each vSMC electrode using normalized reverse correlation ( see Materials and methods ) , which describes the spectrotemporal properties of speech acoustics that predict the activity of a single site in motor cortex .\",\n \"To compute the STRF , we calculate the correlation between the neural response at an electrode and the stimulus spectrogram at multiple time lags .\",\n \"The result is then normalized by the auto-correlation in the stimulus .\",\n \"This results in a linear filter for each electrode ( the STRF ) , which , when convolved with the stimulus spectrogram , produces a predicted neural response to that stimulus .\",\n \"The prediction performance of each STRF was determined by calculating the correlation between the activity predicted by the STRF and the actual response on held out data .\",\n \"A fraction of vSMC sites ( 16/98 sites total ) were reasonably well-predicted with a linear STRF ( r>=0 . 10 and p<0 . 01 , permutation test ) ( Theunissen et al . , 2001 ) .\",\n \"STRFs with significant correlation coefficients were localized to superior and inferior vSMC ( primarily precentral gyrus ) in addition to STG ( Figure 5a ) .\",\n \"Still , the prediction performance of STRFs in vSMC was generally lower than that of the STG ( Figure 5b ) .\",\n \"Furthermore , the majority of STRFs in both regions showed strong low frequency tuning ( 100–200 Hz ) properties related to voicing ( Figure 5c ) , though some also showed high frequency tuning consistent with selectivity for fricatives and stop consonants by visual inspection ( Mesgarani et al . , 2014 ) .\",\n \"We also estimated the mean cortical response at each motor site to every phoneme in English and found a diverse set of responses ( Figure 5—figure supplement 1a ) that were notably weaker in magnitude compared to STG responses ( Figure 5—figure supplement 1b ) .\",\n \"Weak selectivity to phonetic features measured by the Phoneme Selectivity Index ( PSI ) was also observed ( Figure 5—figure supplement 1c ) ( Mesgarani et al . , 2014 ) .\",\n \"These findings reveal that individual sites in motor cortex reflect sensory responses to definable spectrotemporal features speech acoustics , including voicing attributes .\",\n \"Presumably , this tuning gives rise to the acoustic organization found in the previous analysis of distributed spatial patterns of neural activity . 10 . 7554/eLife . 12577 . 013Figure 5 . Acoustic spectrotemporal tuning in vSMC .\",\n \"( a ) All STRF correlations and locations are plotted with opacity signifying the strength of the correlation .\",\n \"CS denotes the central sulcus; SF denotes the Sylvian fissure .\",\n \"( b ) Distribution of STRF prediction correlations for significantly active vSMC and STG sites .\",\n \"Cut-off at r = 0 . 1 is shown as a dashed line .\",\n \"( c ) Individual STRFs from all subjects ( S1-S5 , STRF correlation>0 . 1 ) plotted as a function of distance from the central sulcus and Sylvian fissure , with opacity signifying the strength of the STRF correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 01310 . 7554/eLife . 12577 . 014Figure 5—figure supplement 1 . Summary and comparison of vSMC and STG responses to syllables .\",\n \"( a ) Average responses at sample electrodes to all English phonemes and their PSI vectors .\",\n \"( b ) Peak high-gamma z-score distributions for significantly active vSMC and STG sites when listening ( p<0 . 01 , comparing silence to stimulus activity ) .\",\n \"( c ) Average PSI distributions for significantly active vSMC and STG sites . DOI: http://dx . doi . org/10 . 7554/eLife . 12577 . 014\"\n ],\n [\n \"Our principal objective was to determine the vSMC motor cortex representation of auditory speech sounds .\",\n \"We used high-resolution cortical recordings and a wide array of speech sounds to determine how the vSMC structure of speech sounds compared to the structure of motor commands in vSMC and sensory processing in STG .\",\n \"We found evidence for both spatially local and distributed activity correlated to speech acoustics , which suggests an auditory representation of speech in motor cortex .\",\n \"The proposal that the motor cortex critically integrates observations with motor commands largely stems from the discovery of mirror neurons ( in area F5 of macaques ) that fire both when a monkey produced an action and observed a similar action ( di Pellegrino et al . , 1992; Rizzolatti and Craighero , 2004; Pulvermüller and Fadiga , 2010 ) .\",\n \"This 'integrative' view is reminiscent of linguistic production-referencing theories , including the motor theory of speech perception , which propose that motor circuits are involved in speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) .\",\n \"In line with these theories , human neuroimaging studies have showed mirror activity in ventral premotor cortex during listening ( Wilson et al . , 2004; Pulvermüller et al . , 2006; Edwards et al . , 2010 ) , and modulated premotor activity in phoneme categorization tasks ( Alho et al . , 2012; Chevillet et al . , 2013 ) .\",\n \"Our results extend these findings by detailing the representational selectivity and encoding of vSMC in perception .\",\n \"Consistent with previous findings , we demonstrated local ‘audiomotor’ responses to speech sounds in vSMC .\",\n \"When the responses were further examined for phonetic structure , we found major motor articulatory place features , such as labial , alveolar , and velar , were not represented with single site activity or distributed spatial activity .\",\n \"This observation is in direct contrast with structural predictions made by the original motor theory of speech perception ( Liberman et al . , 1967; Liberman and Mattingly , 1985 ) , while confirming that motor cortex plays a role in perception ( Lindblom , 1996; Hickok and Poeppel , 2007 ) .\",\n \"We localized activity during speech perception to regions of the vSMC that have been implicated in phonation and laryngeal control ( Penfield and Boldrey , 1937; Brown et al . , 2008 ) .\",\n \"When listening to speech , we observed these regions reflected acoustic sensory properties of speech , with individual sites tuned for spectrotemporal acoustic properties .\",\n \"The tuning properties of responsive sites in vSMC are similar to properties observed in STG during listening ( Mesgarani et al . , 2014 ) and appear to give rise to an acoustic sensory organization of speech sounds ( rather than purely motor organization ) in motor cortex during listening .\",\n \"There is an emerging consensus that frontal and motor regions are recruited during effortful listening ( Du et al . , 2014 ) .\",\n \"For example , previous studies have demonstrated that frontal areas come online to process degraded speech for the attentional enhancement of auditory processing ( Wild et al . , 2012 ) .\",\n \"Our results may complement this interpretation in that the audiomotor cortex enhancement is specific to an auditory representation , without transforming information to a motor articulatory representation .\",\n \"That being said , the auditory encoding that we observed in the motor cortex did not appear to be as strong as that as that observed in the STG , and exhibited comparatively weaker activity and weaker phoneme selectivity ( Figure 5—figure supplement 1b and c , and see ( Mesgarani et al . , 2014 ) .\",\n \"In addition to having implications for perceptual models , we speculate that these results have strong implications for speech production , as auditory feedback is potentially processed directly in the vSMC in addition to the canonical auditory cortex .\",\n \"Speech production models currently propose a complex role for sensory feedback , where pathways exist for the activation of auditory cortex from vSMC activation ( the forward prediction of production consequences ) , and the activation of vSMC from auditory and somatosensory input ( the error correction signal ) ( Guenther et al . , 2006; Houde and Nagarajan , 2011 ) .\",\n \"In the current study , it appears that the motor cortex contains both sensory and motor representations , where the sensory representations are active during passive listening , whereas motor representations dominate during speech production .\",\n \"Analysis of the time course of vSMC and STG responses revealed a heterogeneous population of both short- and longer-latencies in the inferior and superior vSMC that are generally slower than the STG ( Figure 3a–c ) .\",\n \"Early responses in vSMC may reflect bidirectional connections from STG ( Zatorre et al . , 2007 ) , primary auditory cortex ( Nelson et al . , 2013; Schneider et al . , 2014 ) or auditory thalamus ( Henschke et al . , 2014 ) , whereas later responses might reflect indirect connectivity in areas downstream from the STG ( Rauschecker and Scott , 2009 ) .\",\n \"Indeed , our cross-correlation analysis revealed bidirectional dynamical relationships between vSMC and STG responses , in which STG responses led vSMC responses and vice versa ( Figure 3d–f ) .\",\n \"Still , this analysis was independent of the diverse tuning properties in the vSMC and STG electrode sets , so longer latency responses likely reflect the later responses to vowels relative to consonants .\",\n \"Even so , we found a wide variety of tuning and dynamical profiles in the vSMC electrodes that responded during listening .\",\n \"Given these proposed functional connections , activity in vSMC from speech sounds may be a consequence of sounds activating the sensory feedback circuit ( Hickok et al . , 2011 ) .\",\n \"Alternatively , evoked responses in the motor cortex during passive listening may directly reflect auditory inputs arising from aggregated activity picked up by the electrode .\",\n \"We believe the latter scenario to be less likely , however , given that auditory responses were observed in dorsal vSMC on electrode contacts several centimeters away from auditory inputs in the STG .\",\n \"In addition , the spatial spread of neural signals in the high gamma range is substantially smaller than this difference – high gamma signal correlations at <2 mm spacing are only around r=0 . 5 , and at distances of 1 cm reach a noise floor ( Chang , 2015 ) ; Muller et al , unpublished findings ) .\",\n \"Given the observed acoustic rather than place selectivity observed during listening in the vSMC , our results suggest that motor theories of speech perception may need to be revised to incorporate a novel sensorimotor representation of sound in the vSMC .\"\n ],\n [\n \"Nine human participants were implanted with high-density multi-electrode cortical surface arrays as part of their clinical evaluation for epilepsy surgery .\",\n \"The array contained 256 electrodes with 4 mm pitch .\",\n \"Arrays were implanted on the lateral left hemispheres over the peri-Sylvian cortex , but exact placement was determined entirely by clinical indications ( Figure 1—figure supplement 1 and Figure 1—figure supplement 2 ) .\",\n \"Using anatomic image fusion software from BrainLab ( Munich , Germany ) , electrode positions were extracted from the computed tomography ( CT ) scan , co-registered with the patient’s MRI and then superimposed on the participant’s 3D MRI surface reconstruction image .\",\n \"All participants were left hemisphere language dominant , as assessed by the Wada test .\",\n \"Participants had self-reported normal hearing .\",\n \"The study protocol was approved by the UC San Francisco Committee on Human Research , and all participants provided written informed consent .\",\n \"Participants completed three separate tasks that were designed to sample a range of phonetic features .\",\n \"First , participants listened to eight consonant-vowel ( CV ) syllables ( /ba/ , /da/ , /ga/ , /pa/ , /ta/ , /ka/ , /∫a/ , /sa/ ) produced by a male speaker unknown to the participant .\",\n \"Stimuli were presented randomly , with 4–21 repetitions of each CV syllable for 5 out of the 9 subjects included in all subsequent analyses , and one repetition of each CV syllable for 4 subjects shown only in Figure 1—figure supplement 2 .\",\n \"To remain alert , participants were asked to identify the syllable they heard by selecting from a multiple-choice question on a computer with their ipsilateral ( left ) hand .\",\n \"In the second task , participants spoke aloud the same CV syllables prompted by a visual cue on the laptop computer display .\",\n \"In the third task , participants passively listened to natural speech samples from a phonemically transcribed continuous speech corpus ( TIMIT ) .\",\n \"We chose 499 unique sentences from 400 different male and female speakers .\",\n \"Each sentence was repeated two times .\",\n \"For the phoneme selectivity analysis , we chose a subset of TIMIT phonemes that occurred more than 30 times .\",\n \"This resulted in an analysis of 33 phonemes .\",\n \"For spectrotemporal receptive field analysis ( see below ) , data from all sentences were used .\",\n \"Electrocorticographic ( ECoG ) signals were recorded with a multichannel PZ2 amplifier connected to an RZ2 digital signal acquisition system ( Tucker-Davis Technologies , Alachua , FL , USA ) sampling at 3 , 052 Hz .\",\n \"The produced speech was recorded with a microphone , digitized , and simultaneously recorded .\",\n \"The speech sound signals were presented monaurally from loudspeakers at a comfortable level , digitized , and also simultaneously recorded with the ECoG signals .\",\n \"Line noise ( 60 Hz and harmonics at 120 and 180 Hz ) was next removed from the signal with a notch filter .\",\n \"Each time series was visually and quantitatively inspected for excessive noise , and was subsequently removed from further analyses if its periodogram deviated more than two standard deviations away from the average periodogram of all other time series .\",\n \"The remaining time series were then common-average referenced ( CAR ) and used for analyses .\",\n \"The CAR was taken across 16 channel banks in order to remove non-neural electrical noise from shared inputs to the PZ2 .\",\n \"We find that this method of CAR significantly reduces movement-related and other non-neural artifacts while not adversely affecting our signals of interest .\",\n \"The analytic amplitude of each time series was extracted using eight bandpass filters ( Gaussian filters , logarithmically increasing center frequencies ( 70–150 Hz ) with semi-logarithmically increasing bandwidths ) with the Hilbert transform .\",\n \"The high-gamma power was calculated by averaging the analytic amplitude across these eight bands , and downsampling the signal to 100 Hz .\",\n \"The signal was finally z-scored relative to the mean and standard deviation of baseline rest data for each channel .\",\n \"Supra-Sylvian cortical sites with robust evoked responses to both speech sounds and speech production were selected for this analysis .\",\n \"To identify if a site was responsive to speech sounds , we implemented a bootstrap t-test comparing a site's responses randomly sampled over time during speech sound presentations to responses randomly sampled over time during pre-stimulus silent intervals ( p<0 . 01 ) .\",\n \"This resulted in 10 , 22 , 29 , 27 , and 27 sites for the five participants ( n=115 ) .\",\n \"Next we implemented a bootstrap t-test comparing neural responses during speech production and pre-stimulus silence ( p<0 . 01 ) , resulting in 25 , 74 , 87 , 92 , and 84 sites ( n=362 ) .\",\n \"Finally , we took the intersection of these two groups to arrive at our final supra-Sylvian sites set of 8 , 16 , 28 , 22 , and 24 sites active during listening and speaking ( n=98 ) .\",\n \"To analyze the responses of the auditory cortex , we restricted the infra-Sylvian cortical sites to those that were reliably evoked by speech sounds ( p<0 . 01 , t-test between silence and speech sounds neural responses ) .\",\n \"This resulted in 73 , 61 , 40 , 77 , and 89 infra-Sylvian temporal cortical sites ( n=340 ) responsive to speech sounds .\",\n \"To investigate the degree of spatial clustering in the vSMC electrodes responsive during listening , we used the Dip-means method ( Kalogeratos and Likas , 2012 ) , which allows us to test whether data shows any form of clustering .\",\n \"Importantly , unlike the silhouette index , this allows us to distinguish between k=1 and k>1 clusters .\",\n \"For each subject , the pairwise distances between the spatial locations of all electrodes in a single subject were computed .\",\n \"Using each electrode in turn as a 'viewer' ( Kalogeratos and Likas , 2012 ) , we tested to see whether the distribution of distances to that electrode significantly deviated from unimodality ( Hartigan and Hartigan , 1985 ) .\",\n \"If one or more electrodes showed a signficantly non-unimodal pairwise distance histogram , then the data were considered to be clustered .\",\n \"Following this procedure , k-means clustering was performed with k=2 through k=6 clusters , and the silhouette index was used to determine the best number of clusters for a given subject .\",\n \"The silhouette index for a given data point is defined ass ( i ) =b ( i ) −aimax{a ( i ) , b ( i ) } where b ( i ) is the lowest average distance of i to any other cluster of which i is not a member , and a ( i ) is the average distance between i and any other data point assigned to the same cluster .\",\n \"The silhouette index ranges from −1 to 1 , with higher positive values indicating good clustering .\",\n \"For the speaking and listening CV syllable tasks , the start of the syllable acoustics was used to align the responses of each electrode site .\",\n \"For the phoneme responses , the TIMIT phonetic transcriptions were used to align responses to the phoneme onset .\",\n \"Once responses were aligned to a stimulus , the average activity for each site to each stimulus was measured by taking the mean response over different trials of the same stimuli .\",\n \"The maximum of the mean responses to different stimuli were then used to measure the peak-high gamma distributions between different tasks and sites .\",\n \"We measured the onset latencies for responses to listening in STG and vSMC by calculating the average z-scored high gamma activity across all CV syllables , and then calculating the first time at which activity was significantly higher than the 500-ms pre-stimulus silent rest period ( one-tailed Wilcoxon rank sum test , p<0 . 001 ) .\",\n \"We also calculated the peak latency as the time at which the average z-scored response reached its maximum value .\",\n \"Differences in onset and peak latencies were compared across STG , inferior , and superior vSMC using the a two-tailed Wilcoxon rank sum test at a significance level of p<0 . 05 ( uncorrected ) .\",\n \"To measure the timing/dynamics between pairs of vSMC and STG sites during CV syllable listening , we performed a cross-correlation analysis between pairs of electrodes in these two regions .\",\n \"The cross-correlation measures the similarity of two time series at different time lags by taking pairs of electrode responses and calculating the correlation between one response and a time-shifted version of the second response .\",\n \"If the peak in the cross-correlation between an STG electrode and a vSMC electrode occurs at a negative lag , this indicates that the STG response leads ( occurs earlier than ) the vSMC response and that STG activity in the past is predictive of future activity in the vSMC .\",\n \"In contrast , if the peak in the cross-correlation between an STG electrode and a vSMC electrode occurs at a positive lag , this indicates that the vSMC response leads ( occurs earlier than ) the STG response .\",\n \"The cross-correlation at time lag τ is calculated between the response at an STG electrode ( denoted\",\n \"x ) and the response at a vSMC electrode\",\n \"( y ) as follows:x*y[τ] = def∑t=-0 . 5 st=1 sx*[t]y[t+τ] Where the maximum lag τ was chosen to be 0 . 75 s .\",\n \"Cross-correlations were normalized by 1M−|τ| ( where M is the total number of time points in the response ) to obtain an unbiased estimate at each time lag τ .\",\n \"The cross-correlation between vSMC and STG electrodes was calculated separately for each CV syllable trial , and then averaged across trials ( see examples in Figure 3d ) .\",\n \"To determine the incidence of relationships within our electrode population where STG leads vSMC , vSMC leads STG , or both are coactive , we calculated an asymmetry index .\",\n \"This index ranges from −1 to 1 and describes the relative power in the positive versus negative lags for each vSMC electrode .\",\n \"It is calculated for each vSMC electrode by taking the sum of the positive cross-correlations in the negative lags and the sum of the positive cross-correlations in the positive lags , and then computing the ratio:asymmetry index = Ppos−PnegPpos+Pneg For a given vSMC electrode , an asymmetry index of −1 indicates that the cross-correlations lie fully in the negative lags ( indicating that STG responses lead the vSMC response in that electrode ) .\",\n \"In contrast , a value of 1 indicates that the cross-correlations are in the positive lags only , indicating that the vSMC electrode leads all STG electrodes .\",\n \"To examine the relational organization of the neural responses to syllables , we applied unsupervised multidimensional scaling ( MDS ) to the distance matrix of the mean neural responses at the sites of interest described in Materials and methods: Electrode selection .\",\n \"For analysis of speaking and listening responses , the vSMC sites used were those identified as significantly active during both speech production and speech perception ( n=98 , Figure 4b , c , f ) .\",\n \"However , clustering results for speaking were similar when all vSMC sites identified as significantly active during speech production were included ( n=362 , Figure 4—figure supplement 3c ) .\",\n \"The STG sites used were those identified as significantly active during speech perception ( n=340 , Figure 4e , Figure 4—figure supplement 3b ) .\",\n \"Syllables placed closer together in MDS space elicited similar neural response patterns , and those further apart from one another elicited more dissimilar patterns .\",\n \"To calculate the distance between a pair of mean neural responses , a mean neural response to one syllable was linearly correlated to another , and the resulting correlation coefficient was subtracted from 1 .\",\n \"We used unsupervised K-means clustering to examine the grouping of the mean neural activity to syllables of the electrodes of interest described in Methods: Electrode selection .\",\n \"We clustered the mean activity into 3 distinct clusters .\",\n \"This number of clusters was chosen because there are 3 major place of articulations and manner of articulations in the syllable stimuli set ( Figure 4a ) that have been shown to play a major role in the neural organization of motor cortex during speech production and auditory cortex during speech perception .\",\n \"After clustering the neural responses into three distinct groups , we measured the similarity of the grouping to the linguistically defined grouping of consonants by place of articulation and acoustic features ( Figure 4a and Figure 2—figure supplement 1 ) using the adjusted Rand Index ( RIadj ) .\",\n \"The RIadj is frequently used in statistics for cluster validation .\",\n \"It measures the amount of agreement between two clustering schemes: one by a given clustering process ( e . g . K-means ) , and the other by some external criteria , or gold-standard ( e . g . place of articulation linguistic features ) .\",\n \"The RIadj takes an intuitive approach to measuring cluster similarity by counting the number of pairs of objects classified in the same cluster under both clustering schemes , and controlling for chance ( hence , 'adjusted' RI ) .\",\n \"It has an expected value of 0 for independent clusterings , and a maximum value of 1 for identical clustering .\",\n \"It is defined as the following: Let S be a set of n objects , S = ( o1 , o2 , … , on ) .\",\n \"Partitioning the objects in two different ways such that U = ( U1 , … , Ur ) is a partition of S into r subsets , and V = ( V1 , … , Vt ) is a partition of S into t subsets , let: a = number of pair of objects that are in the same set in U and in the same set in V , b = number of pair of objects that are in the same set in U and in different sets in V , c = number of pair of objects that are in different sets in U and in the same set in V , d = number of pair of objects that are in different sets in U and in different sets in V . Without adjusting for chance , the RI is simply:RI = a+da+b+c+d=a+dn2 .\",\n \"Taking into account chance pairings , RIadj becomes:RIadj = n2 ( a+d ) - ( a+b ) ( a+c ) + ( c+d ) ( b+d ) n22- ( a+b ) ( a+c ) + ( c+d ) ( b+d ) .\",\n \"To localize an unbiased time window for analysis , the ΔRIadj metric was derived for all time windows by subtracting the RIadj measured with the place of articulation features gold-standard from the RIadj measured with the acoustic feature gold-standard ( Figure 4—figure supplement 1 ) .\",\n \"An ΔRIadj = 1 denotes organization by acoustic features , and an ΔRIadj = −1 denotes organization by place features .\",\n \"The significance of the ΔRIadj was computed by calculating the RIadj for a randomized labeling of neural responses compared to either acoustic feature or place feature clustering , taking the difference ( ∆RIadj ) , and repeating this procedure 1000 times with different randomized labelings to create a null distribution of ∆RIadj values .\",\n \"The p-value was calculated as the number of times this random ∆RIadj exceeded the observed ∆RIadj , and was thresholded at an FDR-corrected p<0 . 05 using the Benjamini-Hochberg procedure ( Benjamini and Hochberg , 1995 ) .\",\n \"To characterize the phoneme selectivity of each electrode site , we implemented the PSI calculation described by Mesgarani et al . , 2014 .\",\n \"In short , for a single site , we summed the number of responses that were statistically different ( Wilcoxon rank-sum test , p<0 . 01 , corrected for multiple comparisons ) from the response to a particular phoneme .\",\n \"This resulted in a PSI that ranges from 0 to 32 , where a PSI = 32 is an extremely selective electrode and a PSI = 0 is not selective .\",\n \"A PSI describes an electrode’s selectivity to one phoneme , and a vector of PSIs describes an electrode’s selectivity profile to all phonemes .\",\n \"The spectrotemporal representation of speech sounds was first estimated using a cochlear frequency model , consisting of a bank of logarithmically spaced constant Q asymmetric filters .\",\n \"The filter bank output was subjected to nonlinear compression , followed by a first order derivative along the spectral axis modeling a lateral inhibitory network , and an envelope estimation operation ( Wang and Shamma , 1994 ) .\",\n \"This resulted in a two dimensional spectrotemporal representation ( spectrogram ) of speech sounds simulating the pattern of activity on the auditory nerve .\",\n \"We then estimated the spectrotemporal receptive fields ( STRFs ) of the sites from passive listening to TIMIT using normalized reverse correlation ( Aertsen and Johannesma , 1981; Klein et al . , 2000; Theunissen et al . , 2001; Woolley et al . , 2006 ) between spectrotemporal representation of the sentences and the evoked neural activity ( STRFLab software package: http://strflab . berkeley . edu , DirectFit routine ) .\",\n \"The STRF is a linear filter that describes which combinations of spectrotemporal features will elicit a neural response in a given electrode .\",\n \"The relationship between the STRF , H , stimulus spectrogram , S ( as estimated above ) , and the predicted response , r^ ( t ) , of an electrode are given by the following equation:r^ ( t ) =∑i=0M-1 ∑τ=0N-1 H ( τ , f ) S ( t -τ , f ) where N is the number of delays of length τ after which the STRF will be estimated ( reflecting memory for the stimulus ) , and M is the number of frequency bands in the spectrogram .\",\n \"To estimate the STRF , we minimize the mean squared error between the predicted and observed responses .\",\n \"To prevent overfitting , we used an L2 regularization procedure in which a ridge hyperparameter and sparseness hyperparameter were calculated for each electrode's STRF ( details in [Woolley et al . , 2006] ) .\",\n \"The ridge hyperparameter acts as a smoothing factor on the STRF , whereas the sparseness hyperparameter controls the number of non-zero weights in the STRF .\",\n \"These hyperparameters were optimized with a systematic hyperparameter grid search maximizing for mutual information ( bits/s ) .\",\n \"With the optimized hyperparameters , we calculated the final STRF and correlation between the predicted and actual neural response using cross-validation .\",\n \"To do this , a STRF was derived using 9/10 of the stimuli-response pairs , and the Pearson correlation coefficient ( indicating the STRF goodness-of-fit ) was measured by predicting the remaining one-tenth responses .\",\n \"This was repeated 10 times with 10 non-overlapping stimuli-response pair sets .\",\n \"The final STRF and correlation number were derived by averaging the 10 STRFs and correlation coefficients .\",\n \"To assess statistical differences , we used independent sample t-tests when the data were found not to deviate significantly from normality ( KS test ) .\",\n \"When data were not normally distributed , we used the nonparametric Wilcoxon rank sum test .\",\n \"In some cases , a bootstrap t-test was used .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["In humans , listening to speech evokes neural responses in the motor cortex .","This has been controversially interpreted as evidence that speech sounds are processed as articulatory gestures .","However , it is unclear what information is actually encoded by such neural activity .","We used high-density direct human cortical recordings while participants spoke and listened to speech sounds .","Motor cortex neural patterns during listening were substantially different than during articulation of the same sounds .","During listening , we observed neural activity in the superior and inferior regions of ventral motor cortex .","During speaking , responses were distributed throughout somatotopic representations of speech articulators in motor cortex .","The structure of responses in motor cortex during listening was organized along acoustic features similar to auditory cortex , rather than along articulatory features as during speaking .","Motor cortex does not contain articulatory representations of perceived actions in speech , but rather , represents auditory vocal information ."],"string":"[\n \"In humans , listening to speech evokes neural responses in the motor cortex .\",\n \"This has been controversially interpreted as evidence that speech sounds are processed as articulatory gestures .\",\n \"However , it is unclear what information is actually encoded by such neural activity .\",\n \"We used high-density direct human cortical recordings while participants spoke and listened to speech sounds .\",\n \"Motor cortex neural patterns during listening were substantially different than during articulation of the same sounds .\",\n \"During listening , we observed neural activity in the superior and inferior regions of ventral motor cortex .\",\n \"During speaking , responses were distributed throughout somatotopic representations of speech articulators in motor cortex .\",\n \"The structure of responses in motor cortex during listening was organized along acoustic features similar to auditory cortex , rather than along articulatory features as during speaking .\",\n \"Motor cortex does not contain articulatory representations of perceived actions in speech , but rather , represents auditory vocal information .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["When we speak , we force air out of our lungs so that it passes over the vocal cords and causes them to vibrate .","Movements of the jaw , lips and tongue can then shape the resulting sound wave into speech sounds .","The brain’s outer layer , which is called the cortex , controls this process .","More precisely , neighboring areas in the so-called motor cortex trigger the movements in a specific order to produce different sounds .","Brain imaging experiments have also shown that the motor cortex is active when we listen to speech , as well as when we produce it .","One theory is that when we hear a sound , such as the consonant ‘b’ , the sound activates the same areas of motor cortex as those involved in producing that sound .","This could help us to recognize and understand the sounds we hear .","To test this theory , Cheung , Hamilton et al . studied how speech sounds activate the motor cortex by recording electrical signals directly from the brain’s surface in nine human volunteers who were undergoing a clinical evaluation for epilepsy surgery .","This revealed that speaking activates many different areas of motor cortex .","However , listening to the same sounds activates only a small subset of these areas .","Contrary to what was thought , brain activity patterns in motor cortex during listening do not match those during speaking .","Instead , they depend on the properties of the sounds .","Thus , sounds that have similar acoustic properties but which require different movements to produce them , such as ‘b’ and ‘d’ , activate the motor cortex in similar ways during listening , but not during speaking .","Further research is now needed to work out why the motor cortex behaves differently when we hear as opposed to when we speak .","Previous work has suggested that the region increases its activity during listening when the sounds heard are unclear , for example because of background noise .","One testable idea therefore is that the motor cortex helps to enhance the processing of degraded sounds ."],"string":"[\n \"When we speak , we force air out of our lungs so that it passes over the vocal cords and causes them to vibrate .\",\n \"Movements of the jaw , lips and tongue can then shape the resulting sound wave into speech sounds .\",\n \"The brain’s outer layer , which is called the cortex , controls this process .\",\n \"More precisely , neighboring areas in the so-called motor cortex trigger the movements in a specific order to produce different sounds .\",\n \"Brain imaging experiments have also shown that the motor cortex is active when we listen to speech , as well as when we produce it .\",\n \"One theory is that when we hear a sound , such as the consonant ‘b’ , the sound activates the same areas of motor cortex as those involved in producing that sound .\",\n \"This could help us to recognize and understand the sounds we hear .\",\n \"To test this theory , Cheung , Hamilton et al . studied how speech sounds activate the motor cortex by recording electrical signals directly from the brain’s surface in nine human volunteers who were undergoing a clinical evaluation for epilepsy surgery .\",\n \"This revealed that speaking activates many different areas of motor cortex .\",\n \"However , listening to the same sounds activates only a small subset of these areas .\",\n \"Contrary to what was thought , brain activity patterns in motor cortex during listening do not match those during speaking .\",\n \"Instead , they depend on the properties of the sounds .\",\n \"Thus , sounds that have similar acoustic properties but which require different movements to produce them , such as ‘b’ and ‘d’ , activate the motor cortex in similar ways during listening , but not during speaking .\",\n \"Further research is now needed to work out why the motor cortex behaves differently when we hear as opposed to when we speak .\",\n \"Previous work has suggested that the region increases its activity during listening when the sounds heard are unclear , for example because of background noise .\",\n \"One testable idea therefore is that the motor cortex helps to enhance the processing of degraded sounds .\"\n]","isConversational":false},"year":{"kind":"string","value":"2016"}}},{"rowIdx":296,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["biochemistry and chemical biology","microbiology and infectious disease"],"string":"[\n \"biochemistry and chemical biology\",\n \"microbiology and infectious disease\"\n]","isConversational":false},"title":{"kind":"string","value":"Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals"},"id":{"kind":"string","value":"elife-07861-v3"},"sections":{"kind":"list like","value":[["Fungal secondary metabolites ( SMs ) are often encoded by gene clusters and have been the subject of intensive analysis in recent years .","This has resulted from the large number of completed fungal genomes as well as the intrinsic interest of SMs as environmental/pathogenic agents and their potential for pharmacological use .","However , there are few examples where the regulation of the expression of these clusters has been shown to be ecologically/physiologically relevant ( Rohlfs and Churchill , 2011 ) .","Except for penicillin and perhaps aflatoxin and gliotoxin , the potential real-life functions of most SMs are unknown ( Bhatnagar et al . , 2006; Vargas et al . , 2014 ) .","Most clusters contain a pathway-specific transcription factor , and the common approach is to artificially over-express the cluster genes by constitutive expression of this transcription factor and chemically characterise the product ( Yin and Keller , 2011 ) .","In most cases the relevance of the SM is not known .","In this study the biological function of terrein from the filamentous ascomycete Aspergillus terreus was investigated .","A . terreus is a filamentous ascomycete of biotechnological and medical importance since it produces itaconic acid ( Steiger et al . , 2013 ) and lovastatin ( Bizukojc and Ledakowicz , 2015 ) , but it is also a causative agent of life-threatening invasive aspergillosis in immunocompromised patients ( Baddley et al . , 2003; Slesiona et al . , 2012b ) and has recently been described as a pathogen of potato leaves ( Louis et al . , 2013 ) .","While searching for a candidate protein required for pigment synthesis in A . terreus conidia , we serendipitously identified the gene cluster responsible for terrein production ( Zaehle et al . , 2014 ) , which is the major SM formed by A . terreus .","Terrein production is highly pronounced on sugar-rich plant-derived media such as potato dextrose broth ( PDB ) ( Zaehle et al . , 2014 ) and shows phytotoxic activities such as inhibition of seed germination and lesion formation on fruit surfaces ( Kamata et al . , 1983; Zaehle et al . , 2014 ) .","The terrein biosynthesis gene cluster consists of 11 genes ( terA–J , terR ) , whereby terA encodes the key enzyme , which is a non-reducing polyketide synthase ( Zaehle et al . , 2014 ) , and terR codes for the transcriptional activator TerR containing a GAL4-type Zn2Cys6 binuclear cluster DNA-binding domain ( Gressler et al . , 2015a ) , as frequently found in transcriptional activators of fungal SM gene clusters .","Although activation of cluster genes is strictly TerR-dependent , no signals that lead to TerR activation under in vivo conditions have yet been identified .","Since terrein is the major SM produced by A . terreus—gram scales are easily achieved—we assumed a benefit from its production in the natural habitat .","To elucidate this question , a detailed knowledge on the inducing factors stimulating terrein production and analyses of its biological activities were required .","Therefore , we aimed to investigate environmental signals that result in terR expression and , eventually , the production of terrein .","From these analyses , the impact of different global transcription factors on cluster induction was deduced .","Two global transcription factors sense the quality and availability of nitrogen sources and specifically respond to the plant and fruit environment .","Additionally , the iron responsive regulon plays a vital role in cluster induction , which indicates a specific contribution of terrein in modulating iron homeostasis ."],["In previous studies we showed that terrein is produced on plant-derived media such as PDB , which is in agreement with its phytotoxic biological activity ( Zaehle et al . , 2014 ) .","To address the question of specific conditions that induce the gene cluster , an A . terreus reporter strain was generated expressing the β-galactosidase gene lacZ from Escherichia coli under control of the terrein synthase promoter PterA ( PterA:lacZ ) .","Due to the dependence of PterA expression on TerR , this strain served as a direct indicator of terR expression and terrein production .","In agreement with a lack of terrein production , β-galactosidase activity was near the detection limit when the A . terreus PterA:lacZ strain was grown on glucose minimal medium .","In contrast , and in agreement with previous observations , a 200–500 fold induction was observed on PDB medium ( Figure 1A ) .","Similarly , on Sabouraud and yeast extract-peptone-dextrose ( YPD ) medium , induction levels reached 10–30% compared with PDB .","However , potato broth or casamino acids did not induce the cluster without the addition of glucose , indicating that glucose appears to be required for terrein production rather than repressing gene cluster induction as shown for other SM gene clusters ( Theilgaard et al . , 1997; Brakhage et al . , 2004; Gressler et al . , 2011 ) .","Indeed , when glucose medium was supplemented with 1% casamino acids as the nitrogen source , a 20–30 fold activation was detected .","Since terrein can cause lesions on fruit surfaces and inhibits plant seed germination ( Kamata et al . , 1983; Zaehle et al . , 2014 ) , we assumed that sugar-rich fruit and root juices might have a strong stimulatory capacity .","Therefore , we cultivated the reporter strain on banana , carrot , peach , and apple juice .","β-Galactosidase activities from these media exceeded the activity of the already strong inducing PDB medium ( Figure 1B ) by a factor of at least five .","Additionally , in subsequent LC analyses of culture extracts , a distinct ultraviolet signal for terrein was detected ( Figure 1—figure supplement 1A–D ) .","This led us to infect fresh bananas with the A . terreus ΔakuB strain ( the parental strain for gene deletions; Gressler et al . , 2011 ) , a ΔterR mutant lacking the transcriptional activator , and a ΔterA deletion mutant that lacks the key polyketide synthase from the cluster . 10 . 7554/eLife . 07861 . 003Figure 1 . Terrein production and expression of terA on plant-derived media .","( A ) Promoter activity of strain PterA:lacZ after 48 hr and 72 hr on minimal media supplemented with glucose ( Gluc ) , casamino acids ( CA ) , or glucose + casamino acids ( Gluc CA ) or the complex media Sabouraud ( Sab ) , yeast extract-peptone-dextrose ( YPD ) , potato dextrose broth ( PDB ) or potato extract ( PB ) .","( B ) Promoter activity of strain PterA:lacZ grown for 24 , 36 and 48 hr in carrot , banana , apple , and peach juice .","( C ) High performance liquid chromatography analysis of banana extracts infected with Aspergillus terreus SBUG844 strains ΔakuB , ΔakuBΔterA or ΔakuBΔterR .","A mock-infected fruit served as control .","1—terrein .","( D ) Lesion formation on banana surfaces caused by extracts shown in ( C ) .","Photographs were taken after 40 and 60 hr .","Lesions only occur with extracts from the wild-type and the parental strain of the mutants ( ΔakuB ) .","Numbers indicate the serial twofold dilution of the extracts starting from undiluted crude extracts down to 1:256 dilutions . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00310 . 7554/eLife . 07861 . 004Figure 1—figure supplement 1 . High performance liquid chromatography ( HPLC ) analysis of extracts from Aspergillus terreus strains after cultivation in fruit juices and from infected nectarines and apples .","( A–D )","HPLC analyses of culture extracts from fruit juices after inoculation with SBUG844_PterA:lacZ ( blue line ) and mock-inoculated juices that served as negative controls ( green line ) .","Culture supernatants were extracted 48 hr post inoculation: ( A ) carrot juice , ( B ) banana juice , ( C ) apple juice , ( D ) peach juice .","1 = terrein .","( E , F )","HPLC analyses of fruit extracts after infection with A . terreus wild-type SBUG844ΔakuB ( blue line ) and terrein biosynthesis gene cluster mutants ΔterA ( red ) and ΔterR ( violet ) .","Mock-infected fruits served as negative controls ( green line ) : ( E ) nectarine , ( F ) apple .","1 = terrein . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00410 . 7554/eLife . 07861 . 005Figure 1—figure supplement 2 . β-Galactosidase activity of PterA:lacZ grown in banana juice without ( control ) or with different supplementations .","+Fe = 30 µM FeCl3; +N = 70 mM NH4Cl; +Met = 10 mM L-methionine .","Tests were performed in biological triplicates that were analysed in technical triplicates .","Promoter activity of terA is reduced by nitrogen supplementation and is completely repressed in the presence of nitrogen and iron in the medium .","The addition of methionine partially overwrites this repression .","Statistical significance was calculated in comparison to the control condition without supplementation using the Student's paired t-test with a two-tailed distribution: **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 005 Ethyl acetate extraction of bananas infected with ΔakuB strain revealed high amounts of terrein , whereas no terrein was detected after infection with the ΔterR or ΔterA mutant ( Figure 1C ) .","Similar results were obtained when fresh apples or nectarines were infected with the three A . terreus strains ( Figure 1—figure supplement 1E , F ) .","Furthermore , the extracts of bananas infected with A . terreus wild-type ( and ΔakuB ) caused strong lesion formation on fresh banana peels , which was hardly observed with extracts of bananas infected with the mutants ( Figure 1D ) .","Taken together , these results confirm terrein production in a natural habitat of fruit infection , although the specific factors leading to terrein production still remain unclear .","Since casamino acid-supplemented glucose medium revealed moderate gene cluster activation ( Figure 1A ) , we assumed that specific amino acids could act as inducers .","Therefore , we tested the inducing effect of amino acids as sole nitrogen sources by preparing six distinct pools that covered all 20 canonical proteinogenic amino acids ( Figure 2—figure supplement 1 ) .","While most of the pools did not activate terA expression , the pool with aliphatic amino acids caused moderate induction and media containing the sulphur-rich amino acids cysteine and methionine strongly induced gene expression , especially after 72 hr of cultivation ( Figure 2—figure supplement 1 ) .","In the presence of the preferred nitrogen source glutamine , the induction by aliphatic amino acids was mainly lost .","Also , the activation by cysteine was low and strongly concentration-dependent ( Figure 2A ) .","In contrast , methionine provoked a significant induction even at low concentrations .","Therefore , we also analysed the effect of other sulphur-containing compounds in two concentrations ( Figure 2B ) .","However , no induction was observed when homocysteine , cystathionine , glutathione , dimethylsulfoxide , sulfate , or sulfide were tested .","This indicates that methionine itself rather than a sulphur source causes the induction . 10 . 7554/eLife . 07861 . 006Figure 2 . Methionine-dependent terA expression .","( A ) β-Galactosidase activity of SBUG844_PterA:lacZ after 48 hr of cultivation in glutamine-containing minimal media in combination with 5 , 10 , or 20 mM of the aliphatic ( Ala , Ile , Leu , Val ) or sulphur-containing amino acid ( Cys , Met ) .","Significance calculated against the glutamine control .","( B ) β-Galactosidase assay of SBUG844_PterA:lacZ in the presence of various sulphur sources .","Glutamines containing minimal media were supplemented with low ( 5 mM ) or high ( 10 mM ) concentrations of Na2SO4 , Na2S , methionine ( Met ) , cysteine ( Cys ) , homocysteine ( Hcy ) , reduced gluthathione ( GSH ) , and dimethylsulfoxide ( DMSO ) .","Cystathionine ( Cth ) was used in final concentrations of 1 and 3 mM .","Activity was determined after 48 hr of growth .","Significance calculated against medium supplemented with sodium sulfate .","All cultivations were performed in biological triplicates and activity determinations were made in technical duplicates .","Statistical significance was calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00610 . 7554/eLife . 07861 . 007Figure 2—figure supplement 1 . Amino acid-dependent terA expression . All cultivations were performed in biological triplicates and activity determinations were made in technical duplicates .","Statistical significance was calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 .","β-Galactosidase assay of SBUG844_PterA:lacZ grown in AMM ( -N ) G100 with 1% casamino acids ( CA ) or 50 mM of various amino acid pools as indicated in the table on the right .","The promoter activity of terA was determined after 48 and 72 hr .","Significance calculated against CA control . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 007 Methionine and PDB medium induced the terrein biosynthesis gene cluster to a similar extent , but remained approximately five times below that of banana juice .","This suggested that additional or alternative inducing signals might exist .","Fruit juices are rich in sugars and the C:N ratio in fruits is very high , which could result in severe nitrogen limitation at a later growth state .","Therefore , another set of experiments was performed in which the total concentration of amino acids was either high ( 50 mM ) or low ( 10 mM ) and PterA induction from the lacZ reporter strain was determined at different time points ( Figure 3A ) .","While high concentrations of amino acids or inorganic nitrogen sources such as nitrate or ammonia did not induce the cluster , low nitrogen contents resulted in a time-dependent 40–400 fold induction .","To correlate gene expression with nitrogen exhaustion , the nitrogen consumption from a medium supplemented with 10 mM ammonium chloride was monitored and cluster induction was simultaneously analysed .","Indeed , as soon as nitrogen levels reached the detection limit , cluster expression was strongly induced ( Figure 3B ) .","Subsequently , a culture shift experiment was performed in which mycelium was pre-grown in the presence of 70 mM ammonium chloride and shifted to medium either with or without a nitrogen source ( Figure 3C ) .","While no reporter activity and terrein formation was observed from cultures of the nitrogen-rich medium , high β-galactosidase activity accompanied by terrein accumulation was observed in the nitrogen starvation medium . 10 . 7554/eLife . 07861 . 008Figure 3 . Terrein biosynthesis gene cluster activation under nitrogen starvation .","( A ) β-Galactosidase activity of SBUG844_PterA:lacZ was cultivated for 24 , 36 , 48 , 72 , and 96 hr in glucose minimal medium supplemented with different concentrations of various nitrogen sources: 70 mM and 10 mM NaNO3 or NH4Cl , 1% or 0 . 1% of casamino acids ( CA ) , and 50 or 10 mM aspartate ( Asp ) , asparagine ( Asn ) , glutamate ( Glu ) , or glutamine ( Gln ) .","( B ) Correlation of nitrogen consumption and terA promoter activity determined by β-galactosidase activity and ammonia consumption of SBUG844_PterA:lacZ in glucose minimal medium with 10 mM NH4Cl .","( C ) β-Galactosidase activity of SBUG844_PterA:lacZ in nitrogen shift experiments .","Cultures grown for 48 hr in glucose minimal medium with 70 mM NH4Cl ( PC ) were washed and transferred to medium with ( +N ) or without 70 mM NH4Cl ( −N ) and promoter activity was determined after 15 hr of cultivation .","( D ) Carbon source dependent terA promoter activation under nitrogen starved conditions .","Strain SBUG844_PterA:lacZ was pre-cultured for 48 hr on casamino acids without sugar supplementation .","The mycelium was washed and transferred to nitrogen-free media with different carbon sources .","Reporter activity was determined 24 hr after the shift .","( E ) β-Galactosidase activity from bananas infected with conidia suspension of SBUG844 wild-type and PterA:lacZ .","Sections ( red boxes ) were cut from bananas , ground to a fine powder and subjected to β-galactosidase activity determination .","Activity was only detected from the reporter strain .","( F ) Quantification of terrein from wild-type infected bananas with or without ammonium supplementation .","All tests were performed in biological triplicates and technical duplicates; p values were calculated by the Student's paired t-test with a two-tailed distribution: **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 008 These results confirmed that nitrogen starvation acts as an inducing factor that might be responsible for the high terrein production levels on fruit juices .","In agreement , when ammonium chloride was added to banana juice , β-galactosidase activity was strongly reduced ( Figure 1—figure supplement 2 ) .","Similarly , the PterA:lacZ reporter strain displayed high β-galactosidase activity when bananas were directly infected ( Figure 3E ) .","When bananas were supplemented with ammonium chloride prior to infection with the wild-type , ethyl acetate extracts of the bananas revealed a 50% reduction in terrein content compared with bananas without nitrogen supplementation ( Figure 3F ) .","This finding clearly indicates that nitrogen limitation is a major inducer for terrein production under natural conditions .","However , it should be mentioned that the presence of sugars was always required , since especially hexoses from mono- and disaccharides provoked strong expression under nitrogen-limited conditions ( Figure 3D ) , which is in direct contrast to the production of dihydroisoflavipucine in A . terreus that belongs to the class of fruit and root rot toxins .","This metabolite is only produced in the strict absence of sugars and requires preferred nitrogen sources such as glutamine or asparagine for induction ( Gressler et al . , 2011 ) .","Nitrogen starvation and methionine marked important signals for terrein cluster induction .","To unveil the global regulators that could be involved in signal transduction , the A . terreus genome was analysed for the presence of transcription factors known to play a role in nitrogen sensing , cross pathway control of amino acid synthesis , and stress response .","The global nitrogen regulator AreA ( ATEG_07264 ) ( Arst and Cove , 1973; Hynes , 1975; Davis et al . , 2005 ) , the cross pathway control regulator CpcA ( ATEG_03131 ) ( Hoffmann et al . , 2001; Krappmann et al . , 2004 ) , the stress response bZIP transcription factor AtfA ( ATEG_04664 ) ( Balazs et al . , 2010; Lara-Rojas et al . , 2011 ) , and the nitrogen starvation-induced ras-protein RhbA ( ATEG_09480 ) ( Panepinto et al . , 2002 ) were selected for gene deletions .","All mutants were tested for their growth properties in the presence of proteinogenic amino acids or ornithine , citrulline , urea , and casamino acids , all of which were used as the sole nitrogen source .","Additionally , several complex media were analysed ( Figure 4—figure supplement 1 ) .","Only minor growth defects were observed with the cpcA and rhbA mutants , with some general growth reduction on selected amino acids ( Asp , His , Ser , Thr ) or complex media .","However , among all the mutants tested , the areA mutant revealed the most severe growth defects .","This mutant grew like the wild-type on glutamine and displayed some reduced growth on the nitrogen-rich amino acids asparagine , aspartate , and histidine as well as on urea and ammonium chloride .","However , the ΔareA strain was unable to use any other proteinogenic amino acid .","As described for other Aspergillus species ( Hunter et al . , 2014 ) , these results confirm an essential role of AreA in nitrogen sensing and utilisation in A . terreus .","In contrast , the atfA mutant was only impaired in growth at high aspartate concentrations ( 50 mM ) .","However , conidia of this mutant lacked the typical yellow-brown pigmentation .","Additionally , the atfA mutant completely lacked the typical red colouration of the medium in the presence of methionine , which has previously been demonstrated to be associated with terrein production ( Zaehle et al . , 2014 ) .","This implied a regulatory role of atfA on SM production in A . terreus .","On the contrary , the atfA mutant did not show increased sensitivity against oxidative or osmotic stress as described for other Aspergillus species ( Balazs et al . , 2010; Lara-Rojas et al . , 2011 ) .","Finally , a double deletion of the areA and atfA genes was generated ( ΔareAΔatfA ) resembling the growth phenotypes of both single mutants , since it was only able to grow on the media that supported growth of the areA mutant where it formed white conidia , as observed for the ΔatfA strain .","To test the effect of transcription factor mutations on terrein biosynthesis gene cluster activation , all mutants were pre-grown on non-inducing glucose medium with glutamine and transferred to medium with or without nitrogen .","Terrein was quantified after 24 hr ( Figure 4A ) .","The wild-type , the ΔcpcA , and the ΔrhbA mutant revealed low terrein production in the presence of nitrogen , but high terrein titers when nitrogen was omitted .","This indicates that cpcA and rhbA are dispensable for terrein production .","In contrast , both the ΔareA and the ΔatfA mutant only produced marginal amounts of terrein under nitrogen-limited conditions ( Figure 4A ) , and this effect was completely cured in the complemented mutants areAc and atfAc .","No terrein was detected in a ΔareAΔatfA double mutant .","Therefore , both areA and atfA appear essential for terrein production under nitrogen limitation .","To confirm this assumption , we expressed the cluster-specific transcription factor gene terR under control of the gpdA promoter in a ΔareAΔatfA mutant background .","A constitutive terrein production was observed , indicating that AreA and AtfA regulate terR expression , but are not essential for expression of the structural genes which directly depend on TerR ( Figure 4A ) .","Second , we analysed the effect of areA and atfA deletion on gene expression by qRT-PCR from strains shifted for 0 hr , 12 hr , and 24 hr to nitrogen starvation .","qRT-PCR was performed on the cluster genes terA , terB , the specific activator terR , and the global transcription factors areA and atfA ( Figure 4B ) . 10 . 7554/eLife . 07861 . 009Figure 4 . Terrein quantification and expression of terrein cluster genes in nitrogen regulator mutants .","( A ) Terrein quantification from the parental strains SBUG844ΔakuB ( ΔakuB ) , regulator mutants ( ΔcpcA ) , ( ΔrhbA ) , ( ΔareA ) , ( ΔatfA ) , ( ΔareAΔatfA ) , complemented mutants ( areAC ) , ( atfAC ) , and strain SBUG844ΔakuBΔareAΔatfAΔ::AnPgpdA:terR with terR overexpression in the ΔareAΔatfA background .","Mycelia were pre-grown in glutamine-supplemented media , washed and transferred to minimal medium with ( +N ) or without ( −N ) 50 mM glutamine .","Terrein was quantified from supernatants 24 hr after the shift .","( B ) qRT-PCR of strains ΔakuB , ΔareA , areAC , ΔatfA , atfAC , and ΔareAΔatfA were pre-cultivated for 40 hr in glutamine-supplemented media and transferred to nitrogen starvation .","RNA was isolated at 0 , 12 , and 24 hr of starvation .","Transcript levels were normalised against the actin gene actA by fold expression = 2^ ( CTtarget − CTactA ) .","( C ) Terrein quantification from strains shown in ( B ) after 72 hr of cultivation in glutamine-containing minimal medium supplemented with 10 mM methionine .","( D ) qRT-PCR from RNA of strains shown in ( C ) isolated after 48 hr of cultivation .","Transcript levels were normalized against the enolase gene enoA by fold expression = 2^ ( CTtarget − CTenoA ) .","( E ) Top and bottom view of colonies of Aspergillus terreus wild-type ( ΔakuB ) and mutants ( ΔterA , ΔatfA , ΔareA ) grown for 72 hr on solid minimal media supplemented with 25 mM methionine as sole nitrogen source .","The red pigmentation of the wild-type ( bottom view ) is lost in the ΔterA and ΔatfA mutants that show some enhanced growth while unable to produce terrein .","The ΔareA strain is unable to grow .","In all experiments biological triplicates with technical duplicates were analysed .","Statistical significances in comparison to the parental ΔakuB strain were calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00910 . 7554/eLife . 07861 . 010Figure 4—source data 1 . Genotypes of strains used in the study . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01010 . 7554/eLife . 07861 . 011Figure 4—source data 2 . List of oligo nucleotides used in the study . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01110 . 7554/eLife . 07861 . 012Figure 4—figure supplement 1 . Analysis of colony formation and growth phenotypes of nitrogen regulator mutants in the presence of different nitrogen sourcesAs basal medium AMM-G100 without nitrogen was used , which was supplemented with 70 mM inorganic nitrogen ( NaNO3 or NH4Cl ) , 50 mM of standard amino acids , ornithine ( Orn ) , citrulline ( Cit ) , urea , or 1% casamino acids ( CA ) .","Additionally , AMM-CA1% ( without glucose ) and the complex media yeast extract-peptone-dextrose ( YPD ) , potato dextrose agar ( PDA ) , and malt extract agar ( MA ) were used .","Photographs were generally taken after 96 hr of incubation at 37°C .","Asterisks denote different incubation times: * = 72 hr; ** = 48 hr .","Note the severely reduced colony formation of the ΔareA strain on virtually all single amino acids except for arginine , glutamine , and histidine .","Additionally , note the loss of conidia colouration in the ΔatfA strain . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01210 . 7554/eLife . 07861 . 013Figure 4—figure supplement 2 . Biochemical characterisation of the recombinant AreA DNA-binding domain and in vitro binding to HGATAR motifs identified in the terR promoter of the Aspergillus terreus terrein biosynthesis gene cluster .","( A ) Amino acid sequence alignment of the AreA DNA binding domains from Aspergillus nidulans and A . terreus .","Underlined amino acids denote the 65-residue peptide used for solving the solution structure of the AreA:DNA complex ( Starich et al . , 1998 ) .","Cysteine residues of the Cys2–Cys2 zinc finger module are highlighted in yellow .","( B ) SDS-PAGE analysis of purified AreA663-797 .","( C ) In solution oligomeric state analysis of AreA663-797 as determined via size exclusion chromatography and multiangle static light scattering .","The light scattering signal ( LS ) is shown overlaid with the calculated molar mass ( Mw ) across the elution profile as monitored by the absorbance at 280 nm ( UV ) and changes of the refractive index ( dRI ) .","( D ) Schematic presentation of putative GATA AreA binding sites ( BS 1 and","2 ) in the terR promoter .","( E–G )","Real-time in vitro surface plasmon resonance ( SPR ) interaction analysis of AreA663-797 with DNA containing the identified GATA motifs from the terR promoter of the terrein biosynthesis gene cluster .","Sequences of DNA duplexes used for SPR analysis are shown on top of the sensorgrams .","Numbers represent the GATA motif positions relative to the start of the open reading frame .","HGATAR sites are highlighted in red .","( H ) Dissociation constants and stoichiometry of analysed AreA663–797:DNA interactions analysed by SPR . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01310 . 7554/eLife . 07861 . 014Figure 4—figure supplement 3 . High performance liquid chromatography analyses from culture filtrates of SBUG844 wild-type and two independent atfA overexpression mutants ( AnPgpdA:atfA; OE 1 and 2 ) .","Strains were cultivated under non-inducing conditions for the wild-type .","( A ) AMM-G100; ( B ) AMM-G100Gln50 .","1 = terrein .","Terrein and its side products are detected from the atfA overexpressing strains but not from the wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01410 . 7554/eLife . 07861 . 015Figure 4—figure supplement 4 . Southern blot analyses for Aspergillus terreus strains generated in this study . Genomic DNA was isolated and restricted with the respective restriction enzymes indicated in each panel .","Digoxygenin-labelled probes were used in all experiments and were amplified by the oligonucleotide couples as indicated .","Signals were detected by the CDPstar chemiluminescent substrate .","Expected fragment sizes are also shown .","( A ) General scheme for generation of gene deletions using the pyrithiamine ( ptrA ) resistance cassette ( ΔGOI ) and for complementation of deletion mutants using the phleomycine ( ble ) resistance cassette ( GOIC ) via homologous recombination .","( B ) Generation of the β-galactosidase reporter strain SBUG844_PterA:lacZ in A . terreus SBUG844 wild-type .","( C ) Overexpression of atfA in SBUG844 under control of the A . nidulans gpdA promoter .","The native atfA locus ( signal at 7008 bp ) is still intact in strains that integrated the overexpression construct .","Strains with ectopic single copy integration of the construct used for downstream experiments are denoted by ‘X’ .","( D ) Deletion of the cpcA locus ( ATEG_03131 ) in SBUG844ΔakuB .","( E ) Deletion of the rhbA locus ( ATEG_09480 ) in SBUG844ΔakuB .","( F ) Deletion of the areA locus ( ATEG_07264 ) in SBUG844ΔakuB and complementation with FGSC A1156 areA .","( G ) PCR and PstI digest of PCR products from the areA upstream fragments amplified from genomic DNA from FGSC A1156 , SBUG844 , and SBUG844/areAC .","The complemented strain shows the identical PstI restriction pattern as the amplified product from FGSC A1156 , whereas no PstI restriction site is present in the SBUG844 product , confirming the complementation of the ΔareA strain by the areA gene from A1156 .","( H ) Deletion of the atfA locus ( ATEG_04664 ) in SBUG844ΔakuB and complementation with SBUG844 atfA .","( I ) Deletion of the atfA locus ( ATEG_04664 ) in the areA negative background of SBUG844ΔakuBΔareA ( Δ ) and replacement of atfA in SBUG844ΔakuBΔareA with the terR overexpression construct under control of the A . nidulans gpdA promoter ( Δ::AnPgpdA:terR ) .","( J ) Partial deletion of the hapX locus ( ATEG_08 , 014 ) in SBUG844ΔakuB and complementation with the Aspergillus nidulans FGSC A4 hapX ( AN08251 ) .","( K ) Deletion of the sreA locus ( ATEG_07714 ) in SBUG844ΔakuB and complementation with SBUG844 sreA .","( L ) Deletion of the sidA locus ( ATEG_06879 ) in SBUG844ΔakuB and complementation with SBUG844 sidA .","( M ) Deletion of sidA ( ATEG_06879 ) in the terA negative background of SBUG844ΔakuBΔterA . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01510 . 7554/eLife . 07861 . 016Figure 4—figure supplement 5 . Terrein production and susceptibility to osmotic stress in a sakA mutant . In all experiments biological triplicates with technical duplicates were analysed .","( A ) Terrein quantification from strains SBUG844ΔakuB ( ΔakuB ) and two sakA deletion strains SBUG844ΔakuBΔsakA ( 1 ) and ( 2 ) ( ΔsakA ) cultivated in AMM ( -N ) G100 for 72 hr in the presence of either 70 mM inorganic ( NaNO3 , NH4Cl ) or 50 mM organic nitrogen ( Glu , Gln , Asp , Asn ) .","( B ) Terrein quantification from strains SBUG844ΔakuB ( ΔakuB ) , SBUG844ΔakuBΔsakA and the complemented strain SBUG844ΔakuBΔsakA/sakAC ( sakAC ) .","Cultures were pre-grown in nitrogen-rich AMM-G100Gln50 , mycelia were washed and transferred to AMM ( -N ) G100 with ( +N ) or without ( −N ) 50 mM Gln .","Terrein was quantified from supernatants 24 hr after the shift .","( C ) Susceptibility assay towards NaCl stress .","105 to 101 conidia of strain SBUG844ΔakuB ( ΔakuB ) , the deletion mutants SBUG844ΔakuBΔsakA ( ΔsakA ) and SBUG844ΔakuBΔatfA ( ΔatfA ) or their complemented strains SBUG844ΔakuBΔsakA/sakAC ( sakAC ) and SBUG844ΔakuBΔatfA/atfAC ( atfAC ) were point inoculated on AMM ( -N ) G100 plates supplemented with 10 mM Gln and 0 , 0 . 5 , 1 . 0 , and 2 . 0 M NaCl .","Plates were incubated 72 hr or 5 d ( * ) at 37°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 016 In the wild-type the regulators areA and atfA showed a time-dependent increase in gene expression .","While the regulator terR was most strongly upregulated after 12 hr , expression of the TerR-controlled terrein biosynthesis genes terA and terB continued to increase after 12 hr and reached 13 . 3 and 3 . 2 times the expression level of the actin control gene at 24 hr ( Figure 4B ) .","On the contrary , while complemented mutants behaved like the wild-type , the areA and atfA deletions strongly reduced activation of terR and , in turn , the expression of terA and terB .","Results from qRT-PCR perfectly coincided with the substantially reduced terrein production rates under nitrogen limitation in these mutants ( Figure 4A ) .","AreA recognises the DNA-binding motif HGATAR , and two adjacent binding sites are generally required for transcriptional activation due to dimer formation of AreA monomers ( Ravagnani et al . , 1997 ) .","In this respect , the terR promoter contains two putative AreA binding sites that match the HGATAR consensus ( BS1and BS2; positions −59 and −72 relative to the translational start point ) .","Surface plasmon resonance ( SPR ) analyses with the Aspergillus nidulans AreA DNA-binding domain , which is 91% identical to the respective A . terreus AreA domain , showed that BS1 and BS2 are recognised with high affinity ( Figure 4—figure supplement 2 ) .","This strengthens the model of a direct involvement of AreA in the activation of terR expression .","However , the reduction of terR expression was less pronounced in the ΔareA than in the ΔatfA background ( Figure 4B ) , which indicates that AreA is not the only activator acting on the terR promoter .","Therefore , we additionally searched for putative palindromic AtfA/Sko1 binding sites ( 5′-TKACGTMA-3′ ) in the promoter regions of the cluster ( Proft et al . , 2005 ) .","Only one hit ( TGACGTCA ) was identified in the promoter of the structural gene terC .","However , if one mismatch is allowed , there is a putative binding site at position −731 relative to the ATG start codon of terR ( 5´-TGGCGTCA-3´ ) , but it remains speculative whether this binding site is recognised by A . terreus AtfA .","Nevertheless , it should be mentioned that even a single half site of the suggested motif could promote transcription factor binding and promoter induction ( Proft et al . , 2005 ) .","We therefore conclude that , although direct evidence for AtfA binding at the terR promoter is lacking , both transcription factors seem to regulate terR expression .","In agreement , terR , terA , and terB expression and terrein production showed the strongest decrease in the ΔareAΔatfA double knock-out mutant ( Figure 4A ) .","In addition , a constitutive expression of atfA by the gpdA promoter led to increased terrein production ( Figure 4—figure supplement 3 ) .","This supports our hypothesis of direct involvement of AtfA in terrein cluster regulation .","Due to the requirement for atfA and areA in terrein biosynthesis gene cluster induction under nitrogen starvation , we also tested their contribution to methionine-dependent induction in nitrogen-supplemented medium ( Figure 4C ) .","While the wild-type strain and complemented mutants showed high terrein production rates of up to 55 mg/g mycelium , the terrein levels in the ΔareA , ΔatfA , or ΔareAΔatfA double mutant remained near the detection limit and qRT-PCR was performed to confirm this result on the transcriptional level .","Although areA and atfA were expressed in the wild-type only at low levels on glutamine/methionine medium ( Figure 4D ) , deletion of areA or atfA reduced terR and , consequentially , terA and terB expression .","In the ΔatfA mutant terR transcription was completely abolished and , in agreement , transcription of terA and terB was no longer detected .","An areA mutant is unable to use methionine as a nitrogen source ( Figure 4E ) , and its uptake may be limited leading to the loss of transcriptional activation .","In contrast , the atfA mutant still uses methionine as a nitrogen source , but neither produces terrein nor pigmented conidia nor the red colouration of the medium which is associated with terrein production in the presence of methionine ( Figure 4E ) .","This implies that atfA may be induced by a methionine-dependent signaling cascade that subsequently leads to terR expression .","While nitrogen starvation stimulated terrein production , starvation of other macroelements such as carbon , sulphur , or phosphate did not result in terrein production ( data not shown ) .","However , this did not exclude limitation of trace elements as inducing signals .","Therefore , the β-galactosidase activity from the PterA:lacZ reporter strain was determined from cultures with reduced amounts of trace elements .","Indeed , a decrease in trace elements was accompanied by increased terA promoter activity ( Figure 5A ) .","To attribute this activation to a specific trace element , media with limited amounts of trace elements were supplemented with each of the single trace elements FeSO4 , ZnSO4 , CuSO4 , MnCl2 , Na2MoO4 , CoCl2 , and H3BO3 .","Cluster induction was observed in all cultures except for that supplemented with FeSO4 ( Figure 5B ) , indicating cluster activation from iron limitation .","Subsequently , a minimal medium was prepared that contained all trace elements but iron which led to strong cluster induction , and the addition of 40 µM FeCl3 completely repressed the induction ( Figure 5C ) .","Thus , besides methionine and the limitation of nitrogen , a lack of iron—but no other trace element—induces the terrein biosynthesis gene cluster . 10 . 7554/eLife . 07861 . 017Figure 5 . Expression of terA , production of siderophores under iron limited conditions and iron reducing properties of terrein . All analyses were performed in biological triplicates and technical duplicates .","Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution .","( A ) β-Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with limited amounts of trace elements .","Asterisks indicate p values vs 100% trace elements: **p<0 . 01; ***p<0 . 001 .","( B ) β -Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with 1% trace elements supplemented with 20 µM of specific trace elements: FeSO4 , ZnSO4 , CuSO4 , MnCl2 , Na2MoO4 , CoCl2 , or H3BO3 .","Asterisks indicate p values vs activity without supplementation: ***p<0 . 001 .","( C ) β-Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with and without 40 µM FeCl3 .","Asterisks indicate p values vs 40 µM FeCl3: ***p<0 . 001 .","( D ) High performance liquid chromatography ( HPLC ) profiles of lyophilised culture supernatants of ΔterA after 72 hr of cultivation in AMM ( -N ) G100Gln50 supplemented with 0 , 20 , or 200 µM FeCl3 .","Peaks for the siderophores ferrichrysin ( 2 ) and coprogen ( 3 ) are indicated .","( E ) Fe ( III ) reduction by terrein determined by the TPTZ assay .","Ascorbic acid served as control and maximum reduction by ascorbic acid was set as 100% .","Analyses were made from duplicates .","( F ) HPLC profiles of terrein after incubation with different oxidised ( upper , red lines ) or reduced ( lower , blue lines ) metal ions .","Left: Cu ( II ) SO4 and Cu ( I ) Ac; right: Fe ( III ) Cl3 and Fe ( II ) SO4 .","1 = terrein; 4 = propylene maleic acid .","( G ) Scheme of terrein oxidation during iron reduction leading to the formation of propylene maleic acid . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01710 . 7554/eLife . 07861 . 018Figure 5—source data 1 . Analytical data of coprogen . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01810 . 7554/eLife . 07861 . 019Figure 5—source data 2 . Analytical data of ferrichrysin . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01910 . 7554/eLife . 07861 . 020Figure 5—figure supplement 1 . qRT-PCR expression analysis of genes from iron acquisition systems under iron-supplemented and limited conditions . All analyses were performed from biological triplicates and technical duplicates .","Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution .","SBUG844 wild-type was grown in AMM-G100Gln50 under iron-supplemented ( 40 µM; +Fe ) and starved conditions ( −Fe ) .","qRT-PCR was performed on genes assumed to be responsible for siderophore biosynthesis , siderophore transport , or reductive iron assimilation .","Transcript quantities were normalised against enoA by fold expression = 2^ ( CTtarget − CTeno ) .","Asterisks indicate p values vs 0 µM FeCl3: **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02010 . 7554/eLife . 07861 . 021Figure 5—figure supplement 2 . Chrome azol S ( CAS ) assay of coprogen and terrein . Both substances were dissolved in methanol and serial dilutions ( 125–15 . 63 µg ) were added to punched holes of CAS plates .","Photographs were taken 48 hr after incubation at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02110 . 7554/eLife . 07861 . 022Figure 5—figure supplement 3 . pH- and time-dependent Fe ( III ) reduction by terrein assayed by TPTZ . 10 µM FeCl3 were incubated with 4 mM fixed concentration of terrein in acetate buffer in a pH range from 3 . 0 to 6 . 0 .","The analyses were performed from duplicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02210 . 7554/eLife . 07861 . 023Figure 5—figure supplement 4 . 1H NMR ( 500 MHz , MeOD; upper panel ) and 13C NMR ( 150 MHz , MeOD; lower panel ) of compound 4 , 2- ( ( E ) -prop-1-en-1-yl ) maleic acid . 1H NMR ( 500 MHz , MeOD ) : δ 6 . 22 ( 1H , d , 3J = 15 . 8 Hz ) , 6 . 15 ( 1H , dq , 3J = 15 . 8 Hz , 3J = 6 . 5 Hz , ) , 5 . 73 ( 1H , s ) , 1 . 85 ppm ( 3H , d , 3J = 6 . 5 Hz ) ; 13C NMR ( 600 MHz , MeOH ) : δ 172 . 6 , 169 . 5 , 151 . 7 , 136 . 4 , 130 . 2 , 118 . 5 , 18 . 8 ppm; HRMS: ( ESI+ ) : m/z calculated for C7H9O4: 157 . 0495 , found 157 . 0495 [M + H]+ .","DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02310 . 7554/eLife . 07861 . 024Figure 5—figure supplement 5 . HSQC NMR ( 600 MHz , MeOD; upper panel ) and HMBC NMR ( 600 MHz , MeOD; lower panel ) of compound 4 , 2- ( ( E ) -prop-1-en-1-yl ) maleic acid . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 024 Fungal iron acquisition has been well investigated in A . nidulans ( Eisendle et al . , 2003 ) and Aspergillus fumigatus ( Haas , 2014 ) , but only limited information was available on the iron acquisition systems from A . terreus .","A BLASTp search for orthologous genes in the genome of A . terreus NIH2624 ( Table","1 ) revealed that all genes required for a reductive iron assimilation ( RIA ) system ( fetC , freB , ftrA ) , siderophore biosynthesis ( sid genes , except sidG ) , siderophore transport ( mirA-D , sitA ) , and regulators of iron homeostasis ( srbA , sreA , and hapX ) are well conserved in the A . terreus genome .","When tested under iron limitation , qRT-PCR analyses confirmed a strong induction of genes from siderophore biosynthesis , siderophore transport , and the RIA pathway ( Figure 5—figure supplement 1 ) .","To confirm siderophore production by A . terreus , we cultivated the wild-type strain under iron-rich ( 200 µM FeCl3 ) and iron-limited conditions ( no or 20 µM FeCl3 ) and identified ferrichrysin ( 2 ) and coprogen ( 3 ) only under iron limitation ( Figure 5D and Figure 5—source data 1 ,","2 ) ( Zähner et al . , 1963; Bertrand et al . , 2010 ) .","Coprogen revealed a higher stability than ferrichrysin .","Subsequent analyses involving siderophores were therefore based only on coprogen .","When terrein was compared with purified coprogen for chelating iron , only coprogen showed the expected iron chelating activity in CAS agar plate assays ( Figure 5—figure supplement 2 ) .","Thus , although terrein is produced under iron limitation , it does not depict a new kind of siderophore . 10 . 7554/eLife . 07861 . 025Table 1 . BLASTp analysis of homologous genes for iron uptake and regulation of iron homeostasis ( adapted by [Haas , 2012] ) DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 025Aspergillus fumigatus functionGene code‡ExpressionAspergillus terreus gene codeIdentity/similarityReductive iron assimilation ( RIA ) FetCFerroxidaseAFUA_5G03790−FeATEG_0803279%/89% FreBFerric reductaseAFUA_1G17270−FeATEG_1032253%/64% FtrAIron permeaseAFUA_5G03800−FeATEG_0803175%/84%Siderophore biosynthesis ( SB ) EstATAFC esteraseAFUA_3G03660−FeATEG_0407244%/58% NpgA/PptAPhosphopantetheinyl transferaseAFUA_2G08590−ATEG_0969556%/65% SidAOrnithine monooxygenaseAFUA_2G07680−FeATEG_0687978%/85% SidCFC NRPSAFUA_1G17200−FeATEG_0507360%/76% SidDFSC NRPSAFUA_3G03420−FeATEG_0748843%/59% SidFTransacylaseAFUA_3G03400−FeATEG_0507552%/67% SidGTransacetylaseAFUA_3G03650−Fenone– SidHMevalonyl hydrataseAFUA_3G03410−FeATEG_0150953%/67% SidIMevalonyl ligaseAFUA_1G17190−FeATEG_0507486%/91% SidLTransacetylaseAFUA_1G04450–ATEG_0377064%/76%Siderophore transporter ( SIT ) MirAEnterobactin transporterAN7800; -−FeATEG_0407168%/77% MirBTAFC transporterAN8540; -AFUA_3G03640−FeATEG_0271150%/68% MirC*AN7485; AFUA_2G05730−FeATEG_0676278%/87% MirD†Trichotecene efflux pumpAFUA_3G03440–ATEG_0748740%/58% SitA/SitT*AN5378; AFUA_7G06060−FeATEG_0632962%/73%Regulatory proteins HapXbZip-TFAFUA_5G03920−FeATEG_0801477%/83% ( hapX re-annotated ) SreAGATA TFAFUA_5G11260+FeATEG_0774167%/75% SrbA†HLH TFAFUA_2G01260−FeATEG_0815672%/82%Genes selected for qPCR analyses are highlighted in bold .","*Genes annotated according to ( Schrettl et al . , 2008 ) .","†Genes annotated according to ( Blatzer et al . , 2011 ) .","‡Gene codes ANxxxx refer to the A . nidulans genome .","Since some antioxidative properties of terrein had previously been described ( Trabolsy et al . , 2014 ) , the ability of terrein in reducing ferric ( Fe3+ ) to ferrous ( Fe2+ ) iron was investigated by using the ferrous iron chelator 2 , 4 , 6-tripyridyl-S-triazine ( TPTZ ) .","The strong antioxidant ascorbic acid ( Elmagirbi et al . , 2012 ) served as control ( Figure 5E ) .","Although ascorbic acid showed a much higher reducing potential , terrein was also able to convert ferric to ferrous iron in a concentration-dependent ( Figure 5E ) and pH-dependent ( Figure 5—figure supplement","3 ) manner with a pH optimum of 3–4 .","In this respect , we noticed a significant fall in the culture pH under iron limitation from 6 . 5 to about 3 . 5 , which agrees with the optimum pH for terrein-mediated iron reduction .","Thus , since terrein is produced during growth under iron limitation , the combination with a fall in pH could indeed increase iron availability .","To elucidate the structure of the terrein oxidation product , the terrein-mediated iron reduction assay was scaled up and extractions were subjected to high performance liquid chromatography ( HPLC ) analysis .","Here , besides a major proportion of terrein that remained in its original structure ( Figure 5F ) , a new peak ( 4 ) was observed , which was identified by NMR analyses ( Figure 5—figure supplements 4 ,","5 ) as 2- ( ( E ) -prop-1-en-1-yl ) maleic acid ( PMA , Figure 5G ) .","The structure of the oxidation product implies that oxidation took place at the two hydroxylated carbon atoms of terrein , leading to a cleavage of the pentenone ring system as proposed by Trabolsy et al . ( 2014 ) .","In contrast , conversion of terrein to PMA was not observed during incubation with Fe2+ , confirming that iron reduction was the cause of PMA formation .","No conversion of terrein was observed with oxidised copper ions ( Cu2+ ) , indicating a limited reductive potential which is sufficient for iron but not for copper ( Figure 5F ) .","In conclusion , while terrein cannot chelate iron to support the siderophore-mediated iron uptake , its reductive potential is sufficient to reduce ferric to ferrous iron , which may increase iron solubility and could ease the direct uptake of iron via the ferrous iron transport system .","Due to the co-regulation with siderophore biosynthesis , we assumed that transcriptional regulators involved in iron homeostasis could also regulate terrein biosynthesis .","In A . nidulans and A . fumigatus , siderophore biosynthesis is regulated by HapX , a transcriptional inducer under iron limitation , and SreA , a repressor in the presence of iron ( Haas , 2012 ) .","To investigate the impact of SreA and HapX on siderophore ( coprogen ) and terrein synthesis , the sreA gene was completely and the hapX gene partially deleted ( the latter due to incomplete sequence information at the hapX locus tag ATEG_08014 ) .","Additionally , while the sreA mutant was complemented with the A . terreus sreA gene , the hapX mutant was complemented with the hapX gene from A . nidulans FGSC A4 .","All strains were cultivated in iron-supplemented and iron-limited media and terrein and coprogen were quantified from culture supernatants ( Figure 6A , B ) .","All complemented mutants behaved similar to the wild-type with production of only marginal amounts of coprogen and terrein in the presence of iron .","Under iron starvation the production rates for both metabolites strongly increased . 10 . 7554/eLife . 07861 . 026Figure 6 . Effect of hapX and sreA deletion on terrein and coprogen biosynthesis .","( A ) Coprogen or ( B ) terrein quantification from SBUG844ΔakuB ( ΔakuB ) , SBUG844ΔakuBΔhapX ( ΔhapX ) , SBUG844ΔakuBΔhapX/AnhapXC ( AnhapXC ) , SBUG844ΔakuBΔsreA ( ΔsreA ) , and SBUG844ΔakuBΔsreA/sreAC ( sreAC ) grown for 72 hr in AMM_G100Gln50 with ( +Fe ) or without 40 µM FeCl3 ( −Fe ) .","Coprogen was quantified from lyophilised culture supernatants and terrein from culture extracts .","( C ) qRT-PCR from strains and media described in ( A ) and ( B ) .","RNA was isolated after 40 hr of cultivation .","Transcript levels were normalised against enoA by fold expression = 2^ ( CTtarget − CTenoA ) .","( $ ) denotes the lack of hapX transcripts from the complemented ΔhapX strain , since the Aspergillus nidulans hapX was used for complementation .","qRT-PCR on the complemented mutant with oligonucleotides specific for AnhapX are shown in Figure 6—figure supplement 1 .","All analyses were performed from biological triplicates and technical duplicates .","Statistical significances were calculated in comparison to the parental ΔakuB strain by the Student's paired t-test with a two-tailed distribution: *p<0 . 05; **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02610 . 7554/eLife . 07861 . 027Figure 6—figure supplement 1 . qRT-PCR analysis of Aspergillus nidulans hapX expression in the Aspergillus terreus wild-type SBUG844ΔakuB , the hapX mutant SBUG844ΔakuBΔhapX and its complemented strain SBUG844ΔakuBΔhapX/AnhapXC . All analyses were performed from biological triplicates and in technical duplicates .","All strains were grown for 40 hr in the presence ( +Fe ) or absence ( −Fe ) of 40 µM FeCl3 .","Transcript levels of AnhapX were normalised against enoA gene from A . terreus by fold expression = 2^ ( CTtarget − CTeno ) .","The heterologously expressed AnhapX gene behaves in a similar way to the native hapX gene from A . terreus ( compare with Figure 6 ) .","Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution: Asterisks indicate p values vs 0 µM FeCl3: ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 027 On the other hand , partial hapX deletion reduced coprogen production under iron limitation by approximately 50% and terrein concentrations by about 90% ( Figure 6A , B ) .","This indicates that HapX directly activates both siderophore and terrein biosynthesis in A . terreus .","In contrast , the ΔsreA mutant produced significantly higher amounts of coprogen in the presence of iron whereas terrein production did not significantly increase ( Figure 6A , B ) .","This confirms SreA as a negative feedback regulator in siderophore biosynthesis , whereas it does not control terrein production .","In contrast , HapX positively controls both pathways .","These results were confirmed on the transcriptional level by qRT-PCR ( Figure 6C and Figure 6—figure supplement 1 ) .","The inactivation of hapX prohibited terR transcription and resulted in an inability to induce terrein cluster genes terA , terB , and terC .","In contrast , deletion of sreA influenced neither terR activation nor expression of other terrein cluster genes in the presence of iron .","We therefore conclude that hapX is the major regulator for terrein cluster induction under iron starvation .","To elucidate the complete sequence of the hapX gene we subsequently used degenerate primers to amplify the main proportion of hapX from cDNA .","The complete sequence of the A . terreus hapX gene is found under accession number KP233834 ( Gressler et al . , 2015b ) .","The sequence of the full-length HapX protein matches with that of HapX proteins from other Aspergillus species such as A . nidulans ( 73% identity ) , A . fumigatus ( 77% ) , Aspergillus niger ( 81% ) , and A . oryzae ( 81% ) .","In general , siderophore-based iron acquisition is highly efficient and assumed to be more important than the reductive iron assimilation pathway .","In A . fumigatus , growth and virulence defects caused by the interruption of the reductive iron assimilation pathway are only observed when the siderophore-based system is also inactivated ( Blatzer et al . , 2011 ) .","Therefore , to elucidate a positive effect of terrein on iron acquisition , we deleted the sidA gene in A . terreus that encodes the L-ornithine-N5-monooxygenase , a key enzyme in hydroxamate siderophore biosynthesis .","Coprogen production was confirmed in the wild-type and a complemented mutant , but was completely lacking from the ΔsidA mutant ( Figure 7—figure supplement 1 ) .","When analysed for growth phenotypes on solid media , all complemented mutants and the ΔterA strain behaved like the wild-type ( Figure 7A and Figure 7—figure supplement 1A ) .","The hapX mutant showed a reduced growth rate without iron supplementation ( Figure 7A ) , which is in agreement with reduced coprogen production as shown above ( Figure 6A ) .","However , severe iron limitation from the addition of the iron chelator bathophenanthroline sulfonate ( BPS ) completely repressed growth of the hapX mutant ( Figure 7A ) .","The ΔsidA mutant displayed the most severe phenotype: while ΔsidA showed normal growth with slightly reduced conidiation in the presence of high to moderate iron concentrations ( 100 or 20 µM FeCl3 ) ( Figure 7A ) , growth was strongly retarded when the iron concentration was reduced to 2 µM FeCl3 .","No growth was observed even after prolonged incubation when iron was omitted ( Figure 7A ) .","These phenotypes were cured when purified coprogen was externally added ( Figure 7—figure supplement 1B ) .","Interestingly , growth of the ΔsidA strain was also partially restored in the presence of ascorbic acid and , although to a lesser extent , by the addition of terrein ( Figure 7A ) .","This result is supported by previous studies on an A . nidulans ΔsidA mutant which was able to grow under iron limitation in the presence of ascorbic acid ( Eisendle et al . , 2003 ) .","Unexpectedly , terrein supplementation inhibited growth of an A . fumigatus sidA mutant , which was also true for an A . fumigatus wild-type strain ( Figure 7—figure supplement 2 ) .","Subsequent analyses showed that growth of the phytopathogen Fusarium graminearum was also inhibited by terrein-containing culture extracts from A . terreus ( Figure 7—figure supplement 2 ) , indicating some antifungal properties of terrein against environmental competitors . 10 . 7554/eLife . 07861 . 028Figure 7 . Siderophore production in sidA mutants and growth-supporting effect of terrein under iron limitation .","( A ) Growth of wild-type ΔakuB , ΔterA , ΔhapX , and ΔsidA on AMM ( −N ) G100Gln10 plates containing different iron concentrations .","Photographs were taken after 4 d and , as indicated , after 17 d of incubation at 37°C .","Iron-free medium was supplemented with 100 , 20 , 2 , or 0 µM FeCl3 .","Additionally , plates without iron were supplemented with bathophenanthroline disulfonate ( BPS; 100 µM ) to simulate harsh starvation conditions or with either ascorbic acid ( 1 mM ) or terrein ( 5 or 10 mM ) .","( B , C )","Impact of terrein production on growth of ΔsidA mutants .","AMM-G100Gln50 plates were supplemented with 0 , 2 , 20 , or 100 µM FeCl3 and either inoculated with ( B ) conidia or ( C ) mycelial pellets from cultures pre-grown for 40 hr in AMM-G100Gln50 with 200 µM FeCl3 .","The parental Aspergillus terreus wild-type ΔakuB , the ΔakuBΔsidA strain , and the ΔakuBΔterAΔsidA are shown .","Mycelia from a 40 hr culture in AMM-G100Gln50 with 200 µM FeCl3 was washed with iron-free medium and three pellets were applied to the plates .","Plates were incubated at 37°C for 5 d . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02810 . 7554/eLife . 07861 . 029Figure 7—figure supplement 1 . Dependence of colony formation of Aspergillus terreus mutants on iron availabilty , ascorbic acid , terrein , and coprogen .","( A ) As basic media AMM ( -N ) G100Gln10 ( -Fe ) agar plates were used that were supplemented either with different iron concentrations ( 100 , 20 , 2 , or 0 µM FeCl3 ) , the iron chelator bathophenanthroline disulfonate ( BPS; 100 µM ) , ascorbic acid ( Asc , 1 mM ) , or terrein ( 10 mM ) .","Plates were incubated for 4 d at 37°C , except for the ΔterAΔsidA mutant that was further incubated for up to 17 d ( B ) Effect of purified coprogen on growth of A . terreus mutants and complemented strains under iron starvation .","Conidia ( 1 × 105 ) of A . terreus SBUG844ΔakuB , SBUG844ΔakuBΔsidA , SBUG844ΔakuBΔsidA/sidAC , SBUG844ΔakuBΔterA , or SBUG844ΔakuBΔterAΔsidA were plated on AMM-G100Gln10 plates supplemented with ( +Fe ) or without 20 µM FeCl3 ( −Fe ) .","To ( −Fe ) media filter disks soaked with either 20 µl methanol ( MeOH , negative control ) or 20 µl of 2 . 5 mg/ml coprogen ( in methanol ) were applied .","Plates were incubated at 37°C for 72 hr until photographed .","( C ) Coprogen quantification from ΔakuB wild-type , ΔsidA mutant and complemented mutant sidAC .","Strains were cultivated for 40 hr in AMM ( -N ) G100Gln50 supplemented with 200 µM FeCl3 .","The mycelium was washed and transferred to iron-free AMM ( −N ) G100Gln50 medium .","Coprogen was quantified after 30 hr of incubation .","Analyses were performed from biological triplicates and technical triplicates .","Statistical significance was calculated in comparison to the parental ΔakuB strain by the Student's paired t-test with a two-tailed distribution: *p<0 . 05; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02910 . 7554/eLife . 07861 . 030Figure 7—figure supplement 2 . Antifungal activity of terrein and potato dextrose broth ( PDB ) medium from Aspergillus terreus wild-type cultivations .","( A ) AMM-G100Gln10 containing no iron addition ( −Fe ) or 2 mM FeCl3 were supplemented with 0 , 1 , 10 , or 20 mM terrein and inoculated with conidia of Aspergillus fumigatus wild-type ATCC46645 .","Plates were incubated for 84 hr at 37°C until photographed .","Terrein inhibits growth of A . fumigatus independently of the available iron concentration .","( B ) Analysis of the effect of A . terreus inoculated PDB culture broth on growth of A . terreus , A . fumigatus , and Fusarium graminearum .","The basal medium for A . terreus and A . fumigatus was AMM-G50 medium with nitrate as the nitrogen source , whereas the medium for F . graminearum was additionally supplemented with 0 . 2% potato broth ( AMM-G50 + 0 . 2%PB ) .","All plates were supplemented either with 150 µl of PDB ethyl acetate extract from a mock-inoculated culture ( PDB extract ) or inoculated with A . terreus SBUG844 and cultivated for 4 d at 30°C ( PDB metabolite extract ) .","Photographs were taken after 4 d of incubation at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 03010 . 7554/eLife . 07861 . 031Figure 7—figure supplement 3 . Terrein determination in siderophor deletion mutants . High performance liquid chromatography profiles of agar plugs were recorded from plates shown in panel Figure 7C .","Wild-type ( A ) and ΔsidA mutant ( B ) produced terrein ( 1 ) under iron-limited conditions , whereas no terrein was formed by the ΔterAΔsidA mutant ( C ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 031 Due to the beneficial effect of terrein on the A . terreus ΔsidA mutant , we investigated the impact of intrinsic terrein production on growth under iron limitation .","For this , we deleted the sidA gene in the ΔterA background and compared growth of wild-type , ΔsidA , and ΔterAΔsidA under iron-supplemented ( 20 and 200 µM FeCl3 ) and iron-limited conditions ( 0 and 2 µM FeCl3 ) ( Figure 7B ) .","Both mutants ( ΔsidA and ΔterAΔsidA ) were unable to grow on media containing 2 µM or less FeCl3 .","However , since terrein is only produced by vegetative mycelium , strains were pre-grown in the presence of 200 µM FeCl3 , washed , and transferred to the plates with different iron contents ( Figure 7C ) .","While growth of mycelium from the ΔakuB strain looked identical to that from conidia , the ΔsidA mutant started to form small colonies even in the absence of iron , whereas the ΔterAΔsidA mutant showed some weak growth only at 2 µM FeCl3 but not in the absence of external iron supplementation .","Terrein production was analysed from extracted agar plugs , which confirmed that the wild-type and ΔsidA mutant produced substantial amounts of terrein under iron-limited conditions whereas terrein production was fully abrogated in the ΔterAΔsidA mutant ( Figure 7—figure supplement 3 ) .","These results indicate that terrein production supports iron acquisition , although the siderophore system is the dominant iron acquisition system ."],["A . terreus is known as a human pathogen that causes severe invasive bronchopulmonary and disseminated aspergillosis ( Lass-Flörl et al . , 2005; Slesiona et al . , 2012a , 2012b ) .","In addition , A . terreus has been described as a causative agent of foliar blight of potato leaves ( Louis et al . , 2013 , 2014a , 2014b ) .","Besides its pathogenic potential , A . terreus can inhibit proliferation of other plant pathogens such as Fusarium udum ( Upadhyay and Rai , 1987 ) and acts as a mycoparasite on sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum ( Melo et al . , 2006 ) .","SMs are assumed to support competition in the plant and soil environment .","The natural biological activities of terrein include the inhibition of plant seed germination ( Kamata et al . , 1983 ) , the induction of fruit surface lesions ( Zaehle et al . , 2014 ) , and the newly discovered reduction of ferric to ferrous iron accompanied by some antifungal activity .","These activities are all appropriate to supporting the competitiveness of A . terreus in the environment .","However , the production of terrein at the correct timing requires coordinated sensing and transduction of environmental signals .","The terrein biosynthesis gene cluster is directly controlled by the Zn2Cys6 transcription factor TerR ( Gressler et al . , 2015a ) .","Therefore , terrein production requires transcriptional activation of terR .","While sugars are indispensable for high terrein production rates ( Zaehle et al . , 2014 ) , three major signals were identified that resulted in terR transcription: methionine-dependent induction , nitrogen limitation , and iron starvation .","These signals resemble the plant and rhizosphere environment .","Plant roots are leaky for carbon compounds that derive from C-fixation during photosynthesis .","Up to one third of plant-assimilated carbon may end up in the rhizosphere , and especially in the extramatrical mycelium of ectomycorrhiza ( Churchland and Grayston , 2014 ) .","Here , at least a fraction of carbohydrates is subsequently exuded by ectomycorrhiza , making it available to other soil microorganisms ( Sun et al . , 1999 ) .","Mycorrhizas mobilize nitrogen to feed their symbiotic plant partner , resulting in nitrogen limitation for surrounding microorganisms .","These conditions stimulate terrein production in A . terreus , which is typically isolated from the rhizosphere ( Gao et al . , 2013; Rajalakshmi and Mahesh , 2014 ) and might affect existing plant–microbe interactions by its phytotoxic and antifungal activity ( Figure 8 ) . 10 . 7554/eLife . 07861 . 032Figure 8 . Scheme of the regulation of terrein biosynthesis gene cluster expression during interactions in the rhizosphere . Plants secrete methionine ( +Met ) with root exudates into the soil .","Additionally , competing microorganisms reduce the available pool of nitrogen sources ( −N ) and iron ( −Fe ) .","Signals from nitrogen limitation and methionine are sensed via AreA and AtfA , whereas iron limitation is sensed via HapX .","All three transcription factors activate the promoter of the terrein biosynthesis gene cluster-specific transcription factor .","TerR leads to transcription of the structural genes required for terrein biosynthesis and terrein is produced from acetyl- and malonyl-CoA units .","The two-cluster specific major facilitator superfamily ( MFS ) transporters export terrein into the rhizosphere .","Here terrein can counteract iron limitation by its ferric iron reducing activity , supports degradation of organic matter by its phytotoxic activities , and reduces growth of competitors by its antifungal activity . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 032 Another inducing signal derives from methionine , which may be of particular importance when A . terreus acts as a potato plant pathogen ( Louis et al . , 2013 ) .","Notably , PDB induces high terrein production rates ( Zaehle et al . , 2014 ) , which we assume to be methionine-dependent .","In plants , methionine is required for ethylene production in environmental stress response ( Wang et al . , 2002 ) , fruit ripening ( Yang and Hoffman , 1984 ) , and polyamine production in plant defence reactions .","Furthermore , methionine is a precursor for iron chelating siderophores such as nicotianamine and mugineic acid ( Roje , 2006 ) and has been identified in plant root exudates ( Dakora and Phillips , 2002 ) .","Therefore , elevated levels of methionine depict a signal that indicates the presence of a plant environment and stimulates terrein production .","However , although further studies are required to elucidate the specific contribution of terrein to plant infection , its ability to induce lesion formation on several fruit surfaces indicates that terrein interacts with plant defence mechanisms ( Zaehle et al . , 2014 ) .","The third—at first sight unrelated—inducing factor is iron starvation .","Although terrein can reduce ferric to ferrous iron , a direct positive growth-promoting effect of terrein during iron limitation in A . terreus monocultures was only observed in the absence of a functional siderophore system .","However , extracellular siderophores show a high affinity for ferric iron but not for ferrous iron ( Hider and Kong , 2010 ) .","Thus , the reduction of ferric iron could reduce the efficiency of iron chelation by xenosiderophores secreted from competing microorganisms .","A similar strategy for iron acquisition has previously been described for the human pathogenic fungus Histoplasma capsulatum ( Timmerman and Woods , 1999 ) .","H . capsulatum secretes glutathione accompanied by the γ-glutamyltransferase Ggt1 .","This enzyme releases the dipeptide cysteinylglycine from gluthathione , which in turn reduces extracellular ferric iron ( Zarnowski et al . , 2008 ) .","While H . capsulatum also secretes hydroxamate siderophores ( Howard et al . , 2000 ) , silencing of ggt1 mRNA reduced virulence in macrophages even in the presence of the siderophore system ( Zarnowski et al . , 2008 ) .","Thus , terrein may interfere with competing siderophore systems to increase the competitiveness of A . terreus in the environment .","In this study we showed that transduction of environmental signals to the promoter of the transcriptional activator TerR requires activation by the global transcription factors AreA , AtfA , and HapX .","While AreA has previously been described to regulate several SM gene clusters in fungi ( Tudzynski et al . , 1999 ) , HapX activates iron acquisition systems ( Schrettl et al . , 2010 ) and AtfA plays a specific role in osmotic and oxidative stress responses ( Lara-Rojas et al . , 2011 ) .","In Aspergillus species and maybe all saprophytic ascomycetes , the GATA transcription factor AreA plays a substantial role in regulation of nitrogen .","Under nitrogen starvation , AreA is transported into the nucleus ( Todd et al . , 2005 ) where it binds a 5′-HGATAR-3′ sequence in its target promoters ( Ravagnani et al . , 1997 ) , as shown for the genes encoding nitrate reductase ( NiaD ) and nitrite reductase ( NiiD ) ( Chang et al . , 1996 ) that are upregulated in the absence of preferred nitrogen sources ( Johnstone et al . , 1990; Punt et al . , 1995 ) .","In Aspergillus parasiticus AreA also binds to the promoter regions of the transcriptional regulator genes aflR and aflJ from the aflatoxin biosynthesis cluster ( Chang et al . , 2000 ) .","In contrast to nitrate utilisation , this binding impedes aflatoxin production probably due to inhibition of transcriptional activation ( Yu et al . , 2004 ) .","We have shown that terrein biosynthesis in A . terreus during nitrogen starvation is AreA-dependent , and at least two areA binding sites in the terR promoter were recognised by the AreA DNA-binding domain .","Therefore , AreA directly binds to the terR promoter and supports the expression of the terrein biosynthesis genes .","Similarly , biosynthesis of various mycotoxins and gibberellins is activated by AreA in phytopathogenic Fusarium species ( Mihlan et al . , 2003; Kim and Woloshuk , 2008; Giese et al . , 2013 ) .","This implies that AreA plays a general role in regulation of SM production of plant pathogens , and the plant-derived environment may be characterised as a habitat with a high C:N ratio .","In several ascomycetes the nitrogen starvation response is also mediated by the basic leucine zipper transcription factor AtfA ( Hong et al . , 2013 ) .","In A . nidulans , nitrogen starvation , oxidative or osmotic stresses induce phosphorylation of the MAP kinase SakA that is subsequently transported into the nucleus to activate transcription of target genes by interaction with the constitutively expressed transcription factor AtfA ( Lara-Rojas et al . , 2011 ) .","Interestingly , in A . terreus the interaction between AtfA and SakA has not been solved in detail .","While a sakA deletion does not affect terrein production levels under nitrogen limitation , it allows terrein production in the presence of some nitrogen sources , indicating that it may possess a repressor function under some conditions .","In addition , a ΔsakA strain still produces pigmented conidia ( Figure 4—figure supplement 5 ) and the sakA mutant—but not the ΔatfA strain—showed some increased osmosensitivity ( Figure 4—figure supplement 5 ) , which agrees with recent data on ΔatfA and ΔsakA mutants of Penicillium marneffei ( Nimmanee et al . , 2014 ) .","Therefore , it is conceivable that terrein biosynthesis gene cluster activation via AtfA does not require SakA .","In A . terreus AtfA seems to act as a general inducer of SM production since its deletion resulted in non-pigmented conidia and in the loss of terrein production under nitrogen starvation or methionine supplementation .","In contrast , deletion of the atf1 gene in Botrytis cinerea resulted in accumulation rather than depletion of the polyketide botcinin A and the sesquiterpene botrydial ( Temme et al . , 2012 ) .","This points to AtfA as a transcriptional inhibitor in B . cinerea whereas AtfA acts as an inducer in A . terreus .","Interestingly , AreA and AtfA sense the same environmental signals to induce the terrein biosynthesis gene cluster , although their global role in cellular physiology differs significantly .","This adaptation of different transcription factors towards terrein production underlines a special importance of terrein for A . terreus during environmental competition .","Independent from AreA and AtfA , activation of the terrein biosynthesis gene cluster was also triggered by the iron response regulator HapX under iron limitation .","To overcome severe iron starvation , microorganisms frequently produce siderophores ( Pollack and Neilands , 1970; Eisendle et al . , 2003; Kreutzer and Nett , 2012 ) or utilise siderophores from competing species ( xenosiderophores ) ( Boukhalfa and Crumbliss , 2002 ) .","In A . fumigatus and A . nidulans siderophore biosynthesis and uptake is regulated by the opposing transcription factors HapX and SreA depending on the presence or absence of iron ( Schrettl et al . , 2007 , 2008 , 2010 ) .","Similarly , A . terreus produces the HapX and SreA regulated siderophores ferrichrysin and coprogen that allow efficient acquisition of iron .","However , in competition with other micoorganisms , terrein-mediated iron reduction could reduce binding of iron to high-affinity siderophores from competitors that cannot be used by A . terreus , as described for the cysteinylglycine iron reduction system from H . capsulatum .","The control of terrein production by HapX depicts an unprecedented example of the activation of a SM gene cluster outside the siderophore biosynthesis system .","Interestingly , terrein production is only under positive HapX control , but not under negative SreA feedback control , which is in agreement with a lack of SreA binding sites in the terR promoter ( Schrettl et al . , 2008 ) , whereas a CCAAT box required for binding of the CCAAT-binding complex ( CBC ) and its subsequent CBC interaction with HapX ( Hortschansky et al . , 2007; Gsaller et al . , 2014 ) is present .","In the HapX target promoter regions of cycA , sreA , acoA , and lysF from Aspergillus species , a bipartite CBC–HapX-DNA binding motif with consensus 5′-C ( C/A ) AATCAN11-12GAT-3′ is present , of which CCAAT is recognised by the CBC complex while the GAT sequence is bound by HapX ( Hortschansky et al . , 2015 ) .","Interestingly , these are all types of promoters that are repressed under iron limitation .","In contrast , the cccA promoter , which encodes for a vacuolar iron transporter , is the only HapX-induced promoter known so far with a confirmed in vitro HapX binding site ( Gsaller et al . , 2014 ) .","HapX binds to the GAT motif of a 5′-CCAATN16GATC-3′ sequence , which is also present in the cccA promoter of A . terreus and—with one mismatch—in the terR promoter ( position −215 ) .","However , further studies will be required to confirm this motif as a HapX binding site .","In conclusion , we have shown that the terrein biosynthesis gene cluster is activated by at least three independent environmental signals: methionine , nitrogen limitation , and iron starvation .","Additionally , the three global transcription factors AreA , AtfA , and HapX are essential to transfer these signals to the terrein biosynthesis gene cluster .","Although one environmental signal is sufficient to induce the gene cluster , a combination of different signals provides an environmental image of higher resolution and allows an adequate adaptation to the ecological niche .","The phytotoxic and antifungal potential of terrein combined with its iron reductive properties enables A . terreus to acquire nutrients that might otherwise be consumed by competitors .","Notably , similar to terrein , the mycotoxin production in Fusarium species is generally associated with sensing of nitrogen availability .","Although AreA plays an important regulatory function , involvement of AtfA as an additional regulator and methionine as a second inducer in these species needs to be tested .","However , from our results on the terrein biosynthesis gene cluster , we propose that manifold regulation of SM gene clusters and the interplay of different global transcriptional activators might depict a general mechanism to regulate the flexibility of gene cluster activation in fungi ."],["All genotypes of strains used in this study are summarised in ( Figure 4—source data 1 ) .","A . terreus strain SBUG844 and its derivative SBUG844ΔakuB that show increased rates of homologous integration served as parental strains ( Gressler et al . , 2011 ) .","If not indicated otherwise , all strains were cultivated in Aspergillus minimal medium ( AMM; http://www . fgsc . net/methods/anidmed . html ) supplemented with different carbon and nitrogen sources: AMM with 100 mM glucose ( AMM-G100 ) , with 100 mM glucose and 50 mM glutamine ( AMM-G100Gln50 ) , with 100 mM glucose , 50 mM glutamine and 10 mM methionine ( AMM-G100Gln50Met10 ) , with 1% casamino acids ( AMM-CA1% ) .","Additionally , the following complex media were used: Sabouraud ( Sab , Sigma Aldrich , Taufkirchen , Germany ) , YPD ( 10 g/l yeast extract , 20 g/l peptone , 20 g/l dextrose; PDB , Sigma Aldrich ) , potato broth ( 2% potato extract , Sigma Aldrich ) , apple juice ( pH adjusted to 6 . 0 with NaOH ) , carrot juice with honey ( both Wiesgart , ALDI Nord , Jena , Germany ) , banana juice or peach juice ( both FruchtOase , Kiberg , Germany ) .","When required , 140 µg/ml hygromycine B ( Carl Roth GmbH , Karlsruhe , Germany ) , 0 . 1 µg/ml pyrithiamine or 80 µg/ml phleomycin ( both Sigma Aldrich ) were added .","For preparation of conidia suspensions , all strains were cultivated for 4 d at 37°C on solid 2% AMM-G50Gln10 agar plates .","Conidia were harvested by overlaying colonies with water and filtering off the suspension over 40 μm cell strainers ( VWR , Darmstadt , Germany ) .","Liquid media were generally inoculated at a final concentration of 1 × 106 conidia per ml .","For the ΔareA strains , inoculation densities were generally doubled to compensate for reduced growth rates .","For metabolite extraction and quantification liquid cultures in 50 ml scale were used and incubated at 30°C for 48 hr or 72 hr depending on the specific experiment .","For nitrogen or iron shift experiments , strains were pre-cultivated for 40 hr in 150 ml AMM-G100Gln50 or AMM-G100Gln50 with 200 µM FeCl3 .","Mycelia were harvested over sterile miracloth ( Merck , Darmstadt , Germany ) washed twice with AMM lacking a nitrogen ( AMM-N ) or iron source ( AMM-Fe ) and aliquots were transferred to fresh 50 ml media with or without 50 mM Gln or with or without 200 µM FeCl3 .","Depending on the specific experiments , samples were analysed after 12 , 24 , or 48 hr of cultivation .","Metabolites were extracted from culture broth as described previously ( Gressler et al . , 2011 ) .","In brief , an equal volume of ethyl acetate was added and collected after defined shaking of the mixture .","The procedure was repeated once .","After evaporation of the solvent , residues were taken up in 1 ml methanol each and filtered .","Standard extract analyses were performed on an Agilent 1100 series HPLC-DAD system coupled with a MSD trap ( Agilent Technologies , Waldbronn , Germany ) operating in alternating ionisation mode .","Terrein quantification was carried out from 50 ml cultures as described elsewhere ( Zaehle et al . , 2014 ) .","For quantification of the siderophore coprogen , the complete 50 ml culture supernatants were filtered and lyophilised to dryness .","The remaining solids were extracted three times with 10 ml MeOH .","The solvent from the combined organic extracts was removed under reduced pressure and residues were re-dissolved in 2 ml MeOH .","The resulting slurries were filtered and the filtrates analysed by HPLC measurements .","HPLC analyses were carried out on an Agilent 1260 device equipped with a quaternary pump and a UV/Vis detector ( Agilent Technologies; Column: Zorbax Eclipse XDB-C8 , 5 µm , 150 × 4 . 6 mm; flow rate 1 ml/min; eluent A: H2O/0 . 1% HCOOH , eluent B: MeOH ) .","The gradient started with 10% B and reached 30% B after 4 min , increased to 55% B within 10 min and reached 100% B after 2 min , where it was retained for an additional 4 min .","Quantification of coprogen was performed from a calibration curve of known coprogen concentrations .","For correlation of coprogen to the fungal biomass , mycelia from the cultures were dried for 48 hr at 37°C and balanced and coprogen concentrations per gram dried mycelium were calculated .","All quantifications were carried out in biological triplicates and technical duplicates .","Isolation of coprogen for generation of the calibration curve was performed by semi-preparative HPLC from culture supernatants of the ΔakuB and ΔakuBΔterA strains and fractions were collected by automatic fraction collection .","Separation was carried out on a Zorbax Eclipse XDB-C8 , 5 µm , 250 × 4 . 6 mm with a flow rate of 4 . 0 ml/min using H2O as eluent A and MeOH as eluent B . The gradient started with 10% B , reached 30% B after 6 . 5 min , increased to 55% B within 16 . 5 min , reached 100% B after 2 min , and was retained at 100% B for an additional 6 min .","For isolation of 2- ( ( E ) -prop-1-en-1-yl ) maleic acid , the crude product from upscaled terrein reduction assays ( see below ) was subjected to semi-preparative HPLC using a Zorbax Eclipse XDB-C8 , 5 µm , 250 × 4 . 6 mm with a flow rate of 4 . 0 ml/min , eluent A: H2O/0 . 1% HCOOH , eluent B: acetonitrile .","The gradient started with 5% B and was held for 14 min , increased to 10% B within 9 min , increased to 100% B within 2 min where it was retained for an additional 7 min .","Fractions from the new metabolite formed from ferric iron reduction were collected and evaporated resulting in a white solid which revealed a m/z value of 150 . 0495 [M + H+] by HRESI-MS that perfectly matched a calculated molecular formula of C7H8O4 containing four double-bond equivalents .","13C-NMR measurements ( Figure 5—figure supplement","4 ) revealed the presence of two carbonyl groups , one terminal methyl group , and four carbons being part of a conjugated system .","Two-dimensional NMR data ( Figure 5—figure supplement","5 ) and analysis of all proton coupling constants from the 1H-NMR spectrum ( Figure 5—figure supplement","4 ) finally confirmed the structure of 2- ( ( E ) -prop-1-en-1-yl ) maleic acid .","NMR spectra were recorded on a Bruker Avance III 500 and a Bruker Avance III 600 spectrometer ( Bruker BioSpin GmbH , Rheinstetten , Germany ) equipped with a cryoprobe head using DMSO-d6 and methanol-d4 as solvents and internal standards .","Fruit surfaces were wiped with a soft tissue saturated with 70% ethanol .","Apples ( ALDI , type: Tenroy Royal/Gala; Germany ) , bananas ( type Bio , Fairverbindet; tegut , Jena , Germany ) , and nectarines ( tegut , type: Sweet Lady , class 1 , size A; Italy ) were cut lengthwise using a sterile scalpel .","The resulting groove was infected with 200 µl of a conidia suspension containing 4 × 107 conidia and fruits were incubated for 7–10 d at room temperature in a humid chamber .","Cut but uninfected fruits served as controls .","For cultivation in the presence of high levels of nitrogen , 200 µl of a 3 . 5 M NH4Cl solution were applied prior to infection .","For terrein quantification , fruits were homogenised and extracted twice with 100 ml ethyl acetate and the solid residues were collected and evaporated for dry weight determination .","Terrein was quantified from extracts as described above and terrein production rates were calculated as mg terrein/g fruit dry weight .","A detailed description on fruit infection , sample preparation and terrein quantification is described in Bio-protocols by Gressler and Brock ( 2016 ) .","To determine the induction of lesion formation on banana fruit surfaces , organic bananas ( type Bio , Fairverbindet; tegut ) were softly cleaned with water and air dried .","5 µl of various sequential dilutions ( 1:2 to 1:512 ) of metabolite crude extracts ( dissolved in PBS ) were added as a single drop to the surface .","Fruits were incubated in the dark at room temperature and photographs were taken after 40 hr and 60 hr .","The determination of ammonia was performed as described by Weichselbaum et al . ( 1969 ) .","From each culture 1 ml aliquots of broth were removed and centrifuged for 5 min at 16 , 000×g to remove residual mycelium .","The supernatant was collected and adjusted to pH 7 . 0 by addition of NaOH .","A 200 µl aliquot was sequentially diluted in a nitrogen-free medium and transferred to a transparent flat-bottom 96-well plate .","After addition of 20 µl SC solution ( 6 . 5 g sodium salicylate ( C7H5O3Na ) , 6 . 5 g trisodium citrate ( C6H5O7Na3 × 2 H2O ) , 48 . 5 mg disodium pentacyanonitrosylferrate ( Na2Fe ( CN ) 5NO × 2 H2O ) in 50 ml water ) , the reaction was started with 20 µl DCIC solution ( 1 . 6 g NaOH , 100 mg sodium dichloroisocyanurate ( C3N3Cl2O3Na ) in 50 ml water ) .","Readings were done after 4 . 5 hr of incubation at room temperature in a FLOUstar Omega microplate reader ( BMG Labtech , Ortenberg , Germany ) .","Plates were shaken for 1 min in a double orbital direction and absorbance at 655 nm was measured ( 50 flashes/well , gain 2000 ) .","Fresh growth medium served as positive and water as negative controls .","A calibration curve was recorded for calculation of ammonia levels from culture broth .","The ferric iron-reducing assay was performed virtually as described by Benzie and Strain ( 1996 ) .","In brief , three different working solutions were prepared by mixing 20 parts of reagent A ( 0 . 4% sodium acetate , 1 . 6% acetic acid; pH","4 ) with 1 part of reagent B ( 0 . 4% 2 , 4 , 6-tripyridyl-S-triazine ( TPTZ; Sigma ) in 0 . 16% HCl and either 1 part of reagent C1 ( 88 mM–5 . 5 mM sequential dilutions of terrein ) , reagent C2 ( 2% ascorbic acid , serving as positive control ) or reagent C3 ( water , serving as negative control ) .","From each working solution , 200 µl were transferred into wells of a 96-well plate .","An iron stock solution ( 10 mg/ml FeCl3 in 0 . 2 M H2SO4 ) was diluted to 0 . 05 mg/ml and 100 µl were added to the working solution , which started the reaction .","Reduction of ferric to ferrous iron was followed by determination of the change in absorbance at 590 nm for up to 10 hr using a FLOUstar Omega microplate reader ( BMG Labtech ) .","For determination of the pH optimum of the terrein mediated reduction , reagent A was adjusted to pH values of 3 . 5–6 . 0 .","Reductive activity was normalised against the activity determined from ascorbic acid , which was set as 100% .","All experiments were carried out in triplicate .","Upscaling of the iron reduction assays for metabolite extraction was performed by incubating 20 mg ( 125 µmol ) of terrein in a 1:2 molar ratio with either FeSO4 , FeCl3 , CH3COOCu , or ( CH3COO ) 2Cu in 10 ml of reagent A . Samples were incubated for 48 hr at room temperature under continuous vertical rotation ( 20 rpm ) .","Metabolites were extracted three times with ethyl acetate ( 10 ml each ) .","The solvent was evaporated and the residues dissolved in MeOH and analysed as described above .","Chrome azol S agar plate assays were prepared as described by Milagres et al . ( 1999 ) .","Holes of 8 mm diameter were punched into plates and inoculated with 100 µl methanolic solutions of coprogen , terrein , or propylene maleic acid in concentrations of 2 . 50 , 1 . 25 , 0 . 625 , and 0 . 3125 mg/ml .","Plates were incubated for 48 hr at 30°C prior to photography .","Mycelia from specified cultivations were briefly washed with water and ground under liquid nitrogen .","RNA was isolated using the RiboPure RNA Purification Kit ( Ambion , Life Technologies , Darmstadt , Germany ) .","Residual genomic DNA ( gDNA ) was removed by the DNA-free kit ( Ambion ) .","cDNA was synthesised by Revert Aid Reverse transcriptase ( Thermo Scientific , Schwerte , Germany ) using anchored oligodT primers .","qRT-PCR was carried out on a CFX384 Touch Real-Time PCR Detection System ( BioRad , Munich , Germany ) using the EvaGreen 5 × QPCR ( ROX ) Mix ( Bio & Sell , Feucht , Germany ) following the manufacturer's protocol and using 1:5 and 1:10 dilutions of cDNA samples serving as templates .","The actin gene ( actA , ATEG_06973 ) and the enolase gene ( enoA , ATEG_02902 ) were used for normalisation of transcript levels yielding similar results .","Normalised transcript levels were calculated as fold expression = 2Δ ( reference − target ) .","Primers used for qRT-PCRs showed a primer efficiency of 1 . 89–2 . 0 and are listed in Figure 4—source data 2 ( Figure 4—source data 2 ) .","In general , all PCR amplifications were performed using the Phusion DNA Polymerase ( Thermo Scientific ) .","Plasmids were amplified in E . coli DH5α .","A detailed description of all cloning procedures is given below .","Oligonucleotides used for plasmid construction are listed in the Figure 4—source data 2 ( Figure 4—source data 2 ) .","Fungal transformation was performed as described previously ( Gressler et al . , 2011 ) and transformants were checked by diagnostic PCR and Southern blot analyses ( see Figure 4—figure supplement 4 ) .","The promoter of the terA gene ( PterA; 1220 bp ) was amplified with oligonucleotides P47/48 from gDNA of A . terreus SBUG844 and ligated into the NotI/BamHI digested plasmid lacZ:trpCT-pJET1 . 2 ( Gressler et al . , 2011 ) containing the E . coli lacZ gene and the trpC terminator sequence .","The plasmid was linearised by NotI digestion and the NotI-excised ptrA cassette from plasmid ptrA-pJET ( Fleck and Brock , 2010 ) was inserted .","The resulting plasmid was used for transformation of A . terreus SBUG844 wild-type resulting in SBUG844_PterA:lacZ .","Constitutive strong expression of the atfA gene was performed by using the strong constitutive gpdA promoter from A . nidulans FGSC A4 ( AnPgpdA , 1387 bp; oligonucleotides P49/50 ) to control expression of A . terreus atfA .","The atfA ORF together with its natural terminator was amplified from gDNA of A . terreus SBUG844 ( atfA + atfAT , 2466 bp ) using oligonucleotides P51/52 .","Both fragments were fused by in vitro recombination with the SpeI- restricted hph-pCRIV vector using the InFusion HD cloning kit ( Clonetech Laboratories , Saint-Germain-en-Laye , France ) .","The resulting plasmid AnPgpdA:atfA:atfAT_hph-pCRIV was used for transformation of A . terreus SBUG844 wild-type resulting in strain SBUG844_AnPgpdA:atfA .","The upstream and downstream fragments of cpcA ( ATEG_03131 ) were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P53/54 ( 943 bp ) and P55/56 ( 853 bp ) .","Similarly , the upstream and downstream flanks of rhbA ( ATEG_09480 ) were amplified with oligonucleotides P57/58 ( 473 bp ) and P59/60 ( 418 bp ) .","The respective fragments were fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) , resulting in cpcAup-ptrA-cpcAdn-pUC19 , rhbAup-ptrA-rhbAdn-pUC19 , and dnmTup-ptrA-dnmTdn-pUC19 .","The deletion cassettes were excised by KpnI restriction and used for transformation of SBUG844ΔakuB .","The upstream and downstream flanks of areA ( ATEG_07264 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P61/62 ( 820 bp ) and P63/64 ( 731 bp ) .","Fragments were fused by fusion PCR and ligated into the pJET1 . 2 cloning vector ( Thermo Scientific ) .","The fragment was excised with SmaI and subcloned into pUC19 .","After restriction with NotI , the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) was inserted resulting in areAup-ptrA-areAdn-pUC19 .","The deletion cassette was excised by SmaI and used for transformation of SBUG844ΔakuB .","For complementation , a fragment spanning the entire areA ORF including its promoter and terminator sequence was amplified from gDNA of the A . terreus type strain FGSC A1156 ( NIH2624 ) using oligonucleotides P65/64 ( 4181 bp ) and ligated into pJET1 . 2 .","gDNA from the type strain was used because its areA promoter sequence contained an additional PstI restriction site that allowed discrimination of complemented strains from the parental SBUG844ΔakuB strain .","The complementation fragment was excised by XhoI restriction from plasmid A1156 ( areAup:areA:areAdn ) -pJET1 . 2 and directly used for the transformation of SBUG844ΔakuBΔareA .","Due to the inability of the ΔareA mutant to utilise various nitrogen sources , no additional resistance marker was required and transformants were regenerated on media containing nitrate as the sole nitrogen source .","Complemented strains were checked by Southern blot analysis and an additional PstI control digest of the PCR-amplified areA upstream flank .","The upstream and downstream fragments of atfA ( ATEG_04664 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P66/67 ( 1102 bp ) and P68/69 ( 890 bp ) and fused by in vitro recombination with the NotI-excised ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-excised pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in atfAup-ptrA-atfAdn-pUC19 .","The deletion cassette was excised by KpnI and used for transformation of SBUG844ΔakuB .","For deletion of atfA in the ΔareA background , the ptrA resistance cassette was replaced by the phleomycin resistance ( ble ) cassette ( NotI digest from ble-pJET1 . 2 ) .","The deletion cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuBΔareA .","For complementation of the ΔatfA phenotype , a fragment spanning the entire atfA gene including its promoter and terminator region were amplified with oligonucleotides P66/70 ( 3319 bp ) and an additional downstream fragment was amplified with P71/69 ( 859 bp ) .","The fragments were fused by in vitro recombination with the NotI-excised ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in atfAup:atfA:atfAT_ble_atfAdn-pUC19 .","The complementation cassette was excised by KpnI and used for transformation of SBUG844ΔakuBΔatfA .","The atfA flanking regions were used to integrate the terR gene under control of the A . nidulans gpdA promoter in the atfA locus , which results in an atfA deletion .","The upstream and downstream flanks of atfA were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P72/73 ( 1125 bp ) and P74/75 ( 913 bp ) .","Subsequently , a fragment already containing the fusion of AnPgpdA with the terR ORF including its native terminator ( 4405 bp ) was amplified with oligonucleotides P76/77 from plasmid hph_AnPgpdA:terR-pUC19 ( Gressler et al . , 2015a ) .","The plasmid hph-pCRIV ( Fleck and Brock , 2010 ) was restricted with EcoRI to remove the hph resistance cassette and all three fragments were fused by in vitro recombination into the EcoRI site using the InFusion HD cloning kit ( Clonetech Laboratories ) .","The resulting plasmid atfAup_AnPgpdA:terR:terRT_atfAdn_pCRIV was linearised with NotI and the ble resistance cassette from ble-pJET1 . 2 was inserted between the terR terminator and the atfA downstream region .","The final plasmid was restricted with EcoRI , and the fragment atfAup_AnPgpdA:terR:terRT_ble_atfAdn was used for transformation of A . terreus SBUG844ΔakuBΔareA to replace the atfA ORF with the terR overexpression construct .","Because the complete sequence information of the hapX locus was lacking at the beginning of this study , only a partial deletion of the A . terreus hapX gene was performed .","Upstream and downstream flanks inside the hapX coding region ( ATEG_08014 ) were amplified with oligonucleotides P82/83 ( 387 bp ) and P84/85 ( 345 bp ) from genomic DNA of SBUG844 and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in hapXup-ptrA-hapXdn-pUC19 .","The deletion construct was excised by KpnI restriction and used for transformation of SBUG844ΔakuB .","For complementation of the partial hapX deletion by the A . nidulans hapX , the upstream and downstream flanks of ATEG_08014 used for generation of the partial deletion construct were amplified with oligonucleotides P82/86 ( 381 bp ) and P87/85 ( 341 bp ) and the complete hapX ORF ( AN8251 ) including its native promoter and terminator sequence was amplified from gDNA of A . nidulans FGSC A4 using oligonucleotides P88/89 ( 2648 bp ) .","All fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 ( 2073 bp ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in hapXup_PAnhapX:AnhapX:AnhapXT_ble_hapXdn-pUC19 .","The complementation cassette was excised by KpnI and used for transformation of SBUG844ΔakuBΔhapX .","To identify the complete coding region of the A . terreus hapX gene , long run PCRs from within the hapX gene and the last nucleotides of the known 5′- and 3′- borders were performed with oligonucleotides P110/111 ( 800 bp 5′ fragment ) and P112/113 ( 2500 bp 3′ fragment ) .","Bands were excised from agarose gels and cloned into the pJET1 . 2 cloning vector .","Fragments were sequenced from both strands using the primer walking method ( oligonucleotides P114/115 for both fragments and additionally P116 , P117 and P78 for the 3′ fragment ) .","Finally , RNA was isolated from iron starvation conditions , transcribed into cDNA , and the hapX ORF was amplified with gene-specific oligonucleotides 113/79 and sequenced with oligonucleotides P114/115 .","The complete hapX locus information was submitted to EMBL and can be found under accession number KP233834 ( Gressler et al . , 2015b ) .","The upstream and downstream flanking regions of sreA ( ATEG_07714 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P90/91 ( 763 bp ) and P92/93 ( 756 bp ) and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sreAup-ptrA-sreAdn-pUC19 .","The deletion cassette was excised by KpnI and used for transformation of SBUG844ΔakuB .","For complementation , a fragment spanning the sreA coding region including its natural promoter and terminator sequence was amplified with oligonucleotides P94/95 ( 3018 bp ) and additional downstream fragment with P96/97 ( 970 bp ) .","The fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sreAup:sreA:sreAT_ble_sreAdn-pUC19 .","The complementation cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuBΔsreA .","The upstream and downstream fragments of the sidA gene ( ATEG_06879 ) were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P98/99 ( 764 bp ) and P100/101 ( 939 bp ) and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sidAup-ptrA-sidAdn-pUC19 .","For transformation of the terA deletion mutant , the ptrA cassette was excised by NotI restriction and replaced by the ble cassette .","The deletion cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuB and SBUG844ΔakuBΔterA .","For complementation of strain SBUG844ΔakuBΔsidA , the sidA ORF including its promoter and terminator sequence was amplified with oligonucleotides P102/103 ( 2393 bp ) and an additional downstream fragment was amplified with P104/105 ( 919 bp ) .","The fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the HindIII-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sidAup:sidA:sidAT_ble_sidAdn-pUC19 .","The complementation cassette was excised by HindIII restriction and used for transformation of SBUG844ΔakuBΔsidA .","The coding sequence for the A . nidulans AreA DNA-binding domain ( amino acids 663–797 ) was amplified from plasmid pGEX4T1-AreA-ZnF ( Muro-Pastor et al . , 2004 ) as BamHI-HindIII fragment using oligonucleotides P80/81 ( Figure 4—source data 2 ) .","The amplified fragment was cloned into a modified pET-43 . 1a vector allowing the addition of a N-terminal ( His ) 6-tag and removable by a tobacco etch virus ( TEV ) protease ( Novagen ) .","( His ) 6-AreA663-797 was produced by E . coli BL21 ( DE3 ) cells grown at 30°C in 1 l Overnight Express Instant TB Medium ( Novagen , Darmstadt , Germany ) in the presence of 1 mM Zn ( OAc ) 2 .","Cells ( 15–20 g wet weight ) were collected by centrifugation , resuspended in 200 ml lysis buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 10% vol/vol glycerol , 40 mM imidazole , 5 mM β-mercaptoethanol , 1 mM AEBSF , pH 8 . 0 ) and disrupted using an Emulsiflex C5 high pressure homogeniser ( Avestin , Mannheim , Germany ) .","Cleared cellular extract was loaded to a 25 ml Ni Sepharose FF ( GE Heathcare , Freiburg , Germany ) column and ( His ) 6-AreA663-797 was eluted with 200 mM imidazole .","After removal of the ( His ) 6 tag by adding 4 μg TEV protease per mg peptide and overnight incubation at room temperature , samples were adjusted to 150 mM NaCl and applied on a 40 ml CellufineSulfate ( Millipore , Darmstatdt , Germany ) column that was equilibrated with 50 mM NaH2PO4 , 150 mM NaCl , 10% vol/vol glycerol , 5 mM β-mercaptoethanol , 10 µM Zn ( OAc ) 2 , pH 7 . 5 , followed by elution of AreA663–797 with a gradient up to 2 M NaCl .","Peak fractions were concentrated with an Amicon Ultra-15 10K centrifugal filter device and purified to homogeneity by size exclusion chromatography on a Superdex 75 prep grade column ( GE Healthcare ) in 20 mM HEPES , 300 mM NaCl , 5 mM β-mercaptoethanol , 10 µM Zn ( OAc ) 2 , pH 7 . 5 as running buffer .","The absolute molecular mass of AreA663–797 was determined in series on a miniDawn TREOS static light scattering monitor and an Optilab T-rEX differential refractometer ( Wyatt , Dernbach , Germany ) .","The molar mass was calculated using ASTRA 6 software ( Wyatt ) .","AreA663–797 was stored in 50% vol/vol glycerol at −20°C .","Real-time analyses were performed on a Biacore 2000 system ( GE Healthcare ) at 25°C .","DNA duplexes were produced by annealing complementary oligonucleotides using a fivefold molar excess of the non-biotinylated oligonucleotide .","The dsDNA was injected on flow cells of a streptavidin ( Sigma ) -coated CM3 sensor chip at a flow rate of 10 µl/min until the calculated amount of DNA that gives a maximum AreA663–797 binding capacity of 100 RU were bound .","AreA663–797 was injected in running buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 0 . 005% ( vol/vol ) surfactant P20 , 5 mM β-mercaptoethanol and 1 µM ZnCl2 ) at concentrations from 3 . 125 to 200 nM .","Sample injection and dissociation times were set to 200 and 400 s at a flow rate of 30 µl/min .","Regeneration was achieved with 10 mM Tris/HCl pH 7 . 5 , 0 . 5 M NaCl , 1 mM EDTA and 0 . 005% ( wt/vol ) SDS for 1 min .","Refractive index errors due to bulk solvent effects were corrected with responses from DNA-free flow cell 1 as well as subtracting blank injections .","Kinetic raw data were processed and globally fitted with Scrubber 2 . 0c ( BioLogic Software ) using a 1:1 interaction model including a mass transport term .","All analyses in which statistical analyses were required were performed in biological triplicates with at least two technical replications .","Significance was calculated by use of the Microsoft Excel 2007 software package using the Student's paired t-test with a two-tailed distribution; p values were marked as follows: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 ."]],"string":"[\n [\n \"Fungal secondary metabolites ( SMs ) are often encoded by gene clusters and have been the subject of intensive analysis in recent years .\",\n \"This has resulted from the large number of completed fungal genomes as well as the intrinsic interest of SMs as environmental/pathogenic agents and their potential for pharmacological use .\",\n \"However , there are few examples where the regulation of the expression of these clusters has been shown to be ecologically/physiologically relevant ( Rohlfs and Churchill , 2011 ) .\",\n \"Except for penicillin and perhaps aflatoxin and gliotoxin , the potential real-life functions of most SMs are unknown ( Bhatnagar et al . , 2006; Vargas et al . , 2014 ) .\",\n \"Most clusters contain a pathway-specific transcription factor , and the common approach is to artificially over-express the cluster genes by constitutive expression of this transcription factor and chemically characterise the product ( Yin and Keller , 2011 ) .\",\n \"In most cases the relevance of the SM is not known .\",\n \"In this study the biological function of terrein from the filamentous ascomycete Aspergillus terreus was investigated .\",\n \"A . terreus is a filamentous ascomycete of biotechnological and medical importance since it produces itaconic acid ( Steiger et al . , 2013 ) and lovastatin ( Bizukojc and Ledakowicz , 2015 ) , but it is also a causative agent of life-threatening invasive aspergillosis in immunocompromised patients ( Baddley et al . , 2003; Slesiona et al . , 2012b ) and has recently been described as a pathogen of potato leaves ( Louis et al . , 2013 ) .\",\n \"While searching for a candidate protein required for pigment synthesis in A . terreus conidia , we serendipitously identified the gene cluster responsible for terrein production ( Zaehle et al . , 2014 ) , which is the major SM formed by A . terreus .\",\n \"Terrein production is highly pronounced on sugar-rich plant-derived media such as potato dextrose broth ( PDB ) ( Zaehle et al . , 2014 ) and shows phytotoxic activities such as inhibition of seed germination and lesion formation on fruit surfaces ( Kamata et al . , 1983; Zaehle et al . , 2014 ) .\",\n \"The terrein biosynthesis gene cluster consists of 11 genes ( terA–J , terR ) , whereby terA encodes the key enzyme , which is a non-reducing polyketide synthase ( Zaehle et al . , 2014 ) , and terR codes for the transcriptional activator TerR containing a GAL4-type Zn2Cys6 binuclear cluster DNA-binding domain ( Gressler et al . , 2015a ) , as frequently found in transcriptional activators of fungal SM gene clusters .\",\n \"Although activation of cluster genes is strictly TerR-dependent , no signals that lead to TerR activation under in vivo conditions have yet been identified .\",\n \"Since terrein is the major SM produced by A . terreus—gram scales are easily achieved—we assumed a benefit from its production in the natural habitat .\",\n \"To elucidate this question , a detailed knowledge on the inducing factors stimulating terrein production and analyses of its biological activities were required .\",\n \"Therefore , we aimed to investigate environmental signals that result in terR expression and , eventually , the production of terrein .\",\n \"From these analyses , the impact of different global transcription factors on cluster induction was deduced .\",\n \"Two global transcription factors sense the quality and availability of nitrogen sources and specifically respond to the plant and fruit environment .\",\n \"Additionally , the iron responsive regulon plays a vital role in cluster induction , which indicates a specific contribution of terrein in modulating iron homeostasis .\"\n ],\n [\n \"In previous studies we showed that terrein is produced on plant-derived media such as PDB , which is in agreement with its phytotoxic biological activity ( Zaehle et al . , 2014 ) .\",\n \"To address the question of specific conditions that induce the gene cluster , an A . terreus reporter strain was generated expressing the β-galactosidase gene lacZ from Escherichia coli under control of the terrein synthase promoter PterA ( PterA:lacZ ) .\",\n \"Due to the dependence of PterA expression on TerR , this strain served as a direct indicator of terR expression and terrein production .\",\n \"In agreement with a lack of terrein production , β-galactosidase activity was near the detection limit when the A . terreus PterA:lacZ strain was grown on glucose minimal medium .\",\n \"In contrast , and in agreement with previous observations , a 200–500 fold induction was observed on PDB medium ( Figure 1A ) .\",\n \"Similarly , on Sabouraud and yeast extract-peptone-dextrose ( YPD ) medium , induction levels reached 10–30% compared with PDB .\",\n \"However , potato broth or casamino acids did not induce the cluster without the addition of glucose , indicating that glucose appears to be required for terrein production rather than repressing gene cluster induction as shown for other SM gene clusters ( Theilgaard et al . , 1997; Brakhage et al . , 2004; Gressler et al . , 2011 ) .\",\n \"Indeed , when glucose medium was supplemented with 1% casamino acids as the nitrogen source , a 20–30 fold activation was detected .\",\n \"Since terrein can cause lesions on fruit surfaces and inhibits plant seed germination ( Kamata et al . , 1983; Zaehle et al . , 2014 ) , we assumed that sugar-rich fruit and root juices might have a strong stimulatory capacity .\",\n \"Therefore , we cultivated the reporter strain on banana , carrot , peach , and apple juice .\",\n \"β-Galactosidase activities from these media exceeded the activity of the already strong inducing PDB medium ( Figure 1B ) by a factor of at least five .\",\n \"Additionally , in subsequent LC analyses of culture extracts , a distinct ultraviolet signal for terrein was detected ( Figure 1—figure supplement 1A–D ) .\",\n \"This led us to infect fresh bananas with the A . terreus ΔakuB strain ( the parental strain for gene deletions; Gressler et al . , 2011 ) , a ΔterR mutant lacking the transcriptional activator , and a ΔterA deletion mutant that lacks the key polyketide synthase from the cluster . 10 . 7554/eLife . 07861 . 003Figure 1 . Terrein production and expression of terA on plant-derived media .\",\n \"( A ) Promoter activity of strain PterA:lacZ after 48 hr and 72 hr on minimal media supplemented with glucose ( Gluc ) , casamino acids ( CA ) , or glucose + casamino acids ( Gluc CA ) or the complex media Sabouraud ( Sab ) , yeast extract-peptone-dextrose ( YPD ) , potato dextrose broth ( PDB ) or potato extract ( PB ) .\",\n \"( B ) Promoter activity of strain PterA:lacZ grown for 24 , 36 and 48 hr in carrot , banana , apple , and peach juice .\",\n \"( C ) High performance liquid chromatography analysis of banana extracts infected with Aspergillus terreus SBUG844 strains ΔakuB , ΔakuBΔterA or ΔakuBΔterR .\",\n \"A mock-infected fruit served as control .\",\n \"1—terrein .\",\n \"( D ) Lesion formation on banana surfaces caused by extracts shown in ( C ) .\",\n \"Photographs were taken after 40 and 60 hr .\",\n \"Lesions only occur with extracts from the wild-type and the parental strain of the mutants ( ΔakuB ) .\",\n \"Numbers indicate the serial twofold dilution of the extracts starting from undiluted crude extracts down to 1:256 dilutions . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00310 . 7554/eLife . 07861 . 004Figure 1—figure supplement 1 . High performance liquid chromatography ( HPLC ) analysis of extracts from Aspergillus terreus strains after cultivation in fruit juices and from infected nectarines and apples .\",\n \"( A–D )\",\n \"HPLC analyses of culture extracts from fruit juices after inoculation with SBUG844_PterA:lacZ ( blue line ) and mock-inoculated juices that served as negative controls ( green line ) .\",\n \"Culture supernatants were extracted 48 hr post inoculation: ( A ) carrot juice , ( B ) banana juice , ( C ) apple juice , ( D ) peach juice .\",\n \"1 = terrein .\",\n \"( E , F )\",\n \"HPLC analyses of fruit extracts after infection with A . terreus wild-type SBUG844ΔakuB ( blue line ) and terrein biosynthesis gene cluster mutants ΔterA ( red ) and ΔterR ( violet ) .\",\n \"Mock-infected fruits served as negative controls ( green line ) : ( E ) nectarine , ( F ) apple .\",\n \"1 = terrein . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00410 . 7554/eLife . 07861 . 005Figure 1—figure supplement 2 . β-Galactosidase activity of PterA:lacZ grown in banana juice without ( control ) or with different supplementations .\",\n \"+Fe = 30 µM FeCl3; +N = 70 mM NH4Cl; +Met = 10 mM L-methionine .\",\n \"Tests were performed in biological triplicates that were analysed in technical triplicates .\",\n \"Promoter activity of terA is reduced by nitrogen supplementation and is completely repressed in the presence of nitrogen and iron in the medium .\",\n \"The addition of methionine partially overwrites this repression .\",\n \"Statistical significance was calculated in comparison to the control condition without supplementation using the Student's paired t-test with a two-tailed distribution: **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 005 Ethyl acetate extraction of bananas infected with ΔakuB strain revealed high amounts of terrein , whereas no terrein was detected after infection with the ΔterR or ΔterA mutant ( Figure 1C ) .\",\n \"Similar results were obtained when fresh apples or nectarines were infected with the three A . terreus strains ( Figure 1—figure supplement 1E , F ) .\",\n \"Furthermore , the extracts of bananas infected with A . terreus wild-type ( and ΔakuB ) caused strong lesion formation on fresh banana peels , which was hardly observed with extracts of bananas infected with the mutants ( Figure 1D ) .\",\n \"Taken together , these results confirm terrein production in a natural habitat of fruit infection , although the specific factors leading to terrein production still remain unclear .\",\n \"Since casamino acid-supplemented glucose medium revealed moderate gene cluster activation ( Figure 1A ) , we assumed that specific amino acids could act as inducers .\",\n \"Therefore , we tested the inducing effect of amino acids as sole nitrogen sources by preparing six distinct pools that covered all 20 canonical proteinogenic amino acids ( Figure 2—figure supplement 1 ) .\",\n \"While most of the pools did not activate terA expression , the pool with aliphatic amino acids caused moderate induction and media containing the sulphur-rich amino acids cysteine and methionine strongly induced gene expression , especially after 72 hr of cultivation ( Figure 2—figure supplement 1 ) .\",\n \"In the presence of the preferred nitrogen source glutamine , the induction by aliphatic amino acids was mainly lost .\",\n \"Also , the activation by cysteine was low and strongly concentration-dependent ( Figure 2A ) .\",\n \"In contrast , methionine provoked a significant induction even at low concentrations .\",\n \"Therefore , we also analysed the effect of other sulphur-containing compounds in two concentrations ( Figure 2B ) .\",\n \"However , no induction was observed when homocysteine , cystathionine , glutathione , dimethylsulfoxide , sulfate , or sulfide were tested .\",\n \"This indicates that methionine itself rather than a sulphur source causes the induction . 10 . 7554/eLife . 07861 . 006Figure 2 . Methionine-dependent terA expression .\",\n \"( A ) β-Galactosidase activity of SBUG844_PterA:lacZ after 48 hr of cultivation in glutamine-containing minimal media in combination with 5 , 10 , or 20 mM of the aliphatic ( Ala , Ile , Leu , Val ) or sulphur-containing amino acid ( Cys , Met ) .\",\n \"Significance calculated against the glutamine control .\",\n \"( B ) β-Galactosidase assay of SBUG844_PterA:lacZ in the presence of various sulphur sources .\",\n \"Glutamines containing minimal media were supplemented with low ( 5 mM ) or high ( 10 mM ) concentrations of Na2SO4 , Na2S , methionine ( Met ) , cysteine ( Cys ) , homocysteine ( Hcy ) , reduced gluthathione ( GSH ) , and dimethylsulfoxide ( DMSO ) .\",\n \"Cystathionine ( Cth ) was used in final concentrations of 1 and 3 mM .\",\n \"Activity was determined after 48 hr of growth .\",\n \"Significance calculated against medium supplemented with sodium sulfate .\",\n \"All cultivations were performed in biological triplicates and activity determinations were made in technical duplicates .\",\n \"Statistical significance was calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00610 . 7554/eLife . 07861 . 007Figure 2—figure supplement 1 . Amino acid-dependent terA expression . All cultivations were performed in biological triplicates and activity determinations were made in technical duplicates .\",\n \"Statistical significance was calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 .\",\n \"β-Galactosidase assay of SBUG844_PterA:lacZ grown in AMM ( -N ) G100 with 1% casamino acids ( CA ) or 50 mM of various amino acid pools as indicated in the table on the right .\",\n \"The promoter activity of terA was determined after 48 and 72 hr .\",\n \"Significance calculated against CA control . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 007 Methionine and PDB medium induced the terrein biosynthesis gene cluster to a similar extent , but remained approximately five times below that of banana juice .\",\n \"This suggested that additional or alternative inducing signals might exist .\",\n \"Fruit juices are rich in sugars and the C:N ratio in fruits is very high , which could result in severe nitrogen limitation at a later growth state .\",\n \"Therefore , another set of experiments was performed in which the total concentration of amino acids was either high ( 50 mM ) or low ( 10 mM ) and PterA induction from the lacZ reporter strain was determined at different time points ( Figure 3A ) .\",\n \"While high concentrations of amino acids or inorganic nitrogen sources such as nitrate or ammonia did not induce the cluster , low nitrogen contents resulted in a time-dependent 40–400 fold induction .\",\n \"To correlate gene expression with nitrogen exhaustion , the nitrogen consumption from a medium supplemented with 10 mM ammonium chloride was monitored and cluster induction was simultaneously analysed .\",\n \"Indeed , as soon as nitrogen levels reached the detection limit , cluster expression was strongly induced ( Figure 3B ) .\",\n \"Subsequently , a culture shift experiment was performed in which mycelium was pre-grown in the presence of 70 mM ammonium chloride and shifted to medium either with or without a nitrogen source ( Figure 3C ) .\",\n \"While no reporter activity and terrein formation was observed from cultures of the nitrogen-rich medium , high β-galactosidase activity accompanied by terrein accumulation was observed in the nitrogen starvation medium . 10 . 7554/eLife . 07861 . 008Figure 3 . Terrein biosynthesis gene cluster activation under nitrogen starvation .\",\n \"( A ) β-Galactosidase activity of SBUG844_PterA:lacZ was cultivated for 24 , 36 , 48 , 72 , and 96 hr in glucose minimal medium supplemented with different concentrations of various nitrogen sources: 70 mM and 10 mM NaNO3 or NH4Cl , 1% or 0 . 1% of casamino acids ( CA ) , and 50 or 10 mM aspartate ( Asp ) , asparagine ( Asn ) , glutamate ( Glu ) , or glutamine ( Gln ) .\",\n \"( B ) Correlation of nitrogen consumption and terA promoter activity determined by β-galactosidase activity and ammonia consumption of SBUG844_PterA:lacZ in glucose minimal medium with 10 mM NH4Cl .\",\n \"( C ) β-Galactosidase activity of SBUG844_PterA:lacZ in nitrogen shift experiments .\",\n \"Cultures grown for 48 hr in glucose minimal medium with 70 mM NH4Cl ( PC ) were washed and transferred to medium with ( +N ) or without 70 mM NH4Cl ( −N ) and promoter activity was determined after 15 hr of cultivation .\",\n \"( D ) Carbon source dependent terA promoter activation under nitrogen starved conditions .\",\n \"Strain SBUG844_PterA:lacZ was pre-cultured for 48 hr on casamino acids without sugar supplementation .\",\n \"The mycelium was washed and transferred to nitrogen-free media with different carbon sources .\",\n \"Reporter activity was determined 24 hr after the shift .\",\n \"( E ) β-Galactosidase activity from bananas infected with conidia suspension of SBUG844 wild-type and PterA:lacZ .\",\n \"Sections ( red boxes ) were cut from bananas , ground to a fine powder and subjected to β-galactosidase activity determination .\",\n \"Activity was only detected from the reporter strain .\",\n \"( F ) Quantification of terrein from wild-type infected bananas with or without ammonium supplementation .\",\n \"All tests were performed in biological triplicates and technical duplicates; p values were calculated by the Student's paired t-test with a two-tailed distribution: **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 008 These results confirmed that nitrogen starvation acts as an inducing factor that might be responsible for the high terrein production levels on fruit juices .\",\n \"In agreement , when ammonium chloride was added to banana juice , β-galactosidase activity was strongly reduced ( Figure 1—figure supplement 2 ) .\",\n \"Similarly , the PterA:lacZ reporter strain displayed high β-galactosidase activity when bananas were directly infected ( Figure 3E ) .\",\n \"When bananas were supplemented with ammonium chloride prior to infection with the wild-type , ethyl acetate extracts of the bananas revealed a 50% reduction in terrein content compared with bananas without nitrogen supplementation ( Figure 3F ) .\",\n \"This finding clearly indicates that nitrogen limitation is a major inducer for terrein production under natural conditions .\",\n \"However , it should be mentioned that the presence of sugars was always required , since especially hexoses from mono- and disaccharides provoked strong expression under nitrogen-limited conditions ( Figure 3D ) , which is in direct contrast to the production of dihydroisoflavipucine in A . terreus that belongs to the class of fruit and root rot toxins .\",\n \"This metabolite is only produced in the strict absence of sugars and requires preferred nitrogen sources such as glutamine or asparagine for induction ( Gressler et al . , 2011 ) .\",\n \"Nitrogen starvation and methionine marked important signals for terrein cluster induction .\",\n \"To unveil the global regulators that could be involved in signal transduction , the A . terreus genome was analysed for the presence of transcription factors known to play a role in nitrogen sensing , cross pathway control of amino acid synthesis , and stress response .\",\n \"The global nitrogen regulator AreA ( ATEG_07264 ) ( Arst and Cove , 1973; Hynes , 1975; Davis et al . , 2005 ) , the cross pathway control regulator CpcA ( ATEG_03131 ) ( Hoffmann et al . , 2001; Krappmann et al . , 2004 ) , the stress response bZIP transcription factor AtfA ( ATEG_04664 ) ( Balazs et al . , 2010; Lara-Rojas et al . , 2011 ) , and the nitrogen starvation-induced ras-protein RhbA ( ATEG_09480 ) ( Panepinto et al . , 2002 ) were selected for gene deletions .\",\n \"All mutants were tested for their growth properties in the presence of proteinogenic amino acids or ornithine , citrulline , urea , and casamino acids , all of which were used as the sole nitrogen source .\",\n \"Additionally , several complex media were analysed ( Figure 4—figure supplement 1 ) .\",\n \"Only minor growth defects were observed with the cpcA and rhbA mutants , with some general growth reduction on selected amino acids ( Asp , His , Ser , Thr ) or complex media .\",\n \"However , among all the mutants tested , the areA mutant revealed the most severe growth defects .\",\n \"This mutant grew like the wild-type on glutamine and displayed some reduced growth on the nitrogen-rich amino acids asparagine , aspartate , and histidine as well as on urea and ammonium chloride .\",\n \"However , the ΔareA strain was unable to use any other proteinogenic amino acid .\",\n \"As described for other Aspergillus species ( Hunter et al . , 2014 ) , these results confirm an essential role of AreA in nitrogen sensing and utilisation in A . terreus .\",\n \"In contrast , the atfA mutant was only impaired in growth at high aspartate concentrations ( 50 mM ) .\",\n \"However , conidia of this mutant lacked the typical yellow-brown pigmentation .\",\n \"Additionally , the atfA mutant completely lacked the typical red colouration of the medium in the presence of methionine , which has previously been demonstrated to be associated with terrein production ( Zaehle et al . , 2014 ) .\",\n \"This implied a regulatory role of atfA on SM production in A . terreus .\",\n \"On the contrary , the atfA mutant did not show increased sensitivity against oxidative or osmotic stress as described for other Aspergillus species ( Balazs et al . , 2010; Lara-Rojas et al . , 2011 ) .\",\n \"Finally , a double deletion of the areA and atfA genes was generated ( ΔareAΔatfA ) resembling the growth phenotypes of both single mutants , since it was only able to grow on the media that supported growth of the areA mutant where it formed white conidia , as observed for the ΔatfA strain .\",\n \"To test the effect of transcription factor mutations on terrein biosynthesis gene cluster activation , all mutants were pre-grown on non-inducing glucose medium with glutamine and transferred to medium with or without nitrogen .\",\n \"Terrein was quantified after 24 hr ( Figure 4A ) .\",\n \"The wild-type , the ΔcpcA , and the ΔrhbA mutant revealed low terrein production in the presence of nitrogen , but high terrein titers when nitrogen was omitted .\",\n \"This indicates that cpcA and rhbA are dispensable for terrein production .\",\n \"In contrast , both the ΔareA and the ΔatfA mutant only produced marginal amounts of terrein under nitrogen-limited conditions ( Figure 4A ) , and this effect was completely cured in the complemented mutants areAc and atfAc .\",\n \"No terrein was detected in a ΔareAΔatfA double mutant .\",\n \"Therefore , both areA and atfA appear essential for terrein production under nitrogen limitation .\",\n \"To confirm this assumption , we expressed the cluster-specific transcription factor gene terR under control of the gpdA promoter in a ΔareAΔatfA mutant background .\",\n \"A constitutive terrein production was observed , indicating that AreA and AtfA regulate terR expression , but are not essential for expression of the structural genes which directly depend on TerR ( Figure 4A ) .\",\n \"Second , we analysed the effect of areA and atfA deletion on gene expression by qRT-PCR from strains shifted for 0 hr , 12 hr , and 24 hr to nitrogen starvation .\",\n \"qRT-PCR was performed on the cluster genes terA , terB , the specific activator terR , and the global transcription factors areA and atfA ( Figure 4B ) . 10 . 7554/eLife . 07861 . 009Figure 4 . Terrein quantification and expression of terrein cluster genes in nitrogen regulator mutants .\",\n \"( A ) Terrein quantification from the parental strains SBUG844ΔakuB ( ΔakuB ) , regulator mutants ( ΔcpcA ) , ( ΔrhbA ) , ( ΔareA ) , ( ΔatfA ) , ( ΔareAΔatfA ) , complemented mutants ( areAC ) , ( atfAC ) , and strain SBUG844ΔakuBΔareAΔatfAΔ::AnPgpdA:terR with terR overexpression in the ΔareAΔatfA background .\",\n \"Mycelia were pre-grown in glutamine-supplemented media , washed and transferred to minimal medium with ( +N ) or without ( −N ) 50 mM glutamine .\",\n \"Terrein was quantified from supernatants 24 hr after the shift .\",\n \"( B ) qRT-PCR of strains ΔakuB , ΔareA , areAC , ΔatfA , atfAC , and ΔareAΔatfA were pre-cultivated for 40 hr in glutamine-supplemented media and transferred to nitrogen starvation .\",\n \"RNA was isolated at 0 , 12 , and 24 hr of starvation .\",\n \"Transcript levels were normalised against the actin gene actA by fold expression = 2^ ( CTtarget − CTactA ) .\",\n \"( C ) Terrein quantification from strains shown in ( B ) after 72 hr of cultivation in glutamine-containing minimal medium supplemented with 10 mM methionine .\",\n \"( D ) qRT-PCR from RNA of strains shown in ( C ) isolated after 48 hr of cultivation .\",\n \"Transcript levels were normalized against the enolase gene enoA by fold expression = 2^ ( CTtarget − CTenoA ) .\",\n \"( E ) Top and bottom view of colonies of Aspergillus terreus wild-type ( ΔakuB ) and mutants ( ΔterA , ΔatfA , ΔareA ) grown for 72 hr on solid minimal media supplemented with 25 mM methionine as sole nitrogen source .\",\n \"The red pigmentation of the wild-type ( bottom view ) is lost in the ΔterA and ΔatfA mutants that show some enhanced growth while unable to produce terrein .\",\n \"The ΔareA strain is unable to grow .\",\n \"In all experiments biological triplicates with technical duplicates were analysed .\",\n \"Statistical significances in comparison to the parental ΔakuB strain were calculated by the Student's paired t-test with a two-tailed distribution: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 00910 . 7554/eLife . 07861 . 010Figure 4—source data 1 . Genotypes of strains used in the study . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01010 . 7554/eLife . 07861 . 011Figure 4—source data 2 . List of oligo nucleotides used in the study . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01110 . 7554/eLife . 07861 . 012Figure 4—figure supplement 1 . Analysis of colony formation and growth phenotypes of nitrogen regulator mutants in the presence of different nitrogen sourcesAs basal medium AMM-G100 without nitrogen was used , which was supplemented with 70 mM inorganic nitrogen ( NaNO3 or NH4Cl ) , 50 mM of standard amino acids , ornithine ( Orn ) , citrulline ( Cit ) , urea , or 1% casamino acids ( CA ) .\",\n \"Additionally , AMM-CA1% ( without glucose ) and the complex media yeast extract-peptone-dextrose ( YPD ) , potato dextrose agar ( PDA ) , and malt extract agar ( MA ) were used .\",\n \"Photographs were generally taken after 96 hr of incubation at 37°C .\",\n \"Asterisks denote different incubation times: * = 72 hr; ** = 48 hr .\",\n \"Note the severely reduced colony formation of the ΔareA strain on virtually all single amino acids except for arginine , glutamine , and histidine .\",\n \"Additionally , note the loss of conidia colouration in the ΔatfA strain . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01210 . 7554/eLife . 07861 . 013Figure 4—figure supplement 2 . Biochemical characterisation of the recombinant AreA DNA-binding domain and in vitro binding to HGATAR motifs identified in the terR promoter of the Aspergillus terreus terrein biosynthesis gene cluster .\",\n \"( A ) Amino acid sequence alignment of the AreA DNA binding domains from Aspergillus nidulans and A . terreus .\",\n \"Underlined amino acids denote the 65-residue peptide used for solving the solution structure of the AreA:DNA complex ( Starich et al . , 1998 ) .\",\n \"Cysteine residues of the Cys2–Cys2 zinc finger module are highlighted in yellow .\",\n \"( B ) SDS-PAGE analysis of purified AreA663-797 .\",\n \"( C ) In solution oligomeric state analysis of AreA663-797 as determined via size exclusion chromatography and multiangle static light scattering .\",\n \"The light scattering signal ( LS ) is shown overlaid with the calculated molar mass ( Mw ) across the elution profile as monitored by the absorbance at 280 nm ( UV ) and changes of the refractive index ( dRI ) .\",\n \"( D ) Schematic presentation of putative GATA AreA binding sites ( BS 1 and\",\n \"2 ) in the terR promoter .\",\n \"( E–G )\",\n \"Real-time in vitro surface plasmon resonance ( SPR ) interaction analysis of AreA663-797 with DNA containing the identified GATA motifs from the terR promoter of the terrein biosynthesis gene cluster .\",\n \"Sequences of DNA duplexes used for SPR analysis are shown on top of the sensorgrams .\",\n \"Numbers represent the GATA motif positions relative to the start of the open reading frame .\",\n \"HGATAR sites are highlighted in red .\",\n \"( H ) Dissociation constants and stoichiometry of analysed AreA663–797:DNA interactions analysed by SPR . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01310 . 7554/eLife . 07861 . 014Figure 4—figure supplement 3 . High performance liquid chromatography analyses from culture filtrates of SBUG844 wild-type and two independent atfA overexpression mutants ( AnPgpdA:atfA; OE 1 and 2 ) .\",\n \"Strains were cultivated under non-inducing conditions for the wild-type .\",\n \"( A ) AMM-G100; ( B ) AMM-G100Gln50 .\",\n \"1 = terrein .\",\n \"Terrein and its side products are detected from the atfA overexpressing strains but not from the wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01410 . 7554/eLife . 07861 . 015Figure 4—figure supplement 4 . Southern blot analyses for Aspergillus terreus strains generated in this study . Genomic DNA was isolated and restricted with the respective restriction enzymes indicated in each panel .\",\n \"Digoxygenin-labelled probes were used in all experiments and were amplified by the oligonucleotide couples as indicated .\",\n \"Signals were detected by the CDPstar chemiluminescent substrate .\",\n \"Expected fragment sizes are also shown .\",\n \"( A ) General scheme for generation of gene deletions using the pyrithiamine ( ptrA ) resistance cassette ( ΔGOI ) and for complementation of deletion mutants using the phleomycine ( ble ) resistance cassette ( GOIC ) via homologous recombination .\",\n \"( B ) Generation of the β-galactosidase reporter strain SBUG844_PterA:lacZ in A . terreus SBUG844 wild-type .\",\n \"( C ) Overexpression of atfA in SBUG844 under control of the A . nidulans gpdA promoter .\",\n \"The native atfA locus ( signal at 7008 bp ) is still intact in strains that integrated the overexpression construct .\",\n \"Strains with ectopic single copy integration of the construct used for downstream experiments are denoted by ‘X’ .\",\n \"( D ) Deletion of the cpcA locus ( ATEG_03131 ) in SBUG844ΔakuB .\",\n \"( E ) Deletion of the rhbA locus ( ATEG_09480 ) in SBUG844ΔakuB .\",\n \"( F ) Deletion of the areA locus ( ATEG_07264 ) in SBUG844ΔakuB and complementation with FGSC A1156 areA .\",\n \"( G ) PCR and PstI digest of PCR products from the areA upstream fragments amplified from genomic DNA from FGSC A1156 , SBUG844 , and SBUG844/areAC .\",\n \"The complemented strain shows the identical PstI restriction pattern as the amplified product from FGSC A1156 , whereas no PstI restriction site is present in the SBUG844 product , confirming the complementation of the ΔareA strain by the areA gene from A1156 .\",\n \"( H ) Deletion of the atfA locus ( ATEG_04664 ) in SBUG844ΔakuB and complementation with SBUG844 atfA .\",\n \"( I ) Deletion of the atfA locus ( ATEG_04664 ) in the areA negative background of SBUG844ΔakuBΔareA ( Δ ) and replacement of atfA in SBUG844ΔakuBΔareA with the terR overexpression construct under control of the A . nidulans gpdA promoter ( Δ::AnPgpdA:terR ) .\",\n \"( J ) Partial deletion of the hapX locus ( ATEG_08 , 014 ) in SBUG844ΔakuB and complementation with the Aspergillus nidulans FGSC A4 hapX ( AN08251 ) .\",\n \"( K ) Deletion of the sreA locus ( ATEG_07714 ) in SBUG844ΔakuB and complementation with SBUG844 sreA .\",\n \"( L ) Deletion of the sidA locus ( ATEG_06879 ) in SBUG844ΔakuB and complementation with SBUG844 sidA .\",\n \"( M ) Deletion of sidA ( ATEG_06879 ) in the terA negative background of SBUG844ΔakuBΔterA . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01510 . 7554/eLife . 07861 . 016Figure 4—figure supplement 5 . Terrein production and susceptibility to osmotic stress in a sakA mutant . In all experiments biological triplicates with technical duplicates were analysed .\",\n \"( A ) Terrein quantification from strains SBUG844ΔakuB ( ΔakuB ) and two sakA deletion strains SBUG844ΔakuBΔsakA ( 1 ) and ( 2 ) ( ΔsakA ) cultivated in AMM ( -N ) G100 for 72 hr in the presence of either 70 mM inorganic ( NaNO3 , NH4Cl ) or 50 mM organic nitrogen ( Glu , Gln , Asp , Asn ) .\",\n \"( B ) Terrein quantification from strains SBUG844ΔakuB ( ΔakuB ) , SBUG844ΔakuBΔsakA and the complemented strain SBUG844ΔakuBΔsakA/sakAC ( sakAC ) .\",\n \"Cultures were pre-grown in nitrogen-rich AMM-G100Gln50 , mycelia were washed and transferred to AMM ( -N ) G100 with ( +N ) or without ( −N ) 50 mM Gln .\",\n \"Terrein was quantified from supernatants 24 hr after the shift .\",\n \"( C ) Susceptibility assay towards NaCl stress .\",\n \"105 to 101 conidia of strain SBUG844ΔakuB ( ΔakuB ) , the deletion mutants SBUG844ΔakuBΔsakA ( ΔsakA ) and SBUG844ΔakuBΔatfA ( ΔatfA ) or their complemented strains SBUG844ΔakuBΔsakA/sakAC ( sakAC ) and SBUG844ΔakuBΔatfA/atfAC ( atfAC ) were point inoculated on AMM ( -N ) G100 plates supplemented with 10 mM Gln and 0 , 0 . 5 , 1 . 0 , and 2 . 0 M NaCl .\",\n \"Plates were incubated 72 hr or 5 d ( * ) at 37°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 016 In the wild-type the regulators areA and atfA showed a time-dependent increase in gene expression .\",\n \"While the regulator terR was most strongly upregulated after 12 hr , expression of the TerR-controlled terrein biosynthesis genes terA and terB continued to increase after 12 hr and reached 13 . 3 and 3 . 2 times the expression level of the actin control gene at 24 hr ( Figure 4B ) .\",\n \"On the contrary , while complemented mutants behaved like the wild-type , the areA and atfA deletions strongly reduced activation of terR and , in turn , the expression of terA and terB .\",\n \"Results from qRT-PCR perfectly coincided with the substantially reduced terrein production rates under nitrogen limitation in these mutants ( Figure 4A ) .\",\n \"AreA recognises the DNA-binding motif HGATAR , and two adjacent binding sites are generally required for transcriptional activation due to dimer formation of AreA monomers ( Ravagnani et al . , 1997 ) .\",\n \"In this respect , the terR promoter contains two putative AreA binding sites that match the HGATAR consensus ( BS1and BS2; positions −59 and −72 relative to the translational start point ) .\",\n \"Surface plasmon resonance ( SPR ) analyses with the Aspergillus nidulans AreA DNA-binding domain , which is 91% identical to the respective A . terreus AreA domain , showed that BS1 and BS2 are recognised with high affinity ( Figure 4—figure supplement 2 ) .\",\n \"This strengthens the model of a direct involvement of AreA in the activation of terR expression .\",\n \"However , the reduction of terR expression was less pronounced in the ΔareA than in the ΔatfA background ( Figure 4B ) , which indicates that AreA is not the only activator acting on the terR promoter .\",\n \"Therefore , we additionally searched for putative palindromic AtfA/Sko1 binding sites ( 5′-TKACGTMA-3′ ) in the promoter regions of the cluster ( Proft et al . , 2005 ) .\",\n \"Only one hit ( TGACGTCA ) was identified in the promoter of the structural gene terC .\",\n \"However , if one mismatch is allowed , there is a putative binding site at position −731 relative to the ATG start codon of terR ( 5´-TGGCGTCA-3´ ) , but it remains speculative whether this binding site is recognised by A . terreus AtfA .\",\n \"Nevertheless , it should be mentioned that even a single half site of the suggested motif could promote transcription factor binding and promoter induction ( Proft et al . , 2005 ) .\",\n \"We therefore conclude that , although direct evidence for AtfA binding at the terR promoter is lacking , both transcription factors seem to regulate terR expression .\",\n \"In agreement , terR , terA , and terB expression and terrein production showed the strongest decrease in the ΔareAΔatfA double knock-out mutant ( Figure 4A ) .\",\n \"In addition , a constitutive expression of atfA by the gpdA promoter led to increased terrein production ( Figure 4—figure supplement 3 ) .\",\n \"This supports our hypothesis of direct involvement of AtfA in terrein cluster regulation .\",\n \"Due to the requirement for atfA and areA in terrein biosynthesis gene cluster induction under nitrogen starvation , we also tested their contribution to methionine-dependent induction in nitrogen-supplemented medium ( Figure 4C ) .\",\n \"While the wild-type strain and complemented mutants showed high terrein production rates of up to 55 mg/g mycelium , the terrein levels in the ΔareA , ΔatfA , or ΔareAΔatfA double mutant remained near the detection limit and qRT-PCR was performed to confirm this result on the transcriptional level .\",\n \"Although areA and atfA were expressed in the wild-type only at low levels on glutamine/methionine medium ( Figure 4D ) , deletion of areA or atfA reduced terR and , consequentially , terA and terB expression .\",\n \"In the ΔatfA mutant terR transcription was completely abolished and , in agreement , transcription of terA and terB was no longer detected .\",\n \"An areA mutant is unable to use methionine as a nitrogen source ( Figure 4E ) , and its uptake may be limited leading to the loss of transcriptional activation .\",\n \"In contrast , the atfA mutant still uses methionine as a nitrogen source , but neither produces terrein nor pigmented conidia nor the red colouration of the medium which is associated with terrein production in the presence of methionine ( Figure 4E ) .\",\n \"This implies that atfA may be induced by a methionine-dependent signaling cascade that subsequently leads to terR expression .\",\n \"While nitrogen starvation stimulated terrein production , starvation of other macroelements such as carbon , sulphur , or phosphate did not result in terrein production ( data not shown ) .\",\n \"However , this did not exclude limitation of trace elements as inducing signals .\",\n \"Therefore , the β-galactosidase activity from the PterA:lacZ reporter strain was determined from cultures with reduced amounts of trace elements .\",\n \"Indeed , a decrease in trace elements was accompanied by increased terA promoter activity ( Figure 5A ) .\",\n \"To attribute this activation to a specific trace element , media with limited amounts of trace elements were supplemented with each of the single trace elements FeSO4 , ZnSO4 , CuSO4 , MnCl2 , Na2MoO4 , CoCl2 , and H3BO3 .\",\n \"Cluster induction was observed in all cultures except for that supplemented with FeSO4 ( Figure 5B ) , indicating cluster activation from iron limitation .\",\n \"Subsequently , a minimal medium was prepared that contained all trace elements but iron which led to strong cluster induction , and the addition of 40 µM FeCl3 completely repressed the induction ( Figure 5C ) .\",\n \"Thus , besides methionine and the limitation of nitrogen , a lack of iron—but no other trace element—induces the terrein biosynthesis gene cluster . 10 . 7554/eLife . 07861 . 017Figure 5 . Expression of terA , production of siderophores under iron limited conditions and iron reducing properties of terrein . All analyses were performed in biological triplicates and technical duplicates .\",\n \"Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution .\",\n \"( A ) β-Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with limited amounts of trace elements .\",\n \"Asterisks indicate p values vs 100% trace elements: **p<0 . 01; ***p<0 . 001 .\",\n \"( B ) β -Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with 1% trace elements supplemented with 20 µM of specific trace elements: FeSO4 , ZnSO4 , CuSO4 , MnCl2 , Na2MoO4 , CoCl2 , or H3BO3 .\",\n \"Asterisks indicate p values vs activity without supplementation: ***p<0 . 001 .\",\n \"( C ) β-Galactosidase activity from SBUG844_PterA:lacZ in AMM ( -N ) G100Gln50 medium with and without 40 µM FeCl3 .\",\n \"Asterisks indicate p values vs 40 µM FeCl3: ***p<0 . 001 .\",\n \"( D ) High performance liquid chromatography ( HPLC ) profiles of lyophilised culture supernatants of ΔterA after 72 hr of cultivation in AMM ( -N ) G100Gln50 supplemented with 0 , 20 , or 200 µM FeCl3 .\",\n \"Peaks for the siderophores ferrichrysin ( 2 ) and coprogen ( 3 ) are indicated .\",\n \"( E ) Fe ( III ) reduction by terrein determined by the TPTZ assay .\",\n \"Ascorbic acid served as control and maximum reduction by ascorbic acid was set as 100% .\",\n \"Analyses were made from duplicates .\",\n \"( F ) HPLC profiles of terrein after incubation with different oxidised ( upper , red lines ) or reduced ( lower , blue lines ) metal ions .\",\n \"Left: Cu ( II ) SO4 and Cu ( I ) Ac; right: Fe ( III ) Cl3 and Fe ( II ) SO4 .\",\n \"1 = terrein; 4 = propylene maleic acid .\",\n \"( G ) Scheme of terrein oxidation during iron reduction leading to the formation of propylene maleic acid . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01710 . 7554/eLife . 07861 . 018Figure 5—source data 1 . Analytical data of coprogen . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01810 . 7554/eLife . 07861 . 019Figure 5—source data 2 . Analytical data of ferrichrysin . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 01910 . 7554/eLife . 07861 . 020Figure 5—figure supplement 1 . qRT-PCR expression analysis of genes from iron acquisition systems under iron-supplemented and limited conditions . All analyses were performed from biological triplicates and technical duplicates .\",\n \"Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution .\",\n \"SBUG844 wild-type was grown in AMM-G100Gln50 under iron-supplemented ( 40 µM; +Fe ) and starved conditions ( −Fe ) .\",\n \"qRT-PCR was performed on genes assumed to be responsible for siderophore biosynthesis , siderophore transport , or reductive iron assimilation .\",\n \"Transcript quantities were normalised against enoA by fold expression = 2^ ( CTtarget − CTeno ) .\",\n \"Asterisks indicate p values vs 0 µM FeCl3: **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02010 . 7554/eLife . 07861 . 021Figure 5—figure supplement 2 . Chrome azol S ( CAS ) assay of coprogen and terrein . Both substances were dissolved in methanol and serial dilutions ( 125–15 . 63 µg ) were added to punched holes of CAS plates .\",\n \"Photographs were taken 48 hr after incubation at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02110 . 7554/eLife . 07861 . 022Figure 5—figure supplement 3 . pH- and time-dependent Fe ( III ) reduction by terrein assayed by TPTZ . 10 µM FeCl3 were incubated with 4 mM fixed concentration of terrein in acetate buffer in a pH range from 3 . 0 to 6 . 0 .\",\n \"The analyses were performed from duplicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02210 . 7554/eLife . 07861 . 023Figure 5—figure supplement 4 . 1H NMR ( 500 MHz , MeOD; upper panel ) and 13C NMR ( 150 MHz , MeOD; lower panel ) of compound 4 , 2- ( ( E ) -prop-1-en-1-yl ) maleic acid . 1H NMR ( 500 MHz , MeOD ) : δ 6 . 22 ( 1H , d , 3J = 15 . 8 Hz ) , 6 . 15 ( 1H , dq , 3J = 15 . 8 Hz , 3J = 6 . 5 Hz , ) , 5 . 73 ( 1H , s ) , 1 . 85 ppm ( 3H , d , 3J = 6 . 5 Hz ) ; 13C NMR ( 600 MHz , MeOH ) : δ 172 . 6 , 169 . 5 , 151 . 7 , 136 . 4 , 130 . 2 , 118 . 5 , 18 . 8 ppm; HRMS: ( ESI+ ) : m/z calculated for C7H9O4: 157 . 0495 , found 157 . 0495 [M + H]+ .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02310 . 7554/eLife . 07861 . 024Figure 5—figure supplement 5 . HSQC NMR ( 600 MHz , MeOD; upper panel ) and HMBC NMR ( 600 MHz , MeOD; lower panel ) of compound 4 , 2- ( ( E ) -prop-1-en-1-yl ) maleic acid . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 024 Fungal iron acquisition has been well investigated in A . nidulans ( Eisendle et al . , 2003 ) and Aspergillus fumigatus ( Haas , 2014 ) , but only limited information was available on the iron acquisition systems from A . terreus .\",\n \"A BLASTp search for orthologous genes in the genome of A . terreus NIH2624 ( Table\",\n \"1 ) revealed that all genes required for a reductive iron assimilation ( RIA ) system ( fetC , freB , ftrA ) , siderophore biosynthesis ( sid genes , except sidG ) , siderophore transport ( mirA-D , sitA ) , and regulators of iron homeostasis ( srbA , sreA , and hapX ) are well conserved in the A . terreus genome .\",\n \"When tested under iron limitation , qRT-PCR analyses confirmed a strong induction of genes from siderophore biosynthesis , siderophore transport , and the RIA pathway ( Figure 5—figure supplement 1 ) .\",\n \"To confirm siderophore production by A . terreus , we cultivated the wild-type strain under iron-rich ( 200 µM FeCl3 ) and iron-limited conditions ( no or 20 µM FeCl3 ) and identified ferrichrysin ( 2 ) and coprogen ( 3 ) only under iron limitation ( Figure 5D and Figure 5—source data 1 ,\",\n \"2 ) ( Zähner et al . , 1963; Bertrand et al . , 2010 ) .\",\n \"Coprogen revealed a higher stability than ferrichrysin .\",\n \"Subsequent analyses involving siderophores were therefore based only on coprogen .\",\n \"When terrein was compared with purified coprogen for chelating iron , only coprogen showed the expected iron chelating activity in CAS agar plate assays ( Figure 5—figure supplement 2 ) .\",\n \"Thus , although terrein is produced under iron limitation , it does not depict a new kind of siderophore . 10 . 7554/eLife . 07861 . 025Table 1 . BLASTp analysis of homologous genes for iron uptake and regulation of iron homeostasis ( adapted by [Haas , 2012] ) DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 025Aspergillus fumigatus functionGene code‡ExpressionAspergillus terreus gene codeIdentity/similarityReductive iron assimilation ( RIA ) FetCFerroxidaseAFUA_5G03790−FeATEG_0803279%/89% FreBFerric reductaseAFUA_1G17270−FeATEG_1032253%/64% FtrAIron permeaseAFUA_5G03800−FeATEG_0803175%/84%Siderophore biosynthesis ( SB ) EstATAFC esteraseAFUA_3G03660−FeATEG_0407244%/58% NpgA/PptAPhosphopantetheinyl transferaseAFUA_2G08590−ATEG_0969556%/65% SidAOrnithine monooxygenaseAFUA_2G07680−FeATEG_0687978%/85% SidCFC NRPSAFUA_1G17200−FeATEG_0507360%/76% SidDFSC NRPSAFUA_3G03420−FeATEG_0748843%/59% SidFTransacylaseAFUA_3G03400−FeATEG_0507552%/67% SidGTransacetylaseAFUA_3G03650−Fenone– SidHMevalonyl hydrataseAFUA_3G03410−FeATEG_0150953%/67% SidIMevalonyl ligaseAFUA_1G17190−FeATEG_0507486%/91% SidLTransacetylaseAFUA_1G04450–ATEG_0377064%/76%Siderophore transporter ( SIT ) MirAEnterobactin transporterAN7800; -−FeATEG_0407168%/77% MirBTAFC transporterAN8540; -AFUA_3G03640−FeATEG_0271150%/68% MirC*AN7485; AFUA_2G05730−FeATEG_0676278%/87% MirD†Trichotecene efflux pumpAFUA_3G03440–ATEG_0748740%/58% SitA/SitT*AN5378; AFUA_7G06060−FeATEG_0632962%/73%Regulatory proteins HapXbZip-TFAFUA_5G03920−FeATEG_0801477%/83% ( hapX re-annotated ) SreAGATA TFAFUA_5G11260+FeATEG_0774167%/75% SrbA†HLH TFAFUA_2G01260−FeATEG_0815672%/82%Genes selected for qPCR analyses are highlighted in bold .\",\n \"*Genes annotated according to ( Schrettl et al . , 2008 ) .\",\n \"†Genes annotated according to ( Blatzer et al . , 2011 ) .\",\n \"‡Gene codes ANxxxx refer to the A . nidulans genome .\",\n \"Since some antioxidative properties of terrein had previously been described ( Trabolsy et al . , 2014 ) , the ability of terrein in reducing ferric ( Fe3+ ) to ferrous ( Fe2+ ) iron was investigated by using the ferrous iron chelator 2 , 4 , 6-tripyridyl-S-triazine ( TPTZ ) .\",\n \"The strong antioxidant ascorbic acid ( Elmagirbi et al . , 2012 ) served as control ( Figure 5E ) .\",\n \"Although ascorbic acid showed a much higher reducing potential , terrein was also able to convert ferric to ferrous iron in a concentration-dependent ( Figure 5E ) and pH-dependent ( Figure 5—figure supplement\",\n \"3 ) manner with a pH optimum of 3–4 .\",\n \"In this respect , we noticed a significant fall in the culture pH under iron limitation from 6 . 5 to about 3 . 5 , which agrees with the optimum pH for terrein-mediated iron reduction .\",\n \"Thus , since terrein is produced during growth under iron limitation , the combination with a fall in pH could indeed increase iron availability .\",\n \"To elucidate the structure of the terrein oxidation product , the terrein-mediated iron reduction assay was scaled up and extractions were subjected to high performance liquid chromatography ( HPLC ) analysis .\",\n \"Here , besides a major proportion of terrein that remained in its original structure ( Figure 5F ) , a new peak ( 4 ) was observed , which was identified by NMR analyses ( Figure 5—figure supplements 4 ,\",\n \"5 ) as 2- ( ( E ) -prop-1-en-1-yl ) maleic acid ( PMA , Figure 5G ) .\",\n \"The structure of the oxidation product implies that oxidation took place at the two hydroxylated carbon atoms of terrein , leading to a cleavage of the pentenone ring system as proposed by Trabolsy et al . ( 2014 ) .\",\n \"In contrast , conversion of terrein to PMA was not observed during incubation with Fe2+ , confirming that iron reduction was the cause of PMA formation .\",\n \"No conversion of terrein was observed with oxidised copper ions ( Cu2+ ) , indicating a limited reductive potential which is sufficient for iron but not for copper ( Figure 5F ) .\",\n \"In conclusion , while terrein cannot chelate iron to support the siderophore-mediated iron uptake , its reductive potential is sufficient to reduce ferric to ferrous iron , which may increase iron solubility and could ease the direct uptake of iron via the ferrous iron transport system .\",\n \"Due to the co-regulation with siderophore biosynthesis , we assumed that transcriptional regulators involved in iron homeostasis could also regulate terrein biosynthesis .\",\n \"In A . nidulans and A . fumigatus , siderophore biosynthesis is regulated by HapX , a transcriptional inducer under iron limitation , and SreA , a repressor in the presence of iron ( Haas , 2012 ) .\",\n \"To investigate the impact of SreA and HapX on siderophore ( coprogen ) and terrein synthesis , the sreA gene was completely and the hapX gene partially deleted ( the latter due to incomplete sequence information at the hapX locus tag ATEG_08014 ) .\",\n \"Additionally , while the sreA mutant was complemented with the A . terreus sreA gene , the hapX mutant was complemented with the hapX gene from A . nidulans FGSC A4 .\",\n \"All strains were cultivated in iron-supplemented and iron-limited media and terrein and coprogen were quantified from culture supernatants ( Figure 6A , B ) .\",\n \"All complemented mutants behaved similar to the wild-type with production of only marginal amounts of coprogen and terrein in the presence of iron .\",\n \"Under iron starvation the production rates for both metabolites strongly increased . 10 . 7554/eLife . 07861 . 026Figure 6 . Effect of hapX and sreA deletion on terrein and coprogen biosynthesis .\",\n \"( A ) Coprogen or ( B ) terrein quantification from SBUG844ΔakuB ( ΔakuB ) , SBUG844ΔakuBΔhapX ( ΔhapX ) , SBUG844ΔakuBΔhapX/AnhapXC ( AnhapXC ) , SBUG844ΔakuBΔsreA ( ΔsreA ) , and SBUG844ΔakuBΔsreA/sreAC ( sreAC ) grown for 72 hr in AMM_G100Gln50 with ( +Fe ) or without 40 µM FeCl3 ( −Fe ) .\",\n \"Coprogen was quantified from lyophilised culture supernatants and terrein from culture extracts .\",\n \"( C ) qRT-PCR from strains and media described in ( A ) and ( B ) .\",\n \"RNA was isolated after 40 hr of cultivation .\",\n \"Transcript levels were normalised against enoA by fold expression = 2^ ( CTtarget − CTenoA ) .\",\n \"( $ ) denotes the lack of hapX transcripts from the complemented ΔhapX strain , since the Aspergillus nidulans hapX was used for complementation .\",\n \"qRT-PCR on the complemented mutant with oligonucleotides specific for AnhapX are shown in Figure 6—figure supplement 1 .\",\n \"All analyses were performed from biological triplicates and technical duplicates .\",\n \"Statistical significances were calculated in comparison to the parental ΔakuB strain by the Student's paired t-test with a two-tailed distribution: *p<0 . 05; **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02610 . 7554/eLife . 07861 . 027Figure 6—figure supplement 1 . qRT-PCR analysis of Aspergillus nidulans hapX expression in the Aspergillus terreus wild-type SBUG844ΔakuB , the hapX mutant SBUG844ΔakuBΔhapX and its complemented strain SBUG844ΔakuBΔhapX/AnhapXC . All analyses were performed from biological triplicates and in technical duplicates .\",\n \"All strains were grown for 40 hr in the presence ( +Fe ) or absence ( −Fe ) of 40 µM FeCl3 .\",\n \"Transcript levels of AnhapX were normalised against enoA gene from A . terreus by fold expression = 2^ ( CTtarget − CTeno ) .\",\n \"The heterologously expressed AnhapX gene behaves in a similar way to the native hapX gene from A . terreus ( compare with Figure 6 ) .\",\n \"Statistical significances were calculated by the Student's paired t-test with a two-tailed distribution: Asterisks indicate p values vs 0 µM FeCl3: ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 027 On the other hand , partial hapX deletion reduced coprogen production under iron limitation by approximately 50% and terrein concentrations by about 90% ( Figure 6A , B ) .\",\n \"This indicates that HapX directly activates both siderophore and terrein biosynthesis in A . terreus .\",\n \"In contrast , the ΔsreA mutant produced significantly higher amounts of coprogen in the presence of iron whereas terrein production did not significantly increase ( Figure 6A , B ) .\",\n \"This confirms SreA as a negative feedback regulator in siderophore biosynthesis , whereas it does not control terrein production .\",\n \"In contrast , HapX positively controls both pathways .\",\n \"These results were confirmed on the transcriptional level by qRT-PCR ( Figure 6C and Figure 6—figure supplement 1 ) .\",\n \"The inactivation of hapX prohibited terR transcription and resulted in an inability to induce terrein cluster genes terA , terB , and terC .\",\n \"In contrast , deletion of sreA influenced neither terR activation nor expression of other terrein cluster genes in the presence of iron .\",\n \"We therefore conclude that hapX is the major regulator for terrein cluster induction under iron starvation .\",\n \"To elucidate the complete sequence of the hapX gene we subsequently used degenerate primers to amplify the main proportion of hapX from cDNA .\",\n \"The complete sequence of the A . terreus hapX gene is found under accession number KP233834 ( Gressler et al . , 2015b ) .\",\n \"The sequence of the full-length HapX protein matches with that of HapX proteins from other Aspergillus species such as A . nidulans ( 73% identity ) , A . fumigatus ( 77% ) , Aspergillus niger ( 81% ) , and A . oryzae ( 81% ) .\",\n \"In general , siderophore-based iron acquisition is highly efficient and assumed to be more important than the reductive iron assimilation pathway .\",\n \"In A . fumigatus , growth and virulence defects caused by the interruption of the reductive iron assimilation pathway are only observed when the siderophore-based system is also inactivated ( Blatzer et al . , 2011 ) .\",\n \"Therefore , to elucidate a positive effect of terrein on iron acquisition , we deleted the sidA gene in A . terreus that encodes the L-ornithine-N5-monooxygenase , a key enzyme in hydroxamate siderophore biosynthesis .\",\n \"Coprogen production was confirmed in the wild-type and a complemented mutant , but was completely lacking from the ΔsidA mutant ( Figure 7—figure supplement 1 ) .\",\n \"When analysed for growth phenotypes on solid media , all complemented mutants and the ΔterA strain behaved like the wild-type ( Figure 7A and Figure 7—figure supplement 1A ) .\",\n \"The hapX mutant showed a reduced growth rate without iron supplementation ( Figure 7A ) , which is in agreement with reduced coprogen production as shown above ( Figure 6A ) .\",\n \"However , severe iron limitation from the addition of the iron chelator bathophenanthroline sulfonate ( BPS ) completely repressed growth of the hapX mutant ( Figure 7A ) .\",\n \"The ΔsidA mutant displayed the most severe phenotype: while ΔsidA showed normal growth with slightly reduced conidiation in the presence of high to moderate iron concentrations ( 100 or 20 µM FeCl3 ) ( Figure 7A ) , growth was strongly retarded when the iron concentration was reduced to 2 µM FeCl3 .\",\n \"No growth was observed even after prolonged incubation when iron was omitted ( Figure 7A ) .\",\n \"These phenotypes were cured when purified coprogen was externally added ( Figure 7—figure supplement 1B ) .\",\n \"Interestingly , growth of the ΔsidA strain was also partially restored in the presence of ascorbic acid and , although to a lesser extent , by the addition of terrein ( Figure 7A ) .\",\n \"This result is supported by previous studies on an A . nidulans ΔsidA mutant which was able to grow under iron limitation in the presence of ascorbic acid ( Eisendle et al . , 2003 ) .\",\n \"Unexpectedly , terrein supplementation inhibited growth of an A . fumigatus sidA mutant , which was also true for an A . fumigatus wild-type strain ( Figure 7—figure supplement 2 ) .\",\n \"Subsequent analyses showed that growth of the phytopathogen Fusarium graminearum was also inhibited by terrein-containing culture extracts from A . terreus ( Figure 7—figure supplement 2 ) , indicating some antifungal properties of terrein against environmental competitors . 10 . 7554/eLife . 07861 . 028Figure 7 . Siderophore production in sidA mutants and growth-supporting effect of terrein under iron limitation .\",\n \"( A ) Growth of wild-type ΔakuB , ΔterA , ΔhapX , and ΔsidA on AMM ( −N ) G100Gln10 plates containing different iron concentrations .\",\n \"Photographs were taken after 4 d and , as indicated , after 17 d of incubation at 37°C .\",\n \"Iron-free medium was supplemented with 100 , 20 , 2 , or 0 µM FeCl3 .\",\n \"Additionally , plates without iron were supplemented with bathophenanthroline disulfonate ( BPS; 100 µM ) to simulate harsh starvation conditions or with either ascorbic acid ( 1 mM ) or terrein ( 5 or 10 mM ) .\",\n \"( B , C )\",\n \"Impact of terrein production on growth of ΔsidA mutants .\",\n \"AMM-G100Gln50 plates were supplemented with 0 , 2 , 20 , or 100 µM FeCl3 and either inoculated with ( B ) conidia or ( C ) mycelial pellets from cultures pre-grown for 40 hr in AMM-G100Gln50 with 200 µM FeCl3 .\",\n \"The parental Aspergillus terreus wild-type ΔakuB , the ΔakuBΔsidA strain , and the ΔakuBΔterAΔsidA are shown .\",\n \"Mycelia from a 40 hr culture in AMM-G100Gln50 with 200 µM FeCl3 was washed with iron-free medium and three pellets were applied to the plates .\",\n \"Plates were incubated at 37°C for 5 d . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02810 . 7554/eLife . 07861 . 029Figure 7—figure supplement 1 . Dependence of colony formation of Aspergillus terreus mutants on iron availabilty , ascorbic acid , terrein , and coprogen .\",\n \"( A ) As basic media AMM ( -N ) G100Gln10 ( -Fe ) agar plates were used that were supplemented either with different iron concentrations ( 100 , 20 , 2 , or 0 µM FeCl3 ) , the iron chelator bathophenanthroline disulfonate ( BPS; 100 µM ) , ascorbic acid ( Asc , 1 mM ) , or terrein ( 10 mM ) .\",\n \"Plates were incubated for 4 d at 37°C , except for the ΔterAΔsidA mutant that was further incubated for up to 17 d ( B ) Effect of purified coprogen on growth of A . terreus mutants and complemented strains under iron starvation .\",\n \"Conidia ( 1 × 105 ) of A . terreus SBUG844ΔakuB , SBUG844ΔakuBΔsidA , SBUG844ΔakuBΔsidA/sidAC , SBUG844ΔakuBΔterA , or SBUG844ΔakuBΔterAΔsidA were plated on AMM-G100Gln10 plates supplemented with ( +Fe ) or without 20 µM FeCl3 ( −Fe ) .\",\n \"To ( −Fe ) media filter disks soaked with either 20 µl methanol ( MeOH , negative control ) or 20 µl of 2 . 5 mg/ml coprogen ( in methanol ) were applied .\",\n \"Plates were incubated at 37°C for 72 hr until photographed .\",\n \"( C ) Coprogen quantification from ΔakuB wild-type , ΔsidA mutant and complemented mutant sidAC .\",\n \"Strains were cultivated for 40 hr in AMM ( -N ) G100Gln50 supplemented with 200 µM FeCl3 .\",\n \"The mycelium was washed and transferred to iron-free AMM ( −N ) G100Gln50 medium .\",\n \"Coprogen was quantified after 30 hr of incubation .\",\n \"Analyses were performed from biological triplicates and technical triplicates .\",\n \"Statistical significance was calculated in comparison to the parental ΔakuB strain by the Student's paired t-test with a two-tailed distribution: *p<0 . 05; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 02910 . 7554/eLife . 07861 . 030Figure 7—figure supplement 2 . Antifungal activity of terrein and potato dextrose broth ( PDB ) medium from Aspergillus terreus wild-type cultivations .\",\n \"( A ) AMM-G100Gln10 containing no iron addition ( −Fe ) or 2 mM FeCl3 were supplemented with 0 , 1 , 10 , or 20 mM terrein and inoculated with conidia of Aspergillus fumigatus wild-type ATCC46645 .\",\n \"Plates were incubated for 84 hr at 37°C until photographed .\",\n \"Terrein inhibits growth of A . fumigatus independently of the available iron concentration .\",\n \"( B ) Analysis of the effect of A . terreus inoculated PDB culture broth on growth of A . terreus , A . fumigatus , and Fusarium graminearum .\",\n \"The basal medium for A . terreus and A . fumigatus was AMM-G50 medium with nitrate as the nitrogen source , whereas the medium for F . graminearum was additionally supplemented with 0 . 2% potato broth ( AMM-G50 + 0 . 2%PB ) .\",\n \"All plates were supplemented either with 150 µl of PDB ethyl acetate extract from a mock-inoculated culture ( PDB extract ) or inoculated with A . terreus SBUG844 and cultivated for 4 d at 30°C ( PDB metabolite extract ) .\",\n \"Photographs were taken after 4 d of incubation at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 03010 . 7554/eLife . 07861 . 031Figure 7—figure supplement 3 . Terrein determination in siderophor deletion mutants . High performance liquid chromatography profiles of agar plugs were recorded from plates shown in panel Figure 7C .\",\n \"Wild-type ( A ) and ΔsidA mutant ( B ) produced terrein ( 1 ) under iron-limited conditions , whereas no terrein was formed by the ΔterAΔsidA mutant ( C ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 031 Due to the beneficial effect of terrein on the A . terreus ΔsidA mutant , we investigated the impact of intrinsic terrein production on growth under iron limitation .\",\n \"For this , we deleted the sidA gene in the ΔterA background and compared growth of wild-type , ΔsidA , and ΔterAΔsidA under iron-supplemented ( 20 and 200 µM FeCl3 ) and iron-limited conditions ( 0 and 2 µM FeCl3 ) ( Figure 7B ) .\",\n \"Both mutants ( ΔsidA and ΔterAΔsidA ) were unable to grow on media containing 2 µM or less FeCl3 .\",\n \"However , since terrein is only produced by vegetative mycelium , strains were pre-grown in the presence of 200 µM FeCl3 , washed , and transferred to the plates with different iron contents ( Figure 7C ) .\",\n \"While growth of mycelium from the ΔakuB strain looked identical to that from conidia , the ΔsidA mutant started to form small colonies even in the absence of iron , whereas the ΔterAΔsidA mutant showed some weak growth only at 2 µM FeCl3 but not in the absence of external iron supplementation .\",\n \"Terrein production was analysed from extracted agar plugs , which confirmed that the wild-type and ΔsidA mutant produced substantial amounts of terrein under iron-limited conditions whereas terrein production was fully abrogated in the ΔterAΔsidA mutant ( Figure 7—figure supplement 3 ) .\",\n \"These results indicate that terrein production supports iron acquisition , although the siderophore system is the dominant iron acquisition system .\"\n ],\n [\n \"A . terreus is known as a human pathogen that causes severe invasive bronchopulmonary and disseminated aspergillosis ( Lass-Flörl et al . , 2005; Slesiona et al . , 2012a , 2012b ) .\",\n \"In addition , A . terreus has been described as a causative agent of foliar blight of potato leaves ( Louis et al . , 2013 , 2014a , 2014b ) .\",\n \"Besides its pathogenic potential , A . terreus can inhibit proliferation of other plant pathogens such as Fusarium udum ( Upadhyay and Rai , 1987 ) and acts as a mycoparasite on sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum ( Melo et al . , 2006 ) .\",\n \"SMs are assumed to support competition in the plant and soil environment .\",\n \"The natural biological activities of terrein include the inhibition of plant seed germination ( Kamata et al . , 1983 ) , the induction of fruit surface lesions ( Zaehle et al . , 2014 ) , and the newly discovered reduction of ferric to ferrous iron accompanied by some antifungal activity .\",\n \"These activities are all appropriate to supporting the competitiveness of A . terreus in the environment .\",\n \"However , the production of terrein at the correct timing requires coordinated sensing and transduction of environmental signals .\",\n \"The terrein biosynthesis gene cluster is directly controlled by the Zn2Cys6 transcription factor TerR ( Gressler et al . , 2015a ) .\",\n \"Therefore , terrein production requires transcriptional activation of terR .\",\n \"While sugars are indispensable for high terrein production rates ( Zaehle et al . , 2014 ) , three major signals were identified that resulted in terR transcription: methionine-dependent induction , nitrogen limitation , and iron starvation .\",\n \"These signals resemble the plant and rhizosphere environment .\",\n \"Plant roots are leaky for carbon compounds that derive from C-fixation during photosynthesis .\",\n \"Up to one third of plant-assimilated carbon may end up in the rhizosphere , and especially in the extramatrical mycelium of ectomycorrhiza ( Churchland and Grayston , 2014 ) .\",\n \"Here , at least a fraction of carbohydrates is subsequently exuded by ectomycorrhiza , making it available to other soil microorganisms ( Sun et al . , 1999 ) .\",\n \"Mycorrhizas mobilize nitrogen to feed their symbiotic plant partner , resulting in nitrogen limitation for surrounding microorganisms .\",\n \"These conditions stimulate terrein production in A . terreus , which is typically isolated from the rhizosphere ( Gao et al . , 2013; Rajalakshmi and Mahesh , 2014 ) and might affect existing plant–microbe interactions by its phytotoxic and antifungal activity ( Figure 8 ) . 10 . 7554/eLife . 07861 . 032Figure 8 . Scheme of the regulation of terrein biosynthesis gene cluster expression during interactions in the rhizosphere . Plants secrete methionine ( +Met ) with root exudates into the soil .\",\n \"Additionally , competing microorganisms reduce the available pool of nitrogen sources ( −N ) and iron ( −Fe ) .\",\n \"Signals from nitrogen limitation and methionine are sensed via AreA and AtfA , whereas iron limitation is sensed via HapX .\",\n \"All three transcription factors activate the promoter of the terrein biosynthesis gene cluster-specific transcription factor .\",\n \"TerR leads to transcription of the structural genes required for terrein biosynthesis and terrein is produced from acetyl- and malonyl-CoA units .\",\n \"The two-cluster specific major facilitator superfamily ( MFS ) transporters export terrein into the rhizosphere .\",\n \"Here terrein can counteract iron limitation by its ferric iron reducing activity , supports degradation of organic matter by its phytotoxic activities , and reduces growth of competitors by its antifungal activity . DOI: http://dx . doi . org/10 . 7554/eLife . 07861 . 032 Another inducing signal derives from methionine , which may be of particular importance when A . terreus acts as a potato plant pathogen ( Louis et al . , 2013 ) .\",\n \"Notably , PDB induces high terrein production rates ( Zaehle et al . , 2014 ) , which we assume to be methionine-dependent .\",\n \"In plants , methionine is required for ethylene production in environmental stress response ( Wang et al . , 2002 ) , fruit ripening ( Yang and Hoffman , 1984 ) , and polyamine production in plant defence reactions .\",\n \"Furthermore , methionine is a precursor for iron chelating siderophores such as nicotianamine and mugineic acid ( Roje , 2006 ) and has been identified in plant root exudates ( Dakora and Phillips , 2002 ) .\",\n \"Therefore , elevated levels of methionine depict a signal that indicates the presence of a plant environment and stimulates terrein production .\",\n \"However , although further studies are required to elucidate the specific contribution of terrein to plant infection , its ability to induce lesion formation on several fruit surfaces indicates that terrein interacts with plant defence mechanisms ( Zaehle et al . , 2014 ) .\",\n \"The third—at first sight unrelated—inducing factor is iron starvation .\",\n \"Although terrein can reduce ferric to ferrous iron , a direct positive growth-promoting effect of terrein during iron limitation in A . terreus monocultures was only observed in the absence of a functional siderophore system .\",\n \"However , extracellular siderophores show a high affinity for ferric iron but not for ferrous iron ( Hider and Kong , 2010 ) .\",\n \"Thus , the reduction of ferric iron could reduce the efficiency of iron chelation by xenosiderophores secreted from competing microorganisms .\",\n \"A similar strategy for iron acquisition has previously been described for the human pathogenic fungus Histoplasma capsulatum ( Timmerman and Woods , 1999 ) .\",\n \"H . capsulatum secretes glutathione accompanied by the γ-glutamyltransferase Ggt1 .\",\n \"This enzyme releases the dipeptide cysteinylglycine from gluthathione , which in turn reduces extracellular ferric iron ( Zarnowski et al . , 2008 ) .\",\n \"While H . capsulatum also secretes hydroxamate siderophores ( Howard et al . , 2000 ) , silencing of ggt1 mRNA reduced virulence in macrophages even in the presence of the siderophore system ( Zarnowski et al . , 2008 ) .\",\n \"Thus , terrein may interfere with competing siderophore systems to increase the competitiveness of A . terreus in the environment .\",\n \"In this study we showed that transduction of environmental signals to the promoter of the transcriptional activator TerR requires activation by the global transcription factors AreA , AtfA , and HapX .\",\n \"While AreA has previously been described to regulate several SM gene clusters in fungi ( Tudzynski et al . , 1999 ) , HapX activates iron acquisition systems ( Schrettl et al . , 2010 ) and AtfA plays a specific role in osmotic and oxidative stress responses ( Lara-Rojas et al . , 2011 ) .\",\n \"In Aspergillus species and maybe all saprophytic ascomycetes , the GATA transcription factor AreA plays a substantial role in regulation of nitrogen .\",\n \"Under nitrogen starvation , AreA is transported into the nucleus ( Todd et al . , 2005 ) where it binds a 5′-HGATAR-3′ sequence in its target promoters ( Ravagnani et al . , 1997 ) , as shown for the genes encoding nitrate reductase ( NiaD ) and nitrite reductase ( NiiD ) ( Chang et al . , 1996 ) that are upregulated in the absence of preferred nitrogen sources ( Johnstone et al . , 1990; Punt et al . , 1995 ) .\",\n \"In Aspergillus parasiticus AreA also binds to the promoter regions of the transcriptional regulator genes aflR and aflJ from the aflatoxin biosynthesis cluster ( Chang et al . , 2000 ) .\",\n \"In contrast to nitrate utilisation , this binding impedes aflatoxin production probably due to inhibition of transcriptional activation ( Yu et al . , 2004 ) .\",\n \"We have shown that terrein biosynthesis in A . terreus during nitrogen starvation is AreA-dependent , and at least two areA binding sites in the terR promoter were recognised by the AreA DNA-binding domain .\",\n \"Therefore , AreA directly binds to the terR promoter and supports the expression of the terrein biosynthesis genes .\",\n \"Similarly , biosynthesis of various mycotoxins and gibberellins is activated by AreA in phytopathogenic Fusarium species ( Mihlan et al . , 2003; Kim and Woloshuk , 2008; Giese et al . , 2013 ) .\",\n \"This implies that AreA plays a general role in regulation of SM production of plant pathogens , and the plant-derived environment may be characterised as a habitat with a high C:N ratio .\",\n \"In several ascomycetes the nitrogen starvation response is also mediated by the basic leucine zipper transcription factor AtfA ( Hong et al . , 2013 ) .\",\n \"In A . nidulans , nitrogen starvation , oxidative or osmotic stresses induce phosphorylation of the MAP kinase SakA that is subsequently transported into the nucleus to activate transcription of target genes by interaction with the constitutively expressed transcription factor AtfA ( Lara-Rojas et al . , 2011 ) .\",\n \"Interestingly , in A . terreus the interaction between AtfA and SakA has not been solved in detail .\",\n \"While a sakA deletion does not affect terrein production levels under nitrogen limitation , it allows terrein production in the presence of some nitrogen sources , indicating that it may possess a repressor function under some conditions .\",\n \"In addition , a ΔsakA strain still produces pigmented conidia ( Figure 4—figure supplement 5 ) and the sakA mutant—but not the ΔatfA strain—showed some increased osmosensitivity ( Figure 4—figure supplement 5 ) , which agrees with recent data on ΔatfA and ΔsakA mutants of Penicillium marneffei ( Nimmanee et al . , 2014 ) .\",\n \"Therefore , it is conceivable that terrein biosynthesis gene cluster activation via AtfA does not require SakA .\",\n \"In A . terreus AtfA seems to act as a general inducer of SM production since its deletion resulted in non-pigmented conidia and in the loss of terrein production under nitrogen starvation or methionine supplementation .\",\n \"In contrast , deletion of the atf1 gene in Botrytis cinerea resulted in accumulation rather than depletion of the polyketide botcinin A and the sesquiterpene botrydial ( Temme et al . , 2012 ) .\",\n \"This points to AtfA as a transcriptional inhibitor in B . cinerea whereas AtfA acts as an inducer in A . terreus .\",\n \"Interestingly , AreA and AtfA sense the same environmental signals to induce the terrein biosynthesis gene cluster , although their global role in cellular physiology differs significantly .\",\n \"This adaptation of different transcription factors towards terrein production underlines a special importance of terrein for A . terreus during environmental competition .\",\n \"Independent from AreA and AtfA , activation of the terrein biosynthesis gene cluster was also triggered by the iron response regulator HapX under iron limitation .\",\n \"To overcome severe iron starvation , microorganisms frequently produce siderophores ( Pollack and Neilands , 1970; Eisendle et al . , 2003; Kreutzer and Nett , 2012 ) or utilise siderophores from competing species ( xenosiderophores ) ( Boukhalfa and Crumbliss , 2002 ) .\",\n \"In A . fumigatus and A . nidulans siderophore biosynthesis and uptake is regulated by the opposing transcription factors HapX and SreA depending on the presence or absence of iron ( Schrettl et al . , 2007 , 2008 , 2010 ) .\",\n \"Similarly , A . terreus produces the HapX and SreA regulated siderophores ferrichrysin and coprogen that allow efficient acquisition of iron .\",\n \"However , in competition with other micoorganisms , terrein-mediated iron reduction could reduce binding of iron to high-affinity siderophores from competitors that cannot be used by A . terreus , as described for the cysteinylglycine iron reduction system from H . capsulatum .\",\n \"The control of terrein production by HapX depicts an unprecedented example of the activation of a SM gene cluster outside the siderophore biosynthesis system .\",\n \"Interestingly , terrein production is only under positive HapX control , but not under negative SreA feedback control , which is in agreement with a lack of SreA binding sites in the terR promoter ( Schrettl et al . , 2008 ) , whereas a CCAAT box required for binding of the CCAAT-binding complex ( CBC ) and its subsequent CBC interaction with HapX ( Hortschansky et al . , 2007; Gsaller et al . , 2014 ) is present .\",\n \"In the HapX target promoter regions of cycA , sreA , acoA , and lysF from Aspergillus species , a bipartite CBC–HapX-DNA binding motif with consensus 5′-C ( C/A ) AATCAN11-12GAT-3′ is present , of which CCAAT is recognised by the CBC complex while the GAT sequence is bound by HapX ( Hortschansky et al . , 2015 ) .\",\n \"Interestingly , these are all types of promoters that are repressed under iron limitation .\",\n \"In contrast , the cccA promoter , which encodes for a vacuolar iron transporter , is the only HapX-induced promoter known so far with a confirmed in vitro HapX binding site ( Gsaller et al . , 2014 ) .\",\n \"HapX binds to the GAT motif of a 5′-CCAATN16GATC-3′ sequence , which is also present in the cccA promoter of A . terreus and—with one mismatch—in the terR promoter ( position −215 ) .\",\n \"However , further studies will be required to confirm this motif as a HapX binding site .\",\n \"In conclusion , we have shown that the terrein biosynthesis gene cluster is activated by at least three independent environmental signals: methionine , nitrogen limitation , and iron starvation .\",\n \"Additionally , the three global transcription factors AreA , AtfA , and HapX are essential to transfer these signals to the terrein biosynthesis gene cluster .\",\n \"Although one environmental signal is sufficient to induce the gene cluster , a combination of different signals provides an environmental image of higher resolution and allows an adequate adaptation to the ecological niche .\",\n \"The phytotoxic and antifungal potential of terrein combined with its iron reductive properties enables A . terreus to acquire nutrients that might otherwise be consumed by competitors .\",\n \"Notably , similar to terrein , the mycotoxin production in Fusarium species is generally associated with sensing of nitrogen availability .\",\n \"Although AreA plays an important regulatory function , involvement of AtfA as an additional regulator and methionine as a second inducer in these species needs to be tested .\",\n \"However , from our results on the terrein biosynthesis gene cluster , we propose that manifold regulation of SM gene clusters and the interplay of different global transcriptional activators might depict a general mechanism to regulate the flexibility of gene cluster activation in fungi .\"\n ],\n [\n \"All genotypes of strains used in this study are summarised in ( Figure 4—source data 1 ) .\",\n \"A . terreus strain SBUG844 and its derivative SBUG844ΔakuB that show increased rates of homologous integration served as parental strains ( Gressler et al . , 2011 ) .\",\n \"If not indicated otherwise , all strains were cultivated in Aspergillus minimal medium ( AMM; http://www . fgsc . net/methods/anidmed . html ) supplemented with different carbon and nitrogen sources: AMM with 100 mM glucose ( AMM-G100 ) , with 100 mM glucose and 50 mM glutamine ( AMM-G100Gln50 ) , with 100 mM glucose , 50 mM glutamine and 10 mM methionine ( AMM-G100Gln50Met10 ) , with 1% casamino acids ( AMM-CA1% ) .\",\n \"Additionally , the following complex media were used: Sabouraud ( Sab , Sigma Aldrich , Taufkirchen , Germany ) , YPD ( 10 g/l yeast extract , 20 g/l peptone , 20 g/l dextrose; PDB , Sigma Aldrich ) , potato broth ( 2% potato extract , Sigma Aldrich ) , apple juice ( pH adjusted to 6 . 0 with NaOH ) , carrot juice with honey ( both Wiesgart , ALDI Nord , Jena , Germany ) , banana juice or peach juice ( both FruchtOase , Kiberg , Germany ) .\",\n \"When required , 140 µg/ml hygromycine B ( Carl Roth GmbH , Karlsruhe , Germany ) , 0 . 1 µg/ml pyrithiamine or 80 µg/ml phleomycin ( both Sigma Aldrich ) were added .\",\n \"For preparation of conidia suspensions , all strains were cultivated for 4 d at 37°C on solid 2% AMM-G50Gln10 agar plates .\",\n \"Conidia were harvested by overlaying colonies with water and filtering off the suspension over 40 μm cell strainers ( VWR , Darmstadt , Germany ) .\",\n \"Liquid media were generally inoculated at a final concentration of 1 × 106 conidia per ml .\",\n \"For the ΔareA strains , inoculation densities were generally doubled to compensate for reduced growth rates .\",\n \"For metabolite extraction and quantification liquid cultures in 50 ml scale were used and incubated at 30°C for 48 hr or 72 hr depending on the specific experiment .\",\n \"For nitrogen or iron shift experiments , strains were pre-cultivated for 40 hr in 150 ml AMM-G100Gln50 or AMM-G100Gln50 with 200 µM FeCl3 .\",\n \"Mycelia were harvested over sterile miracloth ( Merck , Darmstadt , Germany ) washed twice with AMM lacking a nitrogen ( AMM-N ) or iron source ( AMM-Fe ) and aliquots were transferred to fresh 50 ml media with or without 50 mM Gln or with or without 200 µM FeCl3 .\",\n \"Depending on the specific experiments , samples were analysed after 12 , 24 , or 48 hr of cultivation .\",\n \"Metabolites were extracted from culture broth as described previously ( Gressler et al . , 2011 ) .\",\n \"In brief , an equal volume of ethyl acetate was added and collected after defined shaking of the mixture .\",\n \"The procedure was repeated once .\",\n \"After evaporation of the solvent , residues were taken up in 1 ml methanol each and filtered .\",\n \"Standard extract analyses were performed on an Agilent 1100 series HPLC-DAD system coupled with a MSD trap ( Agilent Technologies , Waldbronn , Germany ) operating in alternating ionisation mode .\",\n \"Terrein quantification was carried out from 50 ml cultures as described elsewhere ( Zaehle et al . , 2014 ) .\",\n \"For quantification of the siderophore coprogen , the complete 50 ml culture supernatants were filtered and lyophilised to dryness .\",\n \"The remaining solids were extracted three times with 10 ml MeOH .\",\n \"The solvent from the combined organic extracts was removed under reduced pressure and residues were re-dissolved in 2 ml MeOH .\",\n \"The resulting slurries were filtered and the filtrates analysed by HPLC measurements .\",\n \"HPLC analyses were carried out on an Agilent 1260 device equipped with a quaternary pump and a UV/Vis detector ( Agilent Technologies; Column: Zorbax Eclipse XDB-C8 , 5 µm , 150 × 4 . 6 mm; flow rate 1 ml/min; eluent A: H2O/0 . 1% HCOOH , eluent B: MeOH ) .\",\n \"The gradient started with 10% B and reached 30% B after 4 min , increased to 55% B within 10 min and reached 100% B after 2 min , where it was retained for an additional 4 min .\",\n \"Quantification of coprogen was performed from a calibration curve of known coprogen concentrations .\",\n \"For correlation of coprogen to the fungal biomass , mycelia from the cultures were dried for 48 hr at 37°C and balanced and coprogen concentrations per gram dried mycelium were calculated .\",\n \"All quantifications were carried out in biological triplicates and technical duplicates .\",\n \"Isolation of coprogen for generation of the calibration curve was performed by semi-preparative HPLC from culture supernatants of the ΔakuB and ΔakuBΔterA strains and fractions were collected by automatic fraction collection .\",\n \"Separation was carried out on a Zorbax Eclipse XDB-C8 , 5 µm , 250 × 4 . 6 mm with a flow rate of 4 . 0 ml/min using H2O as eluent A and MeOH as eluent B . The gradient started with 10% B , reached 30% B after 6 . 5 min , increased to 55% B within 16 . 5 min , reached 100% B after 2 min , and was retained at 100% B for an additional 6 min .\",\n \"For isolation of 2- ( ( E ) -prop-1-en-1-yl ) maleic acid , the crude product from upscaled terrein reduction assays ( see below ) was subjected to semi-preparative HPLC using a Zorbax Eclipse XDB-C8 , 5 µm , 250 × 4 . 6 mm with a flow rate of 4 . 0 ml/min , eluent A: H2O/0 . 1% HCOOH , eluent B: acetonitrile .\",\n \"The gradient started with 5% B and was held for 14 min , increased to 10% B within 9 min , increased to 100% B within 2 min where it was retained for an additional 7 min .\",\n \"Fractions from the new metabolite formed from ferric iron reduction were collected and evaporated resulting in a white solid which revealed a m/z value of 150 . 0495 [M + H+] by HRESI-MS that perfectly matched a calculated molecular formula of C7H8O4 containing four double-bond equivalents .\",\n \"13C-NMR measurements ( Figure 5—figure supplement\",\n \"4 ) revealed the presence of two carbonyl groups , one terminal methyl group , and four carbons being part of a conjugated system .\",\n \"Two-dimensional NMR data ( Figure 5—figure supplement\",\n \"5 ) and analysis of all proton coupling constants from the 1H-NMR spectrum ( Figure 5—figure supplement\",\n \"4 ) finally confirmed the structure of 2- ( ( E ) -prop-1-en-1-yl ) maleic acid .\",\n \"NMR spectra were recorded on a Bruker Avance III 500 and a Bruker Avance III 600 spectrometer ( Bruker BioSpin GmbH , Rheinstetten , Germany ) equipped with a cryoprobe head using DMSO-d6 and methanol-d4 as solvents and internal standards .\",\n \"Fruit surfaces were wiped with a soft tissue saturated with 70% ethanol .\",\n \"Apples ( ALDI , type: Tenroy Royal/Gala; Germany ) , bananas ( type Bio , Fairverbindet; tegut , Jena , Germany ) , and nectarines ( tegut , type: Sweet Lady , class 1 , size A; Italy ) were cut lengthwise using a sterile scalpel .\",\n \"The resulting groove was infected with 200 µl of a conidia suspension containing 4 × 107 conidia and fruits were incubated for 7–10 d at room temperature in a humid chamber .\",\n \"Cut but uninfected fruits served as controls .\",\n \"For cultivation in the presence of high levels of nitrogen , 200 µl of a 3 . 5 M NH4Cl solution were applied prior to infection .\",\n \"For terrein quantification , fruits were homogenised and extracted twice with 100 ml ethyl acetate and the solid residues were collected and evaporated for dry weight determination .\",\n \"Terrein was quantified from extracts as described above and terrein production rates were calculated as mg terrein/g fruit dry weight .\",\n \"A detailed description on fruit infection , sample preparation and terrein quantification is described in Bio-protocols by Gressler and Brock ( 2016 ) .\",\n \"To determine the induction of lesion formation on banana fruit surfaces , organic bananas ( type Bio , Fairverbindet; tegut ) were softly cleaned with water and air dried .\",\n \"5 µl of various sequential dilutions ( 1:2 to 1:512 ) of metabolite crude extracts ( dissolved in PBS ) were added as a single drop to the surface .\",\n \"Fruits were incubated in the dark at room temperature and photographs were taken after 40 hr and 60 hr .\",\n \"The determination of ammonia was performed as described by Weichselbaum et al . ( 1969 ) .\",\n \"From each culture 1 ml aliquots of broth were removed and centrifuged for 5 min at 16 , 000×g to remove residual mycelium .\",\n \"The supernatant was collected and adjusted to pH 7 . 0 by addition of NaOH .\",\n \"A 200 µl aliquot was sequentially diluted in a nitrogen-free medium and transferred to a transparent flat-bottom 96-well plate .\",\n \"After addition of 20 µl SC solution ( 6 . 5 g sodium salicylate ( C7H5O3Na ) , 6 . 5 g trisodium citrate ( C6H5O7Na3 × 2 H2O ) , 48 . 5 mg disodium pentacyanonitrosylferrate ( Na2Fe ( CN ) 5NO × 2 H2O ) in 50 ml water ) , the reaction was started with 20 µl DCIC solution ( 1 . 6 g NaOH , 100 mg sodium dichloroisocyanurate ( C3N3Cl2O3Na ) in 50 ml water ) .\",\n \"Readings were done after 4 . 5 hr of incubation at room temperature in a FLOUstar Omega microplate reader ( BMG Labtech , Ortenberg , Germany ) .\",\n \"Plates were shaken for 1 min in a double orbital direction and absorbance at 655 nm was measured ( 50 flashes/well , gain 2000 ) .\",\n \"Fresh growth medium served as positive and water as negative controls .\",\n \"A calibration curve was recorded for calculation of ammonia levels from culture broth .\",\n \"The ferric iron-reducing assay was performed virtually as described by Benzie and Strain ( 1996 ) .\",\n \"In brief , three different working solutions were prepared by mixing 20 parts of reagent A ( 0 . 4% sodium acetate , 1 . 6% acetic acid; pH\",\n \"4 ) with 1 part of reagent B ( 0 . 4% 2 , 4 , 6-tripyridyl-S-triazine ( TPTZ; Sigma ) in 0 . 16% HCl and either 1 part of reagent C1 ( 88 mM–5 . 5 mM sequential dilutions of terrein ) , reagent C2 ( 2% ascorbic acid , serving as positive control ) or reagent C3 ( water , serving as negative control ) .\",\n \"From each working solution , 200 µl were transferred into wells of a 96-well plate .\",\n \"An iron stock solution ( 10 mg/ml FeCl3 in 0 . 2 M H2SO4 ) was diluted to 0 . 05 mg/ml and 100 µl were added to the working solution , which started the reaction .\",\n \"Reduction of ferric to ferrous iron was followed by determination of the change in absorbance at 590 nm for up to 10 hr using a FLOUstar Omega microplate reader ( BMG Labtech ) .\",\n \"For determination of the pH optimum of the terrein mediated reduction , reagent A was adjusted to pH values of 3 . 5–6 . 0 .\",\n \"Reductive activity was normalised against the activity determined from ascorbic acid , which was set as 100% .\",\n \"All experiments were carried out in triplicate .\",\n \"Upscaling of the iron reduction assays for metabolite extraction was performed by incubating 20 mg ( 125 µmol ) of terrein in a 1:2 molar ratio with either FeSO4 , FeCl3 , CH3COOCu , or ( CH3COO ) 2Cu in 10 ml of reagent A . Samples were incubated for 48 hr at room temperature under continuous vertical rotation ( 20 rpm ) .\",\n \"Metabolites were extracted three times with ethyl acetate ( 10 ml each ) .\",\n \"The solvent was evaporated and the residues dissolved in MeOH and analysed as described above .\",\n \"Chrome azol S agar plate assays were prepared as described by Milagres et al . ( 1999 ) .\",\n \"Holes of 8 mm diameter were punched into plates and inoculated with 100 µl methanolic solutions of coprogen , terrein , or propylene maleic acid in concentrations of 2 . 50 , 1 . 25 , 0 . 625 , and 0 . 3125 mg/ml .\",\n \"Plates were incubated for 48 hr at 30°C prior to photography .\",\n \"Mycelia from specified cultivations were briefly washed with water and ground under liquid nitrogen .\",\n \"RNA was isolated using the RiboPure RNA Purification Kit ( Ambion , Life Technologies , Darmstadt , Germany ) .\",\n \"Residual genomic DNA ( gDNA ) was removed by the DNA-free kit ( Ambion ) .\",\n \"cDNA was synthesised by Revert Aid Reverse transcriptase ( Thermo Scientific , Schwerte , Germany ) using anchored oligodT primers .\",\n \"qRT-PCR was carried out on a CFX384 Touch Real-Time PCR Detection System ( BioRad , Munich , Germany ) using the EvaGreen 5 × QPCR ( ROX ) Mix ( Bio & Sell , Feucht , Germany ) following the manufacturer's protocol and using 1:5 and 1:10 dilutions of cDNA samples serving as templates .\",\n \"The actin gene ( actA , ATEG_06973 ) and the enolase gene ( enoA , ATEG_02902 ) were used for normalisation of transcript levels yielding similar results .\",\n \"Normalised transcript levels were calculated as fold expression = 2Δ ( reference − target ) .\",\n \"Primers used for qRT-PCRs showed a primer efficiency of 1 . 89–2 . 0 and are listed in Figure 4—source data 2 ( Figure 4—source data 2 ) .\",\n \"In general , all PCR amplifications were performed using the Phusion DNA Polymerase ( Thermo Scientific ) .\",\n \"Plasmids were amplified in E . coli DH5α .\",\n \"A detailed description of all cloning procedures is given below .\",\n \"Oligonucleotides used for plasmid construction are listed in the Figure 4—source data 2 ( Figure 4—source data 2 ) .\",\n \"Fungal transformation was performed as described previously ( Gressler et al . , 2011 ) and transformants were checked by diagnostic PCR and Southern blot analyses ( see Figure 4—figure supplement 4 ) .\",\n \"The promoter of the terA gene ( PterA; 1220 bp ) was amplified with oligonucleotides P47/48 from gDNA of A . terreus SBUG844 and ligated into the NotI/BamHI digested plasmid lacZ:trpCT-pJET1 . 2 ( Gressler et al . , 2011 ) containing the E . coli lacZ gene and the trpC terminator sequence .\",\n \"The plasmid was linearised by NotI digestion and the NotI-excised ptrA cassette from plasmid ptrA-pJET ( Fleck and Brock , 2010 ) was inserted .\",\n \"The resulting plasmid was used for transformation of A . terreus SBUG844 wild-type resulting in SBUG844_PterA:lacZ .\",\n \"Constitutive strong expression of the atfA gene was performed by using the strong constitutive gpdA promoter from A . nidulans FGSC A4 ( AnPgpdA , 1387 bp; oligonucleotides P49/50 ) to control expression of A . terreus atfA .\",\n \"The atfA ORF together with its natural terminator was amplified from gDNA of A . terreus SBUG844 ( atfA + atfAT , 2466 bp ) using oligonucleotides P51/52 .\",\n \"Both fragments were fused by in vitro recombination with the SpeI- restricted hph-pCRIV vector using the InFusion HD cloning kit ( Clonetech Laboratories , Saint-Germain-en-Laye , France ) .\",\n \"The resulting plasmid AnPgpdA:atfA:atfAT_hph-pCRIV was used for transformation of A . terreus SBUG844 wild-type resulting in strain SBUG844_AnPgpdA:atfA .\",\n \"The upstream and downstream fragments of cpcA ( ATEG_03131 ) were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P53/54 ( 943 bp ) and P55/56 ( 853 bp ) .\",\n \"Similarly , the upstream and downstream flanks of rhbA ( ATEG_09480 ) were amplified with oligonucleotides P57/58 ( 473 bp ) and P59/60 ( 418 bp ) .\",\n \"The respective fragments were fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) , resulting in cpcAup-ptrA-cpcAdn-pUC19 , rhbAup-ptrA-rhbAdn-pUC19 , and dnmTup-ptrA-dnmTdn-pUC19 .\",\n \"The deletion cassettes were excised by KpnI restriction and used for transformation of SBUG844ΔakuB .\",\n \"The upstream and downstream flanks of areA ( ATEG_07264 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P61/62 ( 820 bp ) and P63/64 ( 731 bp ) .\",\n \"Fragments were fused by fusion PCR and ligated into the pJET1 . 2 cloning vector ( Thermo Scientific ) .\",\n \"The fragment was excised with SmaI and subcloned into pUC19 .\",\n \"After restriction with NotI , the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) was inserted resulting in areAup-ptrA-areAdn-pUC19 .\",\n \"The deletion cassette was excised by SmaI and used for transformation of SBUG844ΔakuB .\",\n \"For complementation , a fragment spanning the entire areA ORF including its promoter and terminator sequence was amplified from gDNA of the A . terreus type strain FGSC A1156 ( NIH2624 ) using oligonucleotides P65/64 ( 4181 bp ) and ligated into pJET1 . 2 .\",\n \"gDNA from the type strain was used because its areA promoter sequence contained an additional PstI restriction site that allowed discrimination of complemented strains from the parental SBUG844ΔakuB strain .\",\n \"The complementation fragment was excised by XhoI restriction from plasmid A1156 ( areAup:areA:areAdn ) -pJET1 . 2 and directly used for the transformation of SBUG844ΔakuBΔareA .\",\n \"Due to the inability of the ΔareA mutant to utilise various nitrogen sources , no additional resistance marker was required and transformants were regenerated on media containing nitrate as the sole nitrogen source .\",\n \"Complemented strains were checked by Southern blot analysis and an additional PstI control digest of the PCR-amplified areA upstream flank .\",\n \"The upstream and downstream fragments of atfA ( ATEG_04664 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P66/67 ( 1102 bp ) and P68/69 ( 890 bp ) and fused by in vitro recombination with the NotI-excised ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-excised pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in atfAup-ptrA-atfAdn-pUC19 .\",\n \"The deletion cassette was excised by KpnI and used for transformation of SBUG844ΔakuB .\",\n \"For deletion of atfA in the ΔareA background , the ptrA resistance cassette was replaced by the phleomycin resistance ( ble ) cassette ( NotI digest from ble-pJET1 . 2 ) .\",\n \"The deletion cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuBΔareA .\",\n \"For complementation of the ΔatfA phenotype , a fragment spanning the entire atfA gene including its promoter and terminator region were amplified with oligonucleotides P66/70 ( 3319 bp ) and an additional downstream fragment was amplified with P71/69 ( 859 bp ) .\",\n \"The fragments were fused by in vitro recombination with the NotI-excised ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in atfAup:atfA:atfAT_ble_atfAdn-pUC19 .\",\n \"The complementation cassette was excised by KpnI and used for transformation of SBUG844ΔakuBΔatfA .\",\n \"The atfA flanking regions were used to integrate the terR gene under control of the A . nidulans gpdA promoter in the atfA locus , which results in an atfA deletion .\",\n \"The upstream and downstream flanks of atfA were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P72/73 ( 1125 bp ) and P74/75 ( 913 bp ) .\",\n \"Subsequently , a fragment already containing the fusion of AnPgpdA with the terR ORF including its native terminator ( 4405 bp ) was amplified with oligonucleotides P76/77 from plasmid hph_AnPgpdA:terR-pUC19 ( Gressler et al . , 2015a ) .\",\n \"The plasmid hph-pCRIV ( Fleck and Brock , 2010 ) was restricted with EcoRI to remove the hph resistance cassette and all three fragments were fused by in vitro recombination into the EcoRI site using the InFusion HD cloning kit ( Clonetech Laboratories ) .\",\n \"The resulting plasmid atfAup_AnPgpdA:terR:terRT_atfAdn_pCRIV was linearised with NotI and the ble resistance cassette from ble-pJET1 . 2 was inserted between the terR terminator and the atfA downstream region .\",\n \"The final plasmid was restricted with EcoRI , and the fragment atfAup_AnPgpdA:terR:terRT_ble_atfAdn was used for transformation of A . terreus SBUG844ΔakuBΔareA to replace the atfA ORF with the terR overexpression construct .\",\n \"Because the complete sequence information of the hapX locus was lacking at the beginning of this study , only a partial deletion of the A . terreus hapX gene was performed .\",\n \"Upstream and downstream flanks inside the hapX coding region ( ATEG_08014 ) were amplified with oligonucleotides P82/83 ( 387 bp ) and P84/85 ( 345 bp ) from genomic DNA of SBUG844 and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in hapXup-ptrA-hapXdn-pUC19 .\",\n \"The deletion construct was excised by KpnI restriction and used for transformation of SBUG844ΔakuB .\",\n \"For complementation of the partial hapX deletion by the A . nidulans hapX , the upstream and downstream flanks of ATEG_08014 used for generation of the partial deletion construct were amplified with oligonucleotides P82/86 ( 381 bp ) and P87/85 ( 341 bp ) and the complete hapX ORF ( AN8251 ) including its native promoter and terminator sequence was amplified from gDNA of A . nidulans FGSC A4 using oligonucleotides P88/89 ( 2648 bp ) .\",\n \"All fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 ( 2073 bp ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in hapXup_PAnhapX:AnhapX:AnhapXT_ble_hapXdn-pUC19 .\",\n \"The complementation cassette was excised by KpnI and used for transformation of SBUG844ΔakuBΔhapX .\",\n \"To identify the complete coding region of the A . terreus hapX gene , long run PCRs from within the hapX gene and the last nucleotides of the known 5′- and 3′- borders were performed with oligonucleotides P110/111 ( 800 bp 5′ fragment ) and P112/113 ( 2500 bp 3′ fragment ) .\",\n \"Bands were excised from agarose gels and cloned into the pJET1 . 2 cloning vector .\",\n \"Fragments were sequenced from both strands using the primer walking method ( oligonucleotides P114/115 for both fragments and additionally P116 , P117 and P78 for the 3′ fragment ) .\",\n \"Finally , RNA was isolated from iron starvation conditions , transcribed into cDNA , and the hapX ORF was amplified with gene-specific oligonucleotides 113/79 and sequenced with oligonucleotides P114/115 .\",\n \"The complete hapX locus information was submitted to EMBL and can be found under accession number KP233834 ( Gressler et al . , 2015b ) .\",\n \"The upstream and downstream flanking regions of sreA ( ATEG_07714 ) were amplified from gDNA of A . terreus SBUG844 with oligonucleotides P90/91 ( 763 bp ) and P92/93 ( 756 bp ) and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sreAup-ptrA-sreAdn-pUC19 .\",\n \"The deletion cassette was excised by KpnI and used for transformation of SBUG844ΔakuB .\",\n \"For complementation , a fragment spanning the sreA coding region including its natural promoter and terminator sequence was amplified with oligonucleotides P94/95 ( 3018 bp ) and additional downstream fragment with P96/97 ( 970 bp ) .\",\n \"The fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sreAup:sreA:sreAT_ble_sreAdn-pUC19 .\",\n \"The complementation cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuBΔsreA .\",\n \"The upstream and downstream fragments of the sidA gene ( ATEG_06879 ) were amplified from gDNA of A . terreus SBUG844 using oligonucleotides P98/99 ( 764 bp ) and P100/101 ( 939 bp ) and fused by in vitro recombination with the ptrA resistance cassette ( 1950 bp ) from ptrA-pJET1 ( Fleck and Brock , 2010 ) into the KpnI-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sidAup-ptrA-sidAdn-pUC19 .\",\n \"For transformation of the terA deletion mutant , the ptrA cassette was excised by NotI restriction and replaced by the ble cassette .\",\n \"The deletion cassette was excised by KpnI restriction and used for transformation of SBUG844ΔakuB and SBUG844ΔakuBΔterA .\",\n \"For complementation of strain SBUG844ΔakuBΔsidA , the sidA ORF including its promoter and terminator sequence was amplified with oligonucleotides P102/103 ( 2393 bp ) and an additional downstream fragment was amplified with P104/105 ( 919 bp ) .\",\n \"The fragments were fused by in vitro recombination with the ble resistance cassette ( 2073 bp ) from ble-pJET1 . 2 into the HindIII-restricted pUC19 vector using the InFusion HD cloning kit ( Clonetech Laboratories ) resulting in sidAup:sidA:sidAT_ble_sidAdn-pUC19 .\",\n \"The complementation cassette was excised by HindIII restriction and used for transformation of SBUG844ΔakuBΔsidA .\",\n \"The coding sequence for the A . nidulans AreA DNA-binding domain ( amino acids 663–797 ) was amplified from plasmid pGEX4T1-AreA-ZnF ( Muro-Pastor et al . , 2004 ) as BamHI-HindIII fragment using oligonucleotides P80/81 ( Figure 4—source data 2 ) .\",\n \"The amplified fragment was cloned into a modified pET-43 . 1a vector allowing the addition of a N-terminal ( His ) 6-tag and removable by a tobacco etch virus ( TEV ) protease ( Novagen ) .\",\n \"( His ) 6-AreA663-797 was produced by E . coli BL21 ( DE3 ) cells grown at 30°C in 1 l Overnight Express Instant TB Medium ( Novagen , Darmstadt , Germany ) in the presence of 1 mM Zn ( OAc ) 2 .\",\n \"Cells ( 15–20 g wet weight ) were collected by centrifugation , resuspended in 200 ml lysis buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 10% vol/vol glycerol , 40 mM imidazole , 5 mM β-mercaptoethanol , 1 mM AEBSF , pH 8 . 0 ) and disrupted using an Emulsiflex C5 high pressure homogeniser ( Avestin , Mannheim , Germany ) .\",\n \"Cleared cellular extract was loaded to a 25 ml Ni Sepharose FF ( GE Heathcare , Freiburg , Germany ) column and ( His ) 6-AreA663-797 was eluted with 200 mM imidazole .\",\n \"After removal of the ( His ) 6 tag by adding 4 μg TEV protease per mg peptide and overnight incubation at room temperature , samples were adjusted to 150 mM NaCl and applied on a 40 ml CellufineSulfate ( Millipore , Darmstatdt , Germany ) column that was equilibrated with 50 mM NaH2PO4 , 150 mM NaCl , 10% vol/vol glycerol , 5 mM β-mercaptoethanol , 10 µM Zn ( OAc ) 2 , pH 7 . 5 , followed by elution of AreA663–797 with a gradient up to 2 M NaCl .\",\n \"Peak fractions were concentrated with an Amicon Ultra-15 10K centrifugal filter device and purified to homogeneity by size exclusion chromatography on a Superdex 75 prep grade column ( GE Healthcare ) in 20 mM HEPES , 300 mM NaCl , 5 mM β-mercaptoethanol , 10 µM Zn ( OAc ) 2 , pH 7 . 5 as running buffer .\",\n \"The absolute molecular mass of AreA663–797 was determined in series on a miniDawn TREOS static light scattering monitor and an Optilab T-rEX differential refractometer ( Wyatt , Dernbach , Germany ) .\",\n \"The molar mass was calculated using ASTRA 6 software ( Wyatt ) .\",\n \"AreA663–797 was stored in 50% vol/vol glycerol at −20°C .\",\n \"Real-time analyses were performed on a Biacore 2000 system ( GE Healthcare ) at 25°C .\",\n \"DNA duplexes were produced by annealing complementary oligonucleotides using a fivefold molar excess of the non-biotinylated oligonucleotide .\",\n \"The dsDNA was injected on flow cells of a streptavidin ( Sigma ) -coated CM3 sensor chip at a flow rate of 10 µl/min until the calculated amount of DNA that gives a maximum AreA663–797 binding capacity of 100 RU were bound .\",\n \"AreA663–797 was injected in running buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 0 . 005% ( vol/vol ) surfactant P20 , 5 mM β-mercaptoethanol and 1 µM ZnCl2 ) at concentrations from 3 . 125 to 200 nM .\",\n \"Sample injection and dissociation times were set to 200 and 400 s at a flow rate of 30 µl/min .\",\n \"Regeneration was achieved with 10 mM Tris/HCl pH 7 . 5 , 0 . 5 M NaCl , 1 mM EDTA and 0 . 005% ( wt/vol ) SDS for 1 min .\",\n \"Refractive index errors due to bulk solvent effects were corrected with responses from DNA-free flow cell 1 as well as subtracting blank injections .\",\n \"Kinetic raw data were processed and globally fitted with Scrubber 2 . 0c ( BioLogic Software ) using a 1:1 interaction model including a mass transport term .\",\n \"All analyses in which statistical analyses were required were performed in biological triplicates with at least two technical replications .\",\n \"Significance was calculated by use of the Microsoft Excel 2007 software package using the Student's paired t-test with a two-tailed distribution; p values were marked as follows: *p<0 . 05 , **p<0 . 01; ***p<0 . 001 .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Secondary metabolites have a great potential as pharmaceuticals , but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer .","Here , signals , mediators , and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition .","Terrein causes fruit surface lesions and inhibits plant seed germination .","Additionally , terrein is moderately antifungal and reduces ferric iron , thereby supporting growth of A . terreus under iron starvation .","In accordance , the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX .","Independent signal transduction allows complex sensing of the environment and , combined with its broad spectrum of biological activities , terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition ."],"string":"[\n \"Secondary metabolites have a great potential as pharmaceuticals , but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer .\",\n \"Here , signals , mediators , and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition .\",\n \"Terrein causes fruit surface lesions and inhibits plant seed germination .\",\n \"Additionally , terrein is moderately antifungal and reduces ferric iron , thereby supporting growth of A . terreus under iron starvation .\",\n \"In accordance , the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX .\",\n \"Independent signal transduction allows complex sensing of the environment and , combined with its broad spectrum of biological activities , terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Organisms produce a wide variety of small molecules called metabolites through the break down of food and other chemical reactions .","Some of these molecules—known as primary metabolites—are required for growth , reproduction and other vital processes .","Other molecules called secondary metabolites are not strictly required by the organism , but generally have other roles that may improve the individual’s ability to survive and reproduce .","Fungi and other microbes produce a large variety of secondary metabolites , many of which are used as medicines to treat diseases in humans and other animals .","For example , a molecule called lovastatin—which is produced by a fungus known as Aspergillus terreus—can reduce a human patient's risk of heart disease .","However , it is not known what role many secondary metabolites play in the microbe that produced them .","A . terreus lives in the soil , but it can also infect plants and animals .","In addition to lovastatin , it also makes another secondary metabolite called terrein .","A recent study identified the genes responsible for making terrein , and discovered that this molecule is harmful to plant cells and may help the fungus to colonize and thrive in the area immediately around plant roots , which is known as the rhizosphere .","Here , Gressler et al . studied how terrein may help the fungus to cope with competitors in this environment .","The experiments show that terrein increases the availability of iron and inhibits the growth of competing microbes .","A shortage of iron or nitrogen-containing nutrients can stimulate the fungus to produce terrein , and elevated levels of a molecule called methionine have the same effect .","These conditions are commonly found in the rhizosphere and further experiments identified several proteins in the fungus that are required for sensing them .","Gressler et al . 's findings suggest that terrein helps to ensure that the fungus has sufficient nitrogen and iron to thrive in the rhizosphere .","Also , this study confirms that the production of secondary metabolites in microbes can happen in response to elaborate cues from the environment , which may explain why only a limited number of secondary metabolites are produced by microbes when they are grown in the laboratory .","Future studies will analyze other ways to activate the production of secondary metabolites outside of the microbe's normal environment , which may lead to the discovery of new important drugs ."],"string":"[\n \"Organisms produce a wide variety of small molecules called metabolites through the break down of food and other chemical reactions .\",\n \"Some of these molecules—known as primary metabolites—are required for growth , reproduction and other vital processes .\",\n \"Other molecules called secondary metabolites are not strictly required by the organism , but generally have other roles that may improve the individual’s ability to survive and reproduce .\",\n \"Fungi and other microbes produce a large variety of secondary metabolites , many of which are used as medicines to treat diseases in humans and other animals .\",\n \"For example , a molecule called lovastatin—which is produced by a fungus known as Aspergillus terreus—can reduce a human patient's risk of heart disease .\",\n \"However , it is not known what role many secondary metabolites play in the microbe that produced them .\",\n \"A . terreus lives in the soil , but it can also infect plants and animals .\",\n \"In addition to lovastatin , it also makes another secondary metabolite called terrein .\",\n \"A recent study identified the genes responsible for making terrein , and discovered that this molecule is harmful to plant cells and may help the fungus to colonize and thrive in the area immediately around plant roots , which is known as the rhizosphere .\",\n \"Here , Gressler et al . studied how terrein may help the fungus to cope with competitors in this environment .\",\n \"The experiments show that terrein increases the availability of iron and inhibits the growth of competing microbes .\",\n \"A shortage of iron or nitrogen-containing nutrients can stimulate the fungus to produce terrein , and elevated levels of a molecule called methionine have the same effect .\",\n \"These conditions are commonly found in the rhizosphere and further experiments identified several proteins in the fungus that are required for sensing them .\",\n \"Gressler et al . 's findings suggest that terrein helps to ensure that the fungus has sufficient nitrogen and iron to thrive in the rhizosphere .\",\n \"Also , this study confirms that the production of secondary metabolites in microbes can happen in response to elaborate cues from the environment , which may explain why only a limited number of secondary metabolites are produced by microbes when they are grown in the laboratory .\",\n \"Future studies will analyze other ways to activate the production of secondary metabolites outside of the microbe's normal environment , which may lead to the discovery of new important drugs .\"\n]","isConversational":false},"year":{"kind":"string","value":"2015"}}},{"rowIdx":297,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"Corticostriatal dynamics encode the refinement of specific behavioral variability during skill learning"},"id":{"kind":"string","value":"elife-09423-v2"},"sections":{"kind":"list like","value":[["Animals have the ability to learn novel motor skills , allowing them to perform complex patterns of movement to improve the outcomes of their actions .","Acquiring novel skills usually requires exploration of the behavioral space , which is critical for learning ( Skinner , 1981; Sutton and Barto , 1998; Grunow and Neuringer , 2002; Kao et al . , 2005; Olveczky et al . , 2005; Tumer and Brainard , 2007; Miller et al . , 2010; Wu et al . , 2014 ) .","It also requires the selection of the appropriate behavioral features that lead to the desired outcomes ( Skinner , 1981 ) .","It has been postulated that the motor system can learn complex movements by optimizing motor variability in task-relevant dimensions , correcting only deviations that interfere with the final output of the action ( Todorov and Jordan , 2002; Scott , 2004; Valero-Cuevas et al . , 2009; Diedrichsen et al . , 2010 ) .","By optimizing the precision of an action endpoint , for example , humans can perform smooth movements even in the presence of noise ( Harris and Wolpert , 1998 ) .","Selecting task-relevant features and decreasing task-relevant variability might therefore be a critical component of motor learning ( Franklin and Wolpert , 2008; Cohen and Sternad , 2009; Valero-Cuevas et al . , 2009; Costa , 2011; Shmuelof et al . , 2012 ) .","The reduction of motor variability specifically in relevant domains suggests that the neural activity giving rise to the task-relevant output is selected during learning .","However , it is still unclear how the differential refinement of behavioral variability is encoded at the neural level .","It has been suggested that cortical and basal ganglia circuits are important for the selection of task-relevant features ( Costa et al . , 2004; Barnes et al . , 2005; Kao et al . , 2005; Olveczky et al . , 2005; Jin and Costa , 2010; Woolley et al . , 2014 ) .","Consistently , it has been previously shown that the initial stages of learning have increased behavioral ( Tumer and Brainard , 2007; Jin and Costa , 2010; Miller et al . , 2010 ) and neuronal ( Costa et al . , 2004; Barnes et al . , 2005 ) variability , but as specific movements are consolidated , neural variability is reduced in these circuits ( Costa et al . , 2004; Kao et al . , 2005 ) .","This suggests that after initial motor and neural exploration , specific patterns are selected and consolidated ( Costa , 2011 ) .","In this study , we investigated if the dynamics of neural activity in cortical and striatal circuits reflect the changes of variability in specific behavioral domains , and if corticostriatal plasticity is critical for the refinement of particular behavior features ."],["We trained mice to perform a fast lever-pressing task where they were required to press a lever at increasingly higher frequencies , in order to obtain a 20 mg food pellet .","After introducing the animals ( N = 20 ) to the behavioral apparatus and 1 day of continuous reinforcement , where each lever-press was reinforced , animals were trained intensively with three daily sessions for 3 days to perform fast lever presses .","In the fast press schedules we introduced a covert minimum frequency target , defined by the inverse of three consecutive inter-press intervals ( 3 IPIs , 4 presses ) , which increased across sessions from 0 Hz to a maximum of 4 . 5 Hz ( Figure 1A; see ‘Materials and methods’ ) .","The total number of lever presses per minute increased throughout training ( F8 , 152 = 41 . 34 , p < 0 . 0001; Figure 1—figure supplement 1A ) and animals rapidly started to organize their behavior in self-paced bouts or sequences of lever presses , until there were almost no single presses ( Figure 1C , E and Video 1 ) . 10 . 7554/eLife . 09423 . 003Figure 1 . Mice learn a fast lever-pressing task , shaping their behavior to gradually approach the minimum frequency target .","( A ) Schematic of the training protocol , starting with magazine habituation and CRF training in the first 2 days , followed by 3 days of the fast press schedules ( S1–S9 ) where we introduce an increasingly higher covert target , defined as the inverse of the sum of three consecutive inter-press intervals ( IPIs ) .","( B ) Joint distribution of the frequency ( log scale ) for all individual IPIs , in the first , middle and last session of the fast press schedules , for all the 20 animals .","Vertical dashed lines correspond to the IPI threshold used for sequence definition ( IPI = 2 s , 0 . 5 Hz ) and the final covert target ( IPI = 3/660 ms , 4 . 5 Hz ) .","( C ) Percentage of lever presses comprised within sequences .","( D ) Number of sequences performed per minute .","( E ) Left: Example of sequences performed by a representative animal , aligned at the time of sequence initiation .","Individual lever presses are marked as black ticks , the full sequence duration is shaded in grey and the IPIs that meet the session minimum target are shaded in orange; Top right: Probability of a magazine check immediately after a successful covert target; Bottom right: Probability of a magazine check having occurred after a reinforced lever-press vs a non-reinforced lever-press .","( F ) Distance of all three consecutive IPIs ( summed ) from the final covert target ( ∑ ( 3 IPIs ) <660 ms , ∼4 . 5 Hz ) .","( G ) Spread of the distance between all three consecutive IPIs ( summed ) around the final minimum frequency target .","( H ) Percentage of sequences containing the minimum frequency target of the last session ( end-target: 3 IPIs <660 ms , ∼4 . 5 Hz ) .","Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00310 . 7554/eLife . 09423 . 004Figure 1—figure supplement 1 . Lever-pressing rate increased and shifted towards higher speeds with training , and performance increased or plateaued when task difficulty did not change in consecutive sessions .","( A ) Lever presses per minute ( F8 , 152 = 41 . 34 , p < 0 . 0001 ) .","( B ) Percentage of reinforced sequences ( F8 , 152 = 57 . 31 , p < 0 . 0001 , Post hoc comparisons: Fisher's LSD test , ( 0 . 75 Hz ) Session 3 vs Session 4 t152 = 3 . 847 , p = 0 . 0002; ( 3 Hz ) Session 6 vs Session 7 t152 = 0 . 7681 , p = 0 . 4436; ( 4 . 5 Hz ) Session 8 vs Session 9 t152 = 2 . 639 , p = 0 . 0092 ) .","Shaded areas correspond to mean ± SEM .","( C , D )","Histograms with distribution of instantaneous lever-press frequencies , using linear ( C ) and log scales ( D ) , defined as the inverse of all the individual IPIs from the 20 animals .","Vertical dashed lines correspond to the IPI threshold used for sequence definition ( IPI = 2 s , 0 . 5 Hz ) and the final minimum frequency target ( IPI = 220 ms , 4 . 5 Hz ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00410 . 7554/eLife . 09423 . 005Video 1 . Animal performing sequences of lever-presses , doing magazine checks and obtaining reinforcement during the last training session . A 20 mg food pellet was delivered in the magazine when the animal performed three consecutive presses within 660 ms ( covert target = 4 . 5 Hz ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 005 The distribution of the instantaneous lever press frequencies ( calculated as the inverse of the each IPI ) shows a clear shift from initial sessions , where animals did mostly slow frequency presses ( 0–0 . 5 Hz; but already some higher frequency presses of 0 . 5–4 . 5 Hz and >4 . 5 Hz ) , to latter sessions where the distribution was shifted towards faster pressing speeds ( Figure 1—figure supplement 1C ) .","A clear multimodal distribution became evident in log scale , with long IPIs ( frequencies <0 . 5 Hz , Figure 1B and Figure 1—figure supplement 1D ) representing pauses in pressing or magazine checks .","This allowed us to identify the sequences or bouts of pressing a posteriori , based on behavioral performance ( either by a pause in pressing higher than 2 s or by the occurrence of checking behavior , i . e . , magazine checks between presses; see ‘Materials and methods’ ) , independently of the requirements for a specific training session .","Importantly , reinforcement delivery did not provide an external cue that could be used by the animals to anticipate a reward , as the probability of performing a magazine check immediately after a successful covert target ( instead of performing another press ) was not significantly different from 0 . 5 both on early ( t19 = 0 . 9232 , p = 0 . 3675 ) and late sessions ( t19 = 1 . 763 , p = 0 . 0940 ) , and did not change throughout learning ( F8 , 152 = 1 . 753 , p = 0 . 0907 , Figure 1E , top right ) .","Because a large number of sequences did not contain covert patterns ( were not reinforced ) we have also calculated the probability of a magazine check having occurred after a reinforced lever-press vs a non-reinforced lever-press , and observed that this was rather low ( ∼0 . 25 ) and did not change from early to late sessions ( Post hoc comparison: t144 = 1 . 184 , p = 0 . 283 , Figure 1E , bottom right ) .","The percentage of lever presses performed within a sequence increased significantly from 56 . 98 ± 3 . 98 in the first session of covert target introduction , to 98 . 26 ± 0 . 53 in the last training session ( F8 , 152 = 60 . 22 , p < 0 . 0001; Figure 1C ) , and the number of sequences performed per minute increased with training ( F8 , 152 = 32 . 23 , p < 0 . 0001; Figure 1D ) .","The percentage of reinforced sequences tended to decrease , since the difficulty of the task increased across sessions , but tended to stabilize or increase when the same target difficulty was repeated in two consecutive sessions ( F8 , 152 = 57 . 31 , p < 0 . 0001; Figure 1—figure supplement 1B ) .","Importantly , with training , the distance of consecutive IPIs ( summed in bins of 3 IPIs to mimic the online criteria ) to the final target frequency ( 3 IPIs <660 ms , ∼4 . 5 Hz ) decreased consistently ( F8 , 152 = 25 . 76 , p < 0 . 0001; Figure 1F ) , indicating that animals shaped their behavior gradually to approach the end target .","Not only did the distance to the end target decrease , but the spread around the target also decreased ( F8 , 152 = 9 . 616 , p < 0 . 001; calculated as the standard deviation around the target frequency , Figure 1G ) .","Consistently , animals gradually increased the percentage of press bouts that would achieve the minimum target frequency of the last session ( end-target: 3 IPIs <660 ms , ∼4 . 5 Hz; F8 , 152 = 14 . 15 , p < 0 . 0001; Figure 1H ) .","These data indicate that animals learned to shape their behavior to get closer to the covert target .","The mean frequency of each pressing bout ( sequence frequency ) decreased slightly ( F8 , 152 = 2 . 372 , p = 0 . 0195 , Figure 2A ) , while the duration of each pressing bout ( sequence duration ) increased with training ( F8 , 152 = 22 . 69 , p < 0 . 0001 , Figure 2B ) .","Importantly , the sequence-to-sequence variability of the behavioral parameters ( measured both by the variance and by the Fano factor , Figure 2C–F ) was differentially modulated during training .","While the variability of sequence frequency decreased significantly throughout training ( variance: F8 , 152 = 4 . 450 , p < 0 . 0001 , Figure 2C; Fano factor: F8 , 152 = 5 . 343 , p < 0 . 0001 , Figure 2E ) , the variability of sequence duration significantly increased ( variance: F8 , 152 = 11 . 15 , p < 0 . 0001 , Figure 2D; Fano factor: F8 , 152 = 16 . 86 , p < 0 . 0001 , Figure 2F ) .","The sequence-to-sequence variability of these two behavioral features was independent as there was no correlation between the variability in sequence frequency and the variability in sequence duration ( variance: R2 = 0 . 0135; Fano factor: R2 = 0 . 0119 , Figure 2—figure supplement 1 ) .","This is in contrast with a strong correlation observed between variability in sequence duration and the variability in sequence length—number of presses ( variance: R2 = 0 . 8710; Fano factor: R2 = 0 . 8839 , Figure 2—figure supplement 1 ) .","The decrease in frequency variability cannot be explained by animals reaching a ceiling in pressing frequency , since the average frequency did not increase with training ( it actually decreased slightly ) .","Furthermore , frequency variability started stabilizing after session 4 where the target constrains are still rather loose ( 3 IPIs in less than 4 s ) and this is a frequency that animals can reach in 78 . 91 ± 5 . 09% of the sequences at the end of training . 10 . 7554/eLife . 09423 . 006Figure 2 . Variability of behavioral dimensions evolves independently as animals learn a motor task .","( A , B )","Frequency and duration of lever-press sequences ( C–F ) Variability , measured as the variance and Fano factor , for sequence frequency and sequence duration .","( G–H )","Fano factor of both frequency and duration , normalized to the first session , for the frequency and control tasks .","Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00610 . 7554/eLife . 09423 . 007Figure 2—figure supplement 1 . Significant correlation between variability of number of presses and duration , but not between variability of frequency and duration . Scatter plots of the paired values , variances and Fano factors , for frequency/duration and number of presses/duration .","Each point corresponds to one session of one individual animal , with darker colors depicting later sessions .","Line corresponds to the best linear fit of all the data , with the correspondent R2 value . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 007 In order to test the specificity of these results , a different group of animals ( N = 8 ) was trained on a control task ( Figure 2H ) , where sequences of exactly four consecutive presses were reinforced but where the frequency at which these sequences were performed was not relevant .","In contrast with the results observed for the frequency task , in which the sequence-to-sequence variability in frequency decreased ( F8 , 152 = 5 . 343 , p < 0 . 0001 ) and in duration increased ( F8 , 152 = 16 . 86 , p < 0 . 0001 ) ( Figure 2G ) , in this control task the variability of sequence frequency did not decrease with training ( F8 , 56 = 1 . 049 , p = 0 . 4113 ) , while variability in sequence duration did ( F8 , 56 = 4 . 589 , p = 0 . 0002 ) ( Figure 2H ) .","These data indicate that the decrease in variability in sequence frequency was task-specific .","To further investigate this , we analyzed if the variability of these two behavioral dimensions was different in reinforced vs non-reinforced sequences ( Figure 3 ) .","We verified that sequences leading to reinforcement had indeed significantly lower variability in frequency compared to non-reinforced sequences ( main effect of reinforcement , F1 , 38 = 7 . 608 , p = 0 . 0089 , Figure 3C and F1 , 38 = 28 . 34 , p < 0 . 0001 , Figure 3E ) , but there were no significant differences in the variability of sequence duration between reinforced and non-reinforced sequences ( Figure 3D , F ) .","These results suggest that mice selectively reduced variability in the behavioral domains where variability affected the probability of reinforcement ( sequence frequency ) , but not in domains where variability did not change this probability ( sequence duration ) . 10 . 7554/eLife . 09423 . 008Figure 3 . Behavior variability is differentially modulated during training .","( A , B )","Comparison of frequency and duration between reinforced ( RF ) and non-reinforced ( Non-RF ) sequences .","( C , D )","Variance and ( E , F ) variability , measured as the Fano factor , for reinforced and non-reinforced sequences .","Black lines correspond to mean values for non-reinforced sequences .","Red lines correspond to mean values for reinforced sequences .","Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 008 In order to investigate the dynamics of cortical and striatal circuits during the acquisition and performance of the fast lever pressing task , we continuously recorded extracellular neuronal activity simultaneously in layer 5 of the primary motor cortex ( M1 ) , and in the dorsal striatum ( DS ) of mice during the full duration of training ( 4 days , N = 7 animals , average of 18 M1 units and 10 DS units simultaneously recorded per animal , per session ) .","Non-stop continuous electrophysiological recordings across 4 days encompassing all the sessions of training allowed us to track the activity of a subset of ‘stable’ cells throughout the whole period of training ( 49 M1 units , 21 DS Units ) .","Putative single-units were isolated based on waveform characteristics , inter-spike intervals ( ISI ) and clustering statistics using principal component analysis ( PCA ) .","Units were considered ‘stable’ if the statistics in PCA space and waveform proprieties did not change significantly across sessions ( see ‘Materials and methods’ and Figure 4—figure supplement 1C ) .","We found a high sequence-to-sequence variability in the activity of individual neurons ( measured by the Fano factor of the firing rate ) in the first couple of sessions , that then decreased with training ( DS: F8 , 48 = 2 . 767 , p < 0 . 05; M1: F8 , 48 = 2 . 771 , p < 0 . 05; Figure 4A ) .","These dynamics in neuronal variability were observed during the performance of lever-press sequences , but not during baseline periods ( measured from 5 to 2 s before the initiation of each sequence ) , when the animals were not actively engaged in lever pressing ( DS: F8 , 48 = 1 . 117 , p = 0 . 3324; M1: F8 , 48 = 1 . 459 , p = 0 . 1973; Figure 4B ) , or during periods flanking the sequence ( first press: DS F8 , 48 = 1 . 213 , p = 0 . 3121; M1 F8 , 48 = 0 . 1374 , p = 0 . 9971; last press: DS F8 , 48 = 0 . 5227 , p = 0 . 8335; M1 F8 , 48 = 0 . 8677 , p = 0 . 5499; Figure 4—figure supplement 2 ) .","The decrease in neuronal variability was also observed when using exclusively ‘stable’ cells for this analysis ( DS: F8 , 160 = 5 . 223 , p < 0 . 0001; M1 F8 , 384 = 12 . 72 , p < 0 . 0001; Figure 4C ) , showing that the differences in variability throughout learning could be observed in individual cells , and did not represent a shift in the population of neurons recorded across days .","Importantly , the average firing rate of individual cells did not change significantly , neither across sessions nor across days ( p > 0 . 05 for all conditions , Figure 4E–H ) , suggesting that the reduction in variability was not attributable to overall changes in firing rate , but instead to the selection/refinement of a particular firing patterns related to sequence execution . 10 . 7554/eLife . 09423 . 009Figure 4 . Trial-to-trial variability in corticostriatal circuits decreases throughout training .","( A–D )","Neuronal variability ( measured as the Fano factor of firing rates ) during sequence performance and baseline periods , for all the recorded neuronal units and exclusively for ‘stable units’ , for both M1 ( blue traces ) and dorsal striatum ( DS , red traces ) .","( E–H )","Firing rates during sequence performance and baseline , for all the recorded units and exclusively for stable units , for M1 ( blue traces ) and DS ( red traces ) .","( I , J )","Fano factor ( FF ) and firing rate ( FR ) modulation relative to baseline values , for individual units recorded across the training sessions ( stable units ) within DS ( top colorplots ) and M1 ( bottom colorplots ) .","Right panels depict average modulation .","Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00910 . 7554/eLife . 09423 . 010Figure 4—figure supplement 1 . Histological confirmation of electrode tip position and stable units criteria .","( A ) Depiction of electrode array tip localization for motor cortex ( top ) and DS ( bottom ) for each individual animal .","( B ) Example coronal brain slice magnification using cresyl violet staining for confirmation of electrodes position .","Atlas adapted from Paxinos and Franklin ( 2004 ) .","( C ) Illustration of an example stable cell .","Diagram illustrating the criteria for stability of cells across different recording sessions ( top left , c1: cluster centroid during one session , c2: centroid of the same cluster in the subsequent session , see ‘Materials and methods’ ) .","Average waveform in each session ( bottom left ) .","Cluster projection using principal component analysis ( PCA ) across the training sessions ( right ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01010 . 7554/eLife . 09423 . 011Figure 4—figure supplement 2 . Neuronal variability around the first and last press of a sequence does not change with training . Fano factor calculated for 1 s intervals around the first ( DS F8 , 48 = 1 . 213 , p = 0 . 3121; M1 F8 , 48 = 0 . 1374 , p = 0 . 9971 ) and last presses ( DS F8 , 48 = 0 . 5227 , p = 0 . 8335; M1 F8 , 48 = 0 . 8677 , p = 0 . 5499 ) of a sequence .","Red lines correspond to mean value for DS , blue lines correspond to mean value for M1 .","Shaded areas represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 011 Further analysis of these dynamics for individual stable cells clearly showed higher variability relative to baseline during the initial sessions ( first session DS: W = 134 , p = 0 . 0107; first session M1: W = 1119 , p < 0 . 0001 ) , that decreased throughout training until it reached the same levels of baseline at the end of training ( last session DS: W = 73 , p = 0 . 2157; last session M1: W = 253 , p = 0 . 2121; Figure 4I ) .","Again , average firing rates did not show any significant modulation in relation to baseline throughout the whole period of training ( DS: F8 , 160 = 1 . 031 , p = 0 . 4153; M1: F8 , 384 = 1 . 757 , p = 0 . 084; Figure 4J ) .","This decrease in sequence-to-sequence variability of neural activity did not seem to result from the behavior becoming more stereotyped with training , as variability in behavior decreased for frequency but increased for duration ( Figure 2 ) .","To further control that the decrease in neural variability was due to gross changes in behavior we restricted our analyses to sequences matched for frequency ( t48 = 1 . 800 , p = 0 . 0781 ) and duration ( t48 = 1 . 733 , p = 0 . 0895 ) between early and late sessions ( Figure 5A , B ) .","We observed that neuronal variability was still elevated in early sessions and decreased as training progressed ( DS: F8 , 48 = 2 . 732 , p = 0 . 0144; M1: F8 , 48 = 2 . 491 , p = 0 . 0239; Figure 5C ) .","Again , these dynamics were not observed during baseline periods ( DS: F8 , 48 = 1 . 483 , p = 0 . 1884; M1: F8 , 48 = 1 . 241 , p = 0 . 2965; Figure 5D ) and no changes in firing rates were evident in sequence ( DS: F8 , 48 = 0 . 4684 , p = 0 . 8723; M1: F8 , 48 = 0 . 4040 , p = 0 . 9128; Figure 5E ) or baseline periods ( DS: F8 , 48 = 0 . 2208 , p = 0 . 9855; M1: F8 , 48 = 0 . 3354 , p = 0 . 9479; Figure 5F ) .","Single unit analysis also revealed a significant decrease in Fano factor modulation throughout training ( DS: F8 , 160 = 2 . 688 , p = 0 . 0084; M1:F8 , 384 = 9 . 705 , p < 0 . 0001; Figure 5G ) with no modulation in firing rates ( DS: F8 , 160 = 0 . 3008 , p = 0 . 9648; M1:F8 , 384 = 1 . 406 , p < 0 . 1923; Figure 5H ) . 10 . 7554/eLife . 09423 . 012Figure 5 . Neuronal variability dynamics are still evident when analysis is restricted to sequences with duration and frequency .","( A , B )","Frequency and duration of matched sequences .","( C , D )","Neuronal variability , measured as the Fano factor of the firing rate , for sequences of matched duration and frequency , for both recorded areas , during sequences and baseline .","( E , F )","Firing rates , for sequences of matched duration and frequency , during sequences and baseline .","( G , H )","Fano factor ( FF ) and firing rate ( FR ) modulation relative to baseline values , for individual units recorded across the training sessions ( stable units ) within DS ( top colorplots ) and M1 ( bottom colorplots ) , for sequences of matched duration and frequency .","Right panels depict average modulation .","Error bars correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 012 The results above suggest that the decrease in corticostriatal variability is not related to a general decrease in behavioral variability .","We therefore investigated if the changes in sequence-to-sequence variability in neural activity were related to the changes in sequence-to-sequence variability of specific behavioral dimensions .","We re-calculated the Fano factor of the behavioral features and the neuronal activity using a moving average of a reduced number of trials ( 5 ) to provide a higher within session resolution of the variability dynamics and therefore permit the correlation of behavioral and neuronal dynamics across training for each animal ( Figure 6A , see ‘Materials and methods’ ) .","Analyses of the relationship between the variability of the recorded units and the variability of each independent behavior feature revealed a significant increase in correlation between neuronal and behavior variability , specific for sequence frequency ( Figure 6C ) , but not for duration ( Figure 6D ) .","These results were observed when using only task-relevant or non-task-relevant neurons ( data not shown ) .","They were also observed using different number of trials for calculating the moving average of the Fano factor ( Figure 6—figure supplement 2 ) . 10 . 7554/eLife . 09423 . 013Figure 6 . Correlations between corticostriatal and behavioral variability emerge for specific behavioral features .","( A ) Example traces from a single animal representing variability , calculated as the Fano factor , using a moving window of five consecutive trials shifted by one for sequence frequency ( dark blue trace ) , sequence duration ( green trace ) , M1 units firing rate during sequences ( blue trace ) and baseline ( grey trace ) , and DS units firing rate during sequences ( red trace ) and baseline ( grey trace ) .","Vertical dashed lines represent separation of different training sessions .","Shaded areas correspond to mean ± SEM .","( B ) Correlation between the variability ( FF ) in M1 and DS .","( C , D )","Correlation between variability traces from neuronal firing rates in M1 ( blue bars ) or DS ( red bars ) , and variability of sequence frequency or duration .","Error bars denote correlation coefficient ±standard error of the correlation .","*p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01310 . 7554/eLife . 09423 . 014Figure 6—figure supplement 1 . No significant correlation was found between average firing rate and any of the behavior features . Correlation between the firing rate and ( A ) sequence frequency or ( B ) sequence duration .","Error bars denote correlation coefficient ±standard error of the correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01410 . 7554/eLife . 09423 . 015Figure 6—figure supplement 2 . Changing the number of trials used for Fano factor calculation did not affect the observed corticostriatal and neuronal/behavioral variability correlations .","( A , B )","Correlation between variability traces from neuronal firing rates of cortical and striatal units , and between neuronal variability and variability of sequence frequency and duration using 3 ( A ) or 7 ( B ) consecutive trials .","Error bars denote correlation coefficient ±standard error of the correlation .","*p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 015 These results show that the decrease in variability in M1 and DS is not just a reflection of a more constrained performance of the movement as training progresses; variability of the movement decreased in a specific dimension but it increased in others were no significant correlation with neuronal variability was evident .","Furthermore , no significant correlations were observed between the firing rate of neurons and the variability any of the behavior features ( Figure 6—figure supplement 1 ) , indicating again that the observed relationship between neuronal and behavior dynamics was not the reflex of a general increase in correlation between neuronal activity and behavior .","The data presented above suggested that as training progressed variability in M1 and striatum became more correlated with variability in a specific domain of behavior that changed the probability of reinforcement .","This suggests that neural variability in M1 and striatum could also become more coupled with training .","We verified that at the onset of training the sequence-to-sequence variability of neural activity in DS and M1 in each animal was not correlated .","However , a strong correlation between the variability in DS and M1 rapidly emerged during training ( p < 0 . 05 for all except the first training session , Figure 6B ) , suggesting that as behavioral variability is refined , neural variability in M1 and striatum becomes correlated .","The results presented above show that a coupled reduction in corticostriatal variability accompanies the reduction in variability of sequence frequency , but not of sequence duration , suggesting that corticostriatal plasticity is necessary to select the appropriate motor features and hence reduce variability within specific domains .","We decided to directly test if the observed reduction in sequence frequency variability is dependent on corticostriatal plasticity by using mutant mice with NMDA receptors deleted specifically at glutamatergic synapses of striatal projection neurons ( RGS9-LCre::Grin1tm1Yql; referred to in the figures as striatal projection neuron SPN NR1-KO ) , which have impaired corticostriatal plasticity ( Dang et al . , 2006 ) , and control littermates .","Mutant animals had more difficulty learning the task , so we adapted the training protocol to one session per day for both mutant and littermate controls ( and repeated sessions when needed ) , in order to achieve comparable performance levels ( see ‘Materials and methods’ , Table 1 and Figure 7A ) . 10 . 7554/eLife . 09423 . 016Table 1 . Training protocol and respective number of animals reaching performance criteria for the SPN NR1-KO group and littermate controlsDOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 016Training protocolFree0 . 375 Hz0 . 375/0 . 75 Hz ( 30 reinf ) 0 . 75 Hz1 . 5 Hz1 . 5/3 Hz ( 30 reinf ) 3/6 Hz ( 10 reinf ) 6/7 . 5 Hz ( 10 reinf ) # of subjects reaching criteriaNR1–KO77665411Controls5555555210 . 7554/eLife . 09423 . 017Figure 7 . Corticostriatal plasticity is necessary for the specific refinement of behavioral variability .","( A ) Schematic of the adapted training sessions for mutant animals and littermate controls .","Animals would remain in the same training session until reaching a stable performance .","( B ) Distance of the sum of all three consecutive IPIs from the final covert target ( ∑ ( 3 IPIs ) <660 ms , ∼4 . 5 Hz ) in SPN NR1 mutants and littermate controls ( C ) Spread of the distance between three consecutive IPIs around the final covert target .","( D–G )","Behavior parameters and variability , measured as the Fano factor , during early and late training sessions in SPN NR1 mutants and littermate controls groups .","Bars correspond to mean , with data from individual animals plotted on the background ( red: SPN NR1-KO; black: littermate controls ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01710 . 7554/eLife . 09423 . 018Figure 7—figure supplement 1 . Bootstrapping statistics in the SPN NR1-KO data support the observations from the post hoc planned comparisons . Histograms depicting the sampled statistic ( difference between the means of two groups ) , after sampling with replacement the original data 100 . 000 times .","Red vertical lines correspond to the 5% confidence intervals .","Green vertical line corresponds to the mean of the sampled data .","Blue vertical line corresponds to the difference between the original data groups ( H0 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 018 As expected , the distance to target ( Controls: p = 0 . 0450 , t5 = 2 . 657 , Figure 7B ) and spread around the target ( Controls: p = 0 . 0179 , t5 = 3 . 466 , Figure 7C ) decreased in littermate controls .","However , neither of these measures changed with training in mutants ( Mutants: p = 0 . 3535 , t6 = 1 . 005; and p = 0 . 2817 , t6 = 1 . 183 , respectively; Figure 7B , C ) .","In general , no significant difference was observed for any of the behavior features between the two groups of animals .","However , planned comparisons did show that RGS9-LCre::Grin1tm1Yql mutants did not decrease sequence frequency variability during training , in contrast to littermate controls which did ( significant main effect of training time: F1 , 10 = 10 . 13 , p = 0 . 009; Posthocs: Mutant group: t10 = 1 . 38 , p = 0 . 1964; Control group: t10 = 3 . 00 , p = 0 . 0134 ) .","Importantly , no differences in the modulation of sequence duration variability were observed between the two groups ( no significant main effect for genotype: Duration FF: F1 , 10 = 0 . 02 , p = 0 . 887 ) ( Figure 7D–G ) .","These statistical results were robust as they were confirmed using bootstrapping statistics ( using 100 . 000 random samples of the data , with replacement ) ( Figure 7—figure supplement 1 ) .","These data suggest that corticostriatal plasticity is required for the reduction in variability of specific behavioral features that change the probability of reinforcement ."],["In this study we show that when mice are trained on a difficult operant paradigm they differentially refine specific behavioral features .","When mice were asked to perform progressively faster covert patterns of lever presses to obtain a reinforcer , they reduced variability in sequence frequency , but increased variability in an orthogonal uncorrelated feature ( sequence duration ) .","These results are interesting because both features would be classically considered task-relevant—a covert sequence of four presses , which is the minimum to produce a reinforcer in this task , has to have a minimal duration .","However , although both features could be considered relevant for the task , only changes in frequency variability were differentially reinforced .","Reinforced sequences had lower variability in frequency than non-reinforced sequences , but had equal variability in duration as non-reinforced sequences .","Thus , our results indicate that animals reduced frequency variability because that was what was reinforced throughout training .","Consistent with this interpretation , in a task where the exact number of presses ( correlated with duration ) was reinforced but the frequency at which the sequence was performed was not , variability in duration decreased and in frequency increased .","This in line with data demonstrating differential modulation of the different components of task space during learning ( Todorov and Jordan , 2002; Müller and Sternad , 2004; Cohen and Sternad , 2009 ) .","In previous studies from our group where animals performed operant tasks where the constrains were more relaxed ( Jin and Costa , 2010 ) , animals decreased variability in all behavioral domains ( i . e . , they became more stereotypical overall ) .","However , when faced with a more challenging task as in the present study , they decreased variability in the domain that was critical for getting a reinforcer , but increased variability in orthogonal domains ( i . e . , they were more stereotypical in just a particular domain ) .","It could be that the increase in variability in the orthogonal behavioral domains happens because in difficult tasks animals try to minimize the effort to obtain reinforcers , and hence do not attempt to reduce variability in more than one independent domain .","Alternatively , it could also be that mice increased the duration of the sequence ( and the correlated number of presses ) as a strategy to try to increase the probability of getting a successful covert pattern in that sequence .","However , this second possibility is less likely , given that the two behavioral features were not correlated , and that sequences of different durations were equally likely to get reinforced .","These data suggest that in more challenging motor tasks it is difficult to reduce variability in all domains , and animals seem to differentially refine the motor patterns that led to reinforcement .","Consistently , the number of sequences that comply with the minimum frequency required for the last session ( end-target ) increased with training and the distance to the end-target decreased with training , indicating that mice implicitly learned to shape their behavior to match the task requirements .","At the neural level , we observed initial high sequence-to-sequence variability of neuronal activity in corticostriatal circuits that decreased with training .","Variability in the spike patterns of individual neurons and populations of neurons may be the bases for a process of behavioral exploration ( or trial ) ( Olveczky et al . , 2005; Kao et al . , 2005; Mandelblat-Cerf et al . , 2009 ) , while a decrease in neural variability may reflect a process of selection of specific patterns of neural activity that lead to specific behavioral outputs ( Costa et al . , 2004; Kao et al . , 2005; Fee and Goldberg , 2011 ) .","It has been suggested that a decrease in corticostriatal variability as a motor task is learned ( Costa et al . , 2004; Barnes et al . , 2005 ) could correspond to the process of selection and consolidation of specific motor patterns ( Costa , 2011 ) .","Here , we show that this decrease in neural variability in corticostriatal circuits correlates specifically with the decrease in variability of a particular behavior domain .","These data suggest that the neural patterns in motor cortex and sensorimotor striatum that give rise to the behavioral patterns that are reinforced are progressively selected .","Provocatively , it also suggests that changes in motor variability that are not specifically reinforced but are part of a strategy or driven by effort reduction may be encoded somewhere else .","Finally , we also show that corticostriatal plasticity is important for the refinement of specific behavior features .","Our data therefore suggests an important role for corticostriatal plasticity in selecting the appropriate implicit neural and behavioral patterns that are reinforced ( Costa , 2011 ) .","However corticostriatal plasticity did not seem to be necessary for the increase in behavioral variability in other domains ( Goldberg and Fee , 2011 ) .","Although in this study we don't investigate the mechanisms underlying the generation of variability , several studies have suggested that the basal ganglia , dopaminergic system , specific cortical circuits , or cerebellar circuits could subserve this function ( Olveczky et al . , 2005; Costa et al . , 2006; Leblois et al . , 2010; Costa , 2011; Fee and Goldberg , 2011; Shmuelof and Krakauer , 2011; Woolley et al . , 2014 ) .","Taken together , our findings suggest that corticostriatal plasticity is important to select the neural patterns that lead to the movement patterns that are reinforced .","They highlight that corticostriatal plasticity is not only important for choosing which action to do , but also to shape how to do it to obtain a desired outcome ."],["All experiments were carried in accordance to the ethics committee guidelines of the Champalimaud Foundation and Instituto Gulbenkian de Ciência , and with approval of the Portuguese DGAV ( Ref . 0421 ) .","Experiments were carried out using 20 male , 3 to 5 month old C57BL6/J mice .","From these , 13 animals were used exclusively for behavioral training while the remaining seven underwent microelectrode array implantation for neuronal data recordings .","Animals were maintained on a light–dark cycle of 12 hr:12 hr starting at 7 AM .","All experiments were done during the light cycle .","Mice were housed in groups of four animals prior to surgery and individually after the electrodes were implanted .","3 to 6 months old RGS9-LCre::Grin1tm1Yql homozygous mice ( N = 7 ) and Cre negative littermate controls ( N = 5 ) were used for the mutant mouse behavioral experiments .","Seven C57Bl6/J mice were implanted bilaterally with two micro-electrode arrays ( 2 × 8 ) , 35–50 µm tungsten electrodes with micro-polished tips .","One array targeted the primary motor cortex ( M1 , layer 5 ) while the second was targeting the ( DS , sensorimotor area that receives projections from the same area in M1 ) .","Craniotomies and electrode array positioning were done according to coordinates from the Mouse Brain Atlas ( Paxinos and Franklin , 2008 ) .","M1 array was placed 1 mm rostral and 1 . 6 mm lateral from bregma , and lowered ∼1 mm from the surface of the brain .","DS array was placed 0 . 5 mm rostral and 2 . 1 mm lateral from bregma , and lowered ∼2 . 3 mm from the surface of the brain .","Electrodes were manually lowered at slow rates while constantly monitoring neural activity in all the channels in order to control for proper electrode function and correct positioning .","Final verification of electrode position was done after all the experiments were finished , by perfusing animals with PFA and histological confirmation of Nissl stained 70 µm brain slices ( Figure 4—figure supplement 1A , B ) .","After surgery animals were allowed to recover for at least 2 weeks before starting any other experimental procedure .","Single and multi unit activity was recorded using Blackrock Microsystems Neural Signal Processor , allowing for online sorting of identified units .","Further offline sorting of selected units was done using Plexon Offline Sorter v3 ( Plexon Inc , Dallas , TX , United States ) , based on waveform characteristics , ISI and PCA clustering .","Units stability was assessed from waveforms and PCA cluster proprieties .","For PCA cluster comparison data from all the training sessions was pooled together to calculate common eigen vectors .","Data from individual sessions was then projected into this common PC space , allowing us to determine cluster centroids and dispersion for each session .","Clusters were considered stable whenever the centroid in a given session was comprised within the interval of the centroid of the previous session ±1 . 96 * standard deviation of the cluster , in the first two principal components ( Figure 4—figure supplement 1C for a graphical representation of this criteria ) .","Animals were trained using operant chambers ( MedAssociates Inc , St . Albans , VT , United States ) placed inside sound attenuating boxes .","A retractable lever was extruded in the beginning of each session , simultaneous to the onset of a light .","Animals were required to perform a sequence of presses at a minimum frequency in order to obtain a 20 mg food pellet ( Bio-Serv , Flemington , NJ , United States ) .","24 hr before the first training session animals were placed under a food restriction schedule .","Body weight was constantly monitored in order to be kept above 85% of the initial weight .","In order to facilitate learning , animals were initially exposed to one session of magazine training were food pellets would be available on a random time schedule , and to three sessions of continuous reinforcement schedule ( CRF ) 1 day before training , where single lever presses would be reinforced .","On the following training sessions animals were reinforced if they performed a sequence of consecutive presses at a minimum frequency ( covert target ) , defined by the inverse of three consecutive inter-press intervals ( IPIs ) , which increased with training .","On the first session there was no minimum frequency target , meaning that any consecutive 3 IPIs would lead to reinforcement .","In consecutive sessions the minimum frequency that would lead to reinforcement was increased or maintained in the following order: 0 . 375 Hz , 0 . 75 Hz , 0 . 75 Hz , 1 . 5 Hz , 3 Hz , 3 Hz , 4 . 5 Hz and 4 . 5 Hz .","This constant increase in the minimum frequency of the covert target forced the animals to systematically adapt to the task requirements and perform faster sequences of presses from session to session .","The training protocol for mutant animals and littermate controls was adapted due to difficulties learning the task , to one daily session and using automatic progressive schedules once a minimum number of reinforcements ( 30 or 10 ) was achieved .","( Table 1 for performance summary . )","Sequences of presses were differentiated based on IPI and occurrence of a magazine head entry .","An IPI >2 s ( determined based on the distribution of IPIs ) or a head-entry were used to define the bouts or sequences of presses .","The 2 s cutoff was determined from the joint distribution of the instantaneous IPIs ( and the corresponding log distribution ) from all the animals , by determining the valley between the two main peaks of IPIs ( Figure 1—figure supplement 1C , D ) .","Frequency of each sequence was defined as the inverse of the average IPI of each sequence .","Duration of each sequence was defined as the time between the first and the last press event .","Length of each sequence was defined as the number of press events in each sequence .","For the matched sequences analysis , sequences with a duration of 0 . 2–2 s and a frequency higher than 2 Hz were selected .","Neural activity was averaged in 20-ms bins , shifted by 1 ms , and averaged across trials to construct the peri-event histogram ( PETH ) .","Data from the PETH from 5000 to 2000 ms before lever press were considered as baseline activity .","A positive modulation in firing rate was defined if at least 20 consecutive bins had firing rate larger than a threshold of 99% above baseline activity , and a negative modulation of firing rate was defined if at least 20 consecutive bins had a firing rate smaller than a threshold of 95% below baseline activity ( Belova et al . , 2007 ) .","Paired t-tests between baseline firing rate and sequence firing rate were used to classify individual neurons as sequence-related .","The programs to run the tasks presented in this study can be found at http://tinyurl . com/or7ug72 .","Analyses were done in Matlab ( MathWorks , Natick , MA , United States ) or GraphPad Prism ( GraphPad Software Inc , La Jolla , CA , United States ) .","Normality was verified for all tests using the D'Agostino-Pearson omnibus normality test , or the Kolmogorov–Smirnov test when sample size was too small .","Repeated measures ANOVA were used to evaluate changes in behavior and neuronal features .","Probability of a magazine check after lever-press was evaluated using one-way ANOVA and post hoc comparisons using Fisher's LSD test , but one subject was excluded from these analysis due to a lack of recorded timestamps for magazine head-entries .","Paired t-tests were used to evaluate differences in percentage of lever-presses .","Increases in FF modulation were assessed by the Wilcoxon Rank Signed test .","Repeated measures two-way ANOVA was used to verify the general effect of the RGS9-NR1 mutants experiment .","Bootstrapping statistics were used on the data from the RGS9-NR1 mutants and littermate controls to validate the results from the post hoc tests .","Histograms were built from 100000 randomized samples with replacement .","Sample sizes were calculated based on α = 0 . 05 and power of 0 . 7 .","Trial to trial variability of neuronal and behavior data was assessed using Fano factor .","We calculate the Fano factor of individual units by dividing the variance of firing rates across all the trials of a session by the mean over those trials .","Fano factor and firing rate modulations for individual stable cells were calculated as the ratio between the difference of values for sequence and baseline and the values during baseline ( Fano factor: [FFsequence − FFbaseline]/FFbaseline; firing rate: [FRsequence − FRbaseline]/FRbaseline ) .","Fano factor of the behavioral features was calculated by dividing the variance in the individual features by the mean of the feature for all the trials .","To establish correlations between the variability of the neuronal data and the variability of the behavior , Fano factors were calculated using three , five or seven consecutive trials , allowing us to increase the resolution of the variability measures .","Correlations between neuronal and behavior data were evaluated using Pearson's linear correlations .","To avoid correlations bias due to sample size , statistical significance of all the correlations was assessed using the significance criteria for the session with smaller size .","Within animal correlations averaged using Fisher's z transformation ( Silver and Dunlap , 1987 ) returned similar results to grouped correlations for all the tested conditions ( data not shown ) ."]],"string":"[\n [\n \"Animals have the ability to learn novel motor skills , allowing them to perform complex patterns of movement to improve the outcomes of their actions .\",\n \"Acquiring novel skills usually requires exploration of the behavioral space , which is critical for learning ( Skinner , 1981; Sutton and Barto , 1998; Grunow and Neuringer , 2002; Kao et al . , 2005; Olveczky et al . , 2005; Tumer and Brainard , 2007; Miller et al . , 2010; Wu et al . , 2014 ) .\",\n \"It also requires the selection of the appropriate behavioral features that lead to the desired outcomes ( Skinner , 1981 ) .\",\n \"It has been postulated that the motor system can learn complex movements by optimizing motor variability in task-relevant dimensions , correcting only deviations that interfere with the final output of the action ( Todorov and Jordan , 2002; Scott , 2004; Valero-Cuevas et al . , 2009; Diedrichsen et al . , 2010 ) .\",\n \"By optimizing the precision of an action endpoint , for example , humans can perform smooth movements even in the presence of noise ( Harris and Wolpert , 1998 ) .\",\n \"Selecting task-relevant features and decreasing task-relevant variability might therefore be a critical component of motor learning ( Franklin and Wolpert , 2008; Cohen and Sternad , 2009; Valero-Cuevas et al . , 2009; Costa , 2011; Shmuelof et al . , 2012 ) .\",\n \"The reduction of motor variability specifically in relevant domains suggests that the neural activity giving rise to the task-relevant output is selected during learning .\",\n \"However , it is still unclear how the differential refinement of behavioral variability is encoded at the neural level .\",\n \"It has been suggested that cortical and basal ganglia circuits are important for the selection of task-relevant features ( Costa et al . , 2004; Barnes et al . , 2005; Kao et al . , 2005; Olveczky et al . , 2005; Jin and Costa , 2010; Woolley et al . , 2014 ) .\",\n \"Consistently , it has been previously shown that the initial stages of learning have increased behavioral ( Tumer and Brainard , 2007; Jin and Costa , 2010; Miller et al . , 2010 ) and neuronal ( Costa et al . , 2004; Barnes et al . , 2005 ) variability , but as specific movements are consolidated , neural variability is reduced in these circuits ( Costa et al . , 2004; Kao et al . , 2005 ) .\",\n \"This suggests that after initial motor and neural exploration , specific patterns are selected and consolidated ( Costa , 2011 ) .\",\n \"In this study , we investigated if the dynamics of neural activity in cortical and striatal circuits reflect the changes of variability in specific behavioral domains , and if corticostriatal plasticity is critical for the refinement of particular behavior features .\"\n ],\n [\n \"We trained mice to perform a fast lever-pressing task where they were required to press a lever at increasingly higher frequencies , in order to obtain a 20 mg food pellet .\",\n \"After introducing the animals ( N = 20 ) to the behavioral apparatus and 1 day of continuous reinforcement , where each lever-press was reinforced , animals were trained intensively with three daily sessions for 3 days to perform fast lever presses .\",\n \"In the fast press schedules we introduced a covert minimum frequency target , defined by the inverse of three consecutive inter-press intervals ( 3 IPIs , 4 presses ) , which increased across sessions from 0 Hz to a maximum of 4 . 5 Hz ( Figure 1A; see ‘Materials and methods’ ) .\",\n \"The total number of lever presses per minute increased throughout training ( F8 , 152 = 41 . 34 , p < 0 . 0001; Figure 1—figure supplement 1A ) and animals rapidly started to organize their behavior in self-paced bouts or sequences of lever presses , until there were almost no single presses ( Figure 1C , E and Video 1 ) . 10 . 7554/eLife . 09423 . 003Figure 1 . Mice learn a fast lever-pressing task , shaping their behavior to gradually approach the minimum frequency target .\",\n \"( A ) Schematic of the training protocol , starting with magazine habituation and CRF training in the first 2 days , followed by 3 days of the fast press schedules ( S1–S9 ) where we introduce an increasingly higher covert target , defined as the inverse of the sum of three consecutive inter-press intervals ( IPIs ) .\",\n \"( B ) Joint distribution of the frequency ( log scale ) for all individual IPIs , in the first , middle and last session of the fast press schedules , for all the 20 animals .\",\n \"Vertical dashed lines correspond to the IPI threshold used for sequence definition ( IPI = 2 s , 0 . 5 Hz ) and the final covert target ( IPI = 3/660 ms , 4 . 5 Hz ) .\",\n \"( C ) Percentage of lever presses comprised within sequences .\",\n \"( D ) Number of sequences performed per minute .\",\n \"( E ) Left: Example of sequences performed by a representative animal , aligned at the time of sequence initiation .\",\n \"Individual lever presses are marked as black ticks , the full sequence duration is shaded in grey and the IPIs that meet the session minimum target are shaded in orange; Top right: Probability of a magazine check immediately after a successful covert target; Bottom right: Probability of a magazine check having occurred after a reinforced lever-press vs a non-reinforced lever-press .\",\n \"( F ) Distance of all three consecutive IPIs ( summed ) from the final covert target ( ∑ ( 3 IPIs ) <660 ms , ∼4 . 5 Hz ) .\",\n \"( G ) Spread of the distance between all three consecutive IPIs ( summed ) around the final minimum frequency target .\",\n \"( H ) Percentage of sequences containing the minimum frequency target of the last session ( end-target: 3 IPIs <660 ms , ∼4 . 5 Hz ) .\",\n \"Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00310 . 7554/eLife . 09423 . 004Figure 1—figure supplement 1 . Lever-pressing rate increased and shifted towards higher speeds with training , and performance increased or plateaued when task difficulty did not change in consecutive sessions .\",\n \"( A ) Lever presses per minute ( F8 , 152 = 41 . 34 , p < 0 . 0001 ) .\",\n \"( B ) Percentage of reinforced sequences ( F8 , 152 = 57 . 31 , p < 0 . 0001 , Post hoc comparisons: Fisher's LSD test , ( 0 . 75 Hz ) Session 3 vs Session 4 t152 = 3 . 847 , p = 0 . 0002; ( 3 Hz ) Session 6 vs Session 7 t152 = 0 . 7681 , p = 0 . 4436; ( 4 . 5 Hz ) Session 8 vs Session 9 t152 = 2 . 639 , p = 0 . 0092 ) .\",\n \"Shaded areas correspond to mean ± SEM .\",\n \"( C , D )\",\n \"Histograms with distribution of instantaneous lever-press frequencies , using linear ( C ) and log scales ( D ) , defined as the inverse of all the individual IPIs from the 20 animals .\",\n \"Vertical dashed lines correspond to the IPI threshold used for sequence definition ( IPI = 2 s , 0 . 5 Hz ) and the final minimum frequency target ( IPI = 220 ms , 4 . 5 Hz ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00410 . 7554/eLife . 09423 . 005Video 1 . Animal performing sequences of lever-presses , doing magazine checks and obtaining reinforcement during the last training session . A 20 mg food pellet was delivered in the magazine when the animal performed three consecutive presses within 660 ms ( covert target = 4 . 5 Hz ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 005 The distribution of the instantaneous lever press frequencies ( calculated as the inverse of the each IPI ) shows a clear shift from initial sessions , where animals did mostly slow frequency presses ( 0–0 . 5 Hz; but already some higher frequency presses of 0 . 5–4 . 5 Hz and >4 . 5 Hz ) , to latter sessions where the distribution was shifted towards faster pressing speeds ( Figure 1—figure supplement 1C ) .\",\n \"A clear multimodal distribution became evident in log scale , with long IPIs ( frequencies <0 . 5 Hz , Figure 1B and Figure 1—figure supplement 1D ) representing pauses in pressing or magazine checks .\",\n \"This allowed us to identify the sequences or bouts of pressing a posteriori , based on behavioral performance ( either by a pause in pressing higher than 2 s or by the occurrence of checking behavior , i . e . , magazine checks between presses; see ‘Materials and methods’ ) , independently of the requirements for a specific training session .\",\n \"Importantly , reinforcement delivery did not provide an external cue that could be used by the animals to anticipate a reward , as the probability of performing a magazine check immediately after a successful covert target ( instead of performing another press ) was not significantly different from 0 . 5 both on early ( t19 = 0 . 9232 , p = 0 . 3675 ) and late sessions ( t19 = 1 . 763 , p = 0 . 0940 ) , and did not change throughout learning ( F8 , 152 = 1 . 753 , p = 0 . 0907 , Figure 1E , top right ) .\",\n \"Because a large number of sequences did not contain covert patterns ( were not reinforced ) we have also calculated the probability of a magazine check having occurred after a reinforced lever-press vs a non-reinforced lever-press , and observed that this was rather low ( ∼0 . 25 ) and did not change from early to late sessions ( Post hoc comparison: t144 = 1 . 184 , p = 0 . 283 , Figure 1E , bottom right ) .\",\n \"The percentage of lever presses performed within a sequence increased significantly from 56 . 98 ± 3 . 98 in the first session of covert target introduction , to 98 . 26 ± 0 . 53 in the last training session ( F8 , 152 = 60 . 22 , p < 0 . 0001; Figure 1C ) , and the number of sequences performed per minute increased with training ( F8 , 152 = 32 . 23 , p < 0 . 0001; Figure 1D ) .\",\n \"The percentage of reinforced sequences tended to decrease , since the difficulty of the task increased across sessions , but tended to stabilize or increase when the same target difficulty was repeated in two consecutive sessions ( F8 , 152 = 57 . 31 , p < 0 . 0001; Figure 1—figure supplement 1B ) .\",\n \"Importantly , with training , the distance of consecutive IPIs ( summed in bins of 3 IPIs to mimic the online criteria ) to the final target frequency ( 3 IPIs <660 ms , ∼4 . 5 Hz ) decreased consistently ( F8 , 152 = 25 . 76 , p < 0 . 0001; Figure 1F ) , indicating that animals shaped their behavior gradually to approach the end target .\",\n \"Not only did the distance to the end target decrease , but the spread around the target also decreased ( F8 , 152 = 9 . 616 , p < 0 . 001; calculated as the standard deviation around the target frequency , Figure 1G ) .\",\n \"Consistently , animals gradually increased the percentage of press bouts that would achieve the minimum target frequency of the last session ( end-target: 3 IPIs <660 ms , ∼4 . 5 Hz; F8 , 152 = 14 . 15 , p < 0 . 0001; Figure 1H ) .\",\n \"These data indicate that animals learned to shape their behavior to get closer to the covert target .\",\n \"The mean frequency of each pressing bout ( sequence frequency ) decreased slightly ( F8 , 152 = 2 . 372 , p = 0 . 0195 , Figure 2A ) , while the duration of each pressing bout ( sequence duration ) increased with training ( F8 , 152 = 22 . 69 , p < 0 . 0001 , Figure 2B ) .\",\n \"Importantly , the sequence-to-sequence variability of the behavioral parameters ( measured both by the variance and by the Fano factor , Figure 2C–F ) was differentially modulated during training .\",\n \"While the variability of sequence frequency decreased significantly throughout training ( variance: F8 , 152 = 4 . 450 , p < 0 . 0001 , Figure 2C; Fano factor: F8 , 152 = 5 . 343 , p < 0 . 0001 , Figure 2E ) , the variability of sequence duration significantly increased ( variance: F8 , 152 = 11 . 15 , p < 0 . 0001 , Figure 2D; Fano factor: F8 , 152 = 16 . 86 , p < 0 . 0001 , Figure 2F ) .\",\n \"The sequence-to-sequence variability of these two behavioral features was independent as there was no correlation between the variability in sequence frequency and the variability in sequence duration ( variance: R2 = 0 . 0135; Fano factor: R2 = 0 . 0119 , Figure 2—figure supplement 1 ) .\",\n \"This is in contrast with a strong correlation observed between variability in sequence duration and the variability in sequence length—number of presses ( variance: R2 = 0 . 8710; Fano factor: R2 = 0 . 8839 , Figure 2—figure supplement 1 ) .\",\n \"The decrease in frequency variability cannot be explained by animals reaching a ceiling in pressing frequency , since the average frequency did not increase with training ( it actually decreased slightly ) .\",\n \"Furthermore , frequency variability started stabilizing after session 4 where the target constrains are still rather loose ( 3 IPIs in less than 4 s ) and this is a frequency that animals can reach in 78 . 91 ± 5 . 09% of the sequences at the end of training . 10 . 7554/eLife . 09423 . 006Figure 2 . Variability of behavioral dimensions evolves independently as animals learn a motor task .\",\n \"( A , B )\",\n \"Frequency and duration of lever-press sequences ( C–F ) Variability , measured as the variance and Fano factor , for sequence frequency and sequence duration .\",\n \"( G–H )\",\n \"Fano factor of both frequency and duration , normalized to the first session , for the frequency and control tasks .\",\n \"Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00610 . 7554/eLife . 09423 . 007Figure 2—figure supplement 1 . Significant correlation between variability of number of presses and duration , but not between variability of frequency and duration . Scatter plots of the paired values , variances and Fano factors , for frequency/duration and number of presses/duration .\",\n \"Each point corresponds to one session of one individual animal , with darker colors depicting later sessions .\",\n \"Line corresponds to the best linear fit of all the data , with the correspondent R2 value . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 007 In order to test the specificity of these results , a different group of animals ( N = 8 ) was trained on a control task ( Figure 2H ) , where sequences of exactly four consecutive presses were reinforced but where the frequency at which these sequences were performed was not relevant .\",\n \"In contrast with the results observed for the frequency task , in which the sequence-to-sequence variability in frequency decreased ( F8 , 152 = 5 . 343 , p < 0 . 0001 ) and in duration increased ( F8 , 152 = 16 . 86 , p < 0 . 0001 ) ( Figure 2G ) , in this control task the variability of sequence frequency did not decrease with training ( F8 , 56 = 1 . 049 , p = 0 . 4113 ) , while variability in sequence duration did ( F8 , 56 = 4 . 589 , p = 0 . 0002 ) ( Figure 2H ) .\",\n \"These data indicate that the decrease in variability in sequence frequency was task-specific .\",\n \"To further investigate this , we analyzed if the variability of these two behavioral dimensions was different in reinforced vs non-reinforced sequences ( Figure 3 ) .\",\n \"We verified that sequences leading to reinforcement had indeed significantly lower variability in frequency compared to non-reinforced sequences ( main effect of reinforcement , F1 , 38 = 7 . 608 , p = 0 . 0089 , Figure 3C and F1 , 38 = 28 . 34 , p < 0 . 0001 , Figure 3E ) , but there were no significant differences in the variability of sequence duration between reinforced and non-reinforced sequences ( Figure 3D , F ) .\",\n \"These results suggest that mice selectively reduced variability in the behavioral domains where variability affected the probability of reinforcement ( sequence frequency ) , but not in domains where variability did not change this probability ( sequence duration ) . 10 . 7554/eLife . 09423 . 008Figure 3 . Behavior variability is differentially modulated during training .\",\n \"( A , B )\",\n \"Comparison of frequency and duration between reinforced ( RF ) and non-reinforced ( Non-RF ) sequences .\",\n \"( C , D )\",\n \"Variance and ( E , F ) variability , measured as the Fano factor , for reinforced and non-reinforced sequences .\",\n \"Black lines correspond to mean values for non-reinforced sequences .\",\n \"Red lines correspond to mean values for reinforced sequences .\",\n \"Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 008 In order to investigate the dynamics of cortical and striatal circuits during the acquisition and performance of the fast lever pressing task , we continuously recorded extracellular neuronal activity simultaneously in layer 5 of the primary motor cortex ( M1 ) , and in the dorsal striatum ( DS ) of mice during the full duration of training ( 4 days , N = 7 animals , average of 18 M1 units and 10 DS units simultaneously recorded per animal , per session ) .\",\n \"Non-stop continuous electrophysiological recordings across 4 days encompassing all the sessions of training allowed us to track the activity of a subset of ‘stable’ cells throughout the whole period of training ( 49 M1 units , 21 DS Units ) .\",\n \"Putative single-units were isolated based on waveform characteristics , inter-spike intervals ( ISI ) and clustering statistics using principal component analysis ( PCA ) .\",\n \"Units were considered ‘stable’ if the statistics in PCA space and waveform proprieties did not change significantly across sessions ( see ‘Materials and methods’ and Figure 4—figure supplement 1C ) .\",\n \"We found a high sequence-to-sequence variability in the activity of individual neurons ( measured by the Fano factor of the firing rate ) in the first couple of sessions , that then decreased with training ( DS: F8 , 48 = 2 . 767 , p < 0 . 05; M1: F8 , 48 = 2 . 771 , p < 0 . 05; Figure 4A ) .\",\n \"These dynamics in neuronal variability were observed during the performance of lever-press sequences , but not during baseline periods ( measured from 5 to 2 s before the initiation of each sequence ) , when the animals were not actively engaged in lever pressing ( DS: F8 , 48 = 1 . 117 , p = 0 . 3324; M1: F8 , 48 = 1 . 459 , p = 0 . 1973; Figure 4B ) , or during periods flanking the sequence ( first press: DS F8 , 48 = 1 . 213 , p = 0 . 3121; M1 F8 , 48 = 0 . 1374 , p = 0 . 9971; last press: DS F8 , 48 = 0 . 5227 , p = 0 . 8335; M1 F8 , 48 = 0 . 8677 , p = 0 . 5499; Figure 4—figure supplement 2 ) .\",\n \"The decrease in neuronal variability was also observed when using exclusively ‘stable’ cells for this analysis ( DS: F8 , 160 = 5 . 223 , p < 0 . 0001; M1 F8 , 384 = 12 . 72 , p < 0 . 0001; Figure 4C ) , showing that the differences in variability throughout learning could be observed in individual cells , and did not represent a shift in the population of neurons recorded across days .\",\n \"Importantly , the average firing rate of individual cells did not change significantly , neither across sessions nor across days ( p > 0 . 05 for all conditions , Figure 4E–H ) , suggesting that the reduction in variability was not attributable to overall changes in firing rate , but instead to the selection/refinement of a particular firing patterns related to sequence execution . 10 . 7554/eLife . 09423 . 009Figure 4 . Trial-to-trial variability in corticostriatal circuits decreases throughout training .\",\n \"( A–D )\",\n \"Neuronal variability ( measured as the Fano factor of firing rates ) during sequence performance and baseline periods , for all the recorded neuronal units and exclusively for ‘stable units’ , for both M1 ( blue traces ) and dorsal striatum ( DS , red traces ) .\",\n \"( E–H )\",\n \"Firing rates during sequence performance and baseline , for all the recorded units and exclusively for stable units , for M1 ( blue traces ) and DS ( red traces ) .\",\n \"( I , J )\",\n \"Fano factor ( FF ) and firing rate ( FR ) modulation relative to baseline values , for individual units recorded across the training sessions ( stable units ) within DS ( top colorplots ) and M1 ( bottom colorplots ) .\",\n \"Right panels depict average modulation .\",\n \"Shaded areas correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 00910 . 7554/eLife . 09423 . 010Figure 4—figure supplement 1 . Histological confirmation of electrode tip position and stable units criteria .\",\n \"( A ) Depiction of electrode array tip localization for motor cortex ( top ) and DS ( bottom ) for each individual animal .\",\n \"( B ) Example coronal brain slice magnification using cresyl violet staining for confirmation of electrodes position .\",\n \"Atlas adapted from Paxinos and Franklin ( 2004 ) .\",\n \"( C ) Illustration of an example stable cell .\",\n \"Diagram illustrating the criteria for stability of cells across different recording sessions ( top left , c1: cluster centroid during one session , c2: centroid of the same cluster in the subsequent session , see ‘Materials and methods’ ) .\",\n \"Average waveform in each session ( bottom left ) .\",\n \"Cluster projection using principal component analysis ( PCA ) across the training sessions ( right ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01010 . 7554/eLife . 09423 . 011Figure 4—figure supplement 2 . Neuronal variability around the first and last press of a sequence does not change with training . Fano factor calculated for 1 s intervals around the first ( DS F8 , 48 = 1 . 213 , p = 0 . 3121; M1 F8 , 48 = 0 . 1374 , p = 0 . 9971 ) and last presses ( DS F8 , 48 = 0 . 5227 , p = 0 . 8335; M1 F8 , 48 = 0 . 8677 , p = 0 . 5499 ) of a sequence .\",\n \"Red lines correspond to mean value for DS , blue lines correspond to mean value for M1 .\",\n \"Shaded areas represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 011 Further analysis of these dynamics for individual stable cells clearly showed higher variability relative to baseline during the initial sessions ( first session DS: W = 134 , p = 0 . 0107; first session M1: W = 1119 , p < 0 . 0001 ) , that decreased throughout training until it reached the same levels of baseline at the end of training ( last session DS: W = 73 , p = 0 . 2157; last session M1: W = 253 , p = 0 . 2121; Figure 4I ) .\",\n \"Again , average firing rates did not show any significant modulation in relation to baseline throughout the whole period of training ( DS: F8 , 160 = 1 . 031 , p = 0 . 4153; M1: F8 , 384 = 1 . 757 , p = 0 . 084; Figure 4J ) .\",\n \"This decrease in sequence-to-sequence variability of neural activity did not seem to result from the behavior becoming more stereotyped with training , as variability in behavior decreased for frequency but increased for duration ( Figure 2 ) .\",\n \"To further control that the decrease in neural variability was due to gross changes in behavior we restricted our analyses to sequences matched for frequency ( t48 = 1 . 800 , p = 0 . 0781 ) and duration ( t48 = 1 . 733 , p = 0 . 0895 ) between early and late sessions ( Figure 5A , B ) .\",\n \"We observed that neuronal variability was still elevated in early sessions and decreased as training progressed ( DS: F8 , 48 = 2 . 732 , p = 0 . 0144; M1: F8 , 48 = 2 . 491 , p = 0 . 0239; Figure 5C ) .\",\n \"Again , these dynamics were not observed during baseline periods ( DS: F8 , 48 = 1 . 483 , p = 0 . 1884; M1: F8 , 48 = 1 . 241 , p = 0 . 2965; Figure 5D ) and no changes in firing rates were evident in sequence ( DS: F8 , 48 = 0 . 4684 , p = 0 . 8723; M1: F8 , 48 = 0 . 4040 , p = 0 . 9128; Figure 5E ) or baseline periods ( DS: F8 , 48 = 0 . 2208 , p = 0 . 9855; M1: F8 , 48 = 0 . 3354 , p = 0 . 9479; Figure 5F ) .\",\n \"Single unit analysis also revealed a significant decrease in Fano factor modulation throughout training ( DS: F8 , 160 = 2 . 688 , p = 0 . 0084; M1:F8 , 384 = 9 . 705 , p < 0 . 0001; Figure 5G ) with no modulation in firing rates ( DS: F8 , 160 = 0 . 3008 , p = 0 . 9648; M1:F8 , 384 = 1 . 406 , p < 0 . 1923; Figure 5H ) . 10 . 7554/eLife . 09423 . 012Figure 5 . Neuronal variability dynamics are still evident when analysis is restricted to sequences with duration and frequency .\",\n \"( A , B )\",\n \"Frequency and duration of matched sequences .\",\n \"( C , D )\",\n \"Neuronal variability , measured as the Fano factor of the firing rate , for sequences of matched duration and frequency , for both recorded areas , during sequences and baseline .\",\n \"( E , F )\",\n \"Firing rates , for sequences of matched duration and frequency , during sequences and baseline .\",\n \"( G , H )\",\n \"Fano factor ( FF ) and firing rate ( FR ) modulation relative to baseline values , for individual units recorded across the training sessions ( stable units ) within DS ( top colorplots ) and M1 ( bottom colorplots ) , for sequences of matched duration and frequency .\",\n \"Right panels depict average modulation .\",\n \"Error bars correspond to mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 012 The results above suggest that the decrease in corticostriatal variability is not related to a general decrease in behavioral variability .\",\n \"We therefore investigated if the changes in sequence-to-sequence variability in neural activity were related to the changes in sequence-to-sequence variability of specific behavioral dimensions .\",\n \"We re-calculated the Fano factor of the behavioral features and the neuronal activity using a moving average of a reduced number of trials ( 5 ) to provide a higher within session resolution of the variability dynamics and therefore permit the correlation of behavioral and neuronal dynamics across training for each animal ( Figure 6A , see ‘Materials and methods’ ) .\",\n \"Analyses of the relationship between the variability of the recorded units and the variability of each independent behavior feature revealed a significant increase in correlation between neuronal and behavior variability , specific for sequence frequency ( Figure 6C ) , but not for duration ( Figure 6D ) .\",\n \"These results were observed when using only task-relevant or non-task-relevant neurons ( data not shown ) .\",\n \"They were also observed using different number of trials for calculating the moving average of the Fano factor ( Figure 6—figure supplement 2 ) . 10 . 7554/eLife . 09423 . 013Figure 6 . Correlations between corticostriatal and behavioral variability emerge for specific behavioral features .\",\n \"( A ) Example traces from a single animal representing variability , calculated as the Fano factor , using a moving window of five consecutive trials shifted by one for sequence frequency ( dark blue trace ) , sequence duration ( green trace ) , M1 units firing rate during sequences ( blue trace ) and baseline ( grey trace ) , and DS units firing rate during sequences ( red trace ) and baseline ( grey trace ) .\",\n \"Vertical dashed lines represent separation of different training sessions .\",\n \"Shaded areas correspond to mean ± SEM .\",\n \"( B ) Correlation between the variability ( FF ) in M1 and DS .\",\n \"( C , D )\",\n \"Correlation between variability traces from neuronal firing rates in M1 ( blue bars ) or DS ( red bars ) , and variability of sequence frequency or duration .\",\n \"Error bars denote correlation coefficient ±standard error of the correlation .\",\n \"*p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01310 . 7554/eLife . 09423 . 014Figure 6—figure supplement 1 . No significant correlation was found between average firing rate and any of the behavior features . Correlation between the firing rate and ( A ) sequence frequency or ( B ) sequence duration .\",\n \"Error bars denote correlation coefficient ±standard error of the correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01410 . 7554/eLife . 09423 . 015Figure 6—figure supplement 2 . Changing the number of trials used for Fano factor calculation did not affect the observed corticostriatal and neuronal/behavioral variability correlations .\",\n \"( A , B )\",\n \"Correlation between variability traces from neuronal firing rates of cortical and striatal units , and between neuronal variability and variability of sequence frequency and duration using 3 ( A ) or 7 ( B ) consecutive trials .\",\n \"Error bars denote correlation coefficient ±standard error of the correlation .\",\n \"*p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 015 These results show that the decrease in variability in M1 and DS is not just a reflection of a more constrained performance of the movement as training progresses; variability of the movement decreased in a specific dimension but it increased in others were no significant correlation with neuronal variability was evident .\",\n \"Furthermore , no significant correlations were observed between the firing rate of neurons and the variability any of the behavior features ( Figure 6—figure supplement 1 ) , indicating again that the observed relationship between neuronal and behavior dynamics was not the reflex of a general increase in correlation between neuronal activity and behavior .\",\n \"The data presented above suggested that as training progressed variability in M1 and striatum became more correlated with variability in a specific domain of behavior that changed the probability of reinforcement .\",\n \"This suggests that neural variability in M1 and striatum could also become more coupled with training .\",\n \"We verified that at the onset of training the sequence-to-sequence variability of neural activity in DS and M1 in each animal was not correlated .\",\n \"However , a strong correlation between the variability in DS and M1 rapidly emerged during training ( p < 0 . 05 for all except the first training session , Figure 6B ) , suggesting that as behavioral variability is refined , neural variability in M1 and striatum becomes correlated .\",\n \"The results presented above show that a coupled reduction in corticostriatal variability accompanies the reduction in variability of sequence frequency , but not of sequence duration , suggesting that corticostriatal plasticity is necessary to select the appropriate motor features and hence reduce variability within specific domains .\",\n \"We decided to directly test if the observed reduction in sequence frequency variability is dependent on corticostriatal plasticity by using mutant mice with NMDA receptors deleted specifically at glutamatergic synapses of striatal projection neurons ( RGS9-LCre::Grin1tm1Yql; referred to in the figures as striatal projection neuron SPN NR1-KO ) , which have impaired corticostriatal plasticity ( Dang et al . , 2006 ) , and control littermates .\",\n \"Mutant animals had more difficulty learning the task , so we adapted the training protocol to one session per day for both mutant and littermate controls ( and repeated sessions when needed ) , in order to achieve comparable performance levels ( see ‘Materials and methods’ , Table 1 and Figure 7A ) . 10 . 7554/eLife . 09423 . 016Table 1 . Training protocol and respective number of animals reaching performance criteria for the SPN NR1-KO group and littermate controlsDOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 016Training protocolFree0 . 375 Hz0 . 375/0 . 75 Hz ( 30 reinf ) 0 . 75 Hz1 . 5 Hz1 . 5/3 Hz ( 30 reinf ) 3/6 Hz ( 10 reinf ) 6/7 . 5 Hz ( 10 reinf ) # of subjects reaching criteriaNR1–KO77665411Controls5555555210 . 7554/eLife . 09423 . 017Figure 7 . Corticostriatal plasticity is necessary for the specific refinement of behavioral variability .\",\n \"( A ) Schematic of the adapted training sessions for mutant animals and littermate controls .\",\n \"Animals would remain in the same training session until reaching a stable performance .\",\n \"( B ) Distance of the sum of all three consecutive IPIs from the final covert target ( ∑ ( 3 IPIs ) <660 ms , ∼4 . 5 Hz ) in SPN NR1 mutants and littermate controls ( C ) Spread of the distance between three consecutive IPIs around the final covert target .\",\n \"( D–G )\",\n \"Behavior parameters and variability , measured as the Fano factor , during early and late training sessions in SPN NR1 mutants and littermate controls groups .\",\n \"Bars correspond to mean , with data from individual animals plotted on the background ( red: SPN NR1-KO; black: littermate controls ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 01710 . 7554/eLife . 09423 . 018Figure 7—figure supplement 1 . Bootstrapping statistics in the SPN NR1-KO data support the observations from the post hoc planned comparisons . Histograms depicting the sampled statistic ( difference between the means of two groups ) , after sampling with replacement the original data 100 . 000 times .\",\n \"Red vertical lines correspond to the 5% confidence intervals .\",\n \"Green vertical line corresponds to the mean of the sampled data .\",\n \"Blue vertical line corresponds to the difference between the original data groups ( H0 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 09423 . 018 As expected , the distance to target ( Controls: p = 0 . 0450 , t5 = 2 . 657 , Figure 7B ) and spread around the target ( Controls: p = 0 . 0179 , t5 = 3 . 466 , Figure 7C ) decreased in littermate controls .\",\n \"However , neither of these measures changed with training in mutants ( Mutants: p = 0 . 3535 , t6 = 1 . 005; and p = 0 . 2817 , t6 = 1 . 183 , respectively; Figure 7B , C ) .\",\n \"In general , no significant difference was observed for any of the behavior features between the two groups of animals .\",\n \"However , planned comparisons did show that RGS9-LCre::Grin1tm1Yql mutants did not decrease sequence frequency variability during training , in contrast to littermate controls which did ( significant main effect of training time: F1 , 10 = 10 . 13 , p = 0 . 009; Posthocs: Mutant group: t10 = 1 . 38 , p = 0 . 1964; Control group: t10 = 3 . 00 , p = 0 . 0134 ) .\",\n \"Importantly , no differences in the modulation of sequence duration variability were observed between the two groups ( no significant main effect for genotype: Duration FF: F1 , 10 = 0 . 02 , p = 0 . 887 ) ( Figure 7D–G ) .\",\n \"These statistical results were robust as they were confirmed using bootstrapping statistics ( using 100 . 000 random samples of the data , with replacement ) ( Figure 7—figure supplement 1 ) .\",\n \"These data suggest that corticostriatal plasticity is required for the reduction in variability of specific behavioral features that change the probability of reinforcement .\"\n ],\n [\n \"In this study we show that when mice are trained on a difficult operant paradigm they differentially refine specific behavioral features .\",\n \"When mice were asked to perform progressively faster covert patterns of lever presses to obtain a reinforcer , they reduced variability in sequence frequency , but increased variability in an orthogonal uncorrelated feature ( sequence duration ) .\",\n \"These results are interesting because both features would be classically considered task-relevant—a covert sequence of four presses , which is the minimum to produce a reinforcer in this task , has to have a minimal duration .\",\n \"However , although both features could be considered relevant for the task , only changes in frequency variability were differentially reinforced .\",\n \"Reinforced sequences had lower variability in frequency than non-reinforced sequences , but had equal variability in duration as non-reinforced sequences .\",\n \"Thus , our results indicate that animals reduced frequency variability because that was what was reinforced throughout training .\",\n \"Consistent with this interpretation , in a task where the exact number of presses ( correlated with duration ) was reinforced but the frequency at which the sequence was performed was not , variability in duration decreased and in frequency increased .\",\n \"This in line with data demonstrating differential modulation of the different components of task space during learning ( Todorov and Jordan , 2002; Müller and Sternad , 2004; Cohen and Sternad , 2009 ) .\",\n \"In previous studies from our group where animals performed operant tasks where the constrains were more relaxed ( Jin and Costa , 2010 ) , animals decreased variability in all behavioral domains ( i . e . , they became more stereotypical overall ) .\",\n \"However , when faced with a more challenging task as in the present study , they decreased variability in the domain that was critical for getting a reinforcer , but increased variability in orthogonal domains ( i . e . , they were more stereotypical in just a particular domain ) .\",\n \"It could be that the increase in variability in the orthogonal behavioral domains happens because in difficult tasks animals try to minimize the effort to obtain reinforcers , and hence do not attempt to reduce variability in more than one independent domain .\",\n \"Alternatively , it could also be that mice increased the duration of the sequence ( and the correlated number of presses ) as a strategy to try to increase the probability of getting a successful covert pattern in that sequence .\",\n \"However , this second possibility is less likely , given that the two behavioral features were not correlated , and that sequences of different durations were equally likely to get reinforced .\",\n \"These data suggest that in more challenging motor tasks it is difficult to reduce variability in all domains , and animals seem to differentially refine the motor patterns that led to reinforcement .\",\n \"Consistently , the number of sequences that comply with the minimum frequency required for the last session ( end-target ) increased with training and the distance to the end-target decreased with training , indicating that mice implicitly learned to shape their behavior to match the task requirements .\",\n \"At the neural level , we observed initial high sequence-to-sequence variability of neuronal activity in corticostriatal circuits that decreased with training .\",\n \"Variability in the spike patterns of individual neurons and populations of neurons may be the bases for a process of behavioral exploration ( or trial ) ( Olveczky et al . , 2005; Kao et al . , 2005; Mandelblat-Cerf et al . , 2009 ) , while a decrease in neural variability may reflect a process of selection of specific patterns of neural activity that lead to specific behavioral outputs ( Costa et al . , 2004; Kao et al . , 2005; Fee and Goldberg , 2011 ) .\",\n \"It has been suggested that a decrease in corticostriatal variability as a motor task is learned ( Costa et al . , 2004; Barnes et al . , 2005 ) could correspond to the process of selection and consolidation of specific motor patterns ( Costa , 2011 ) .\",\n \"Here , we show that this decrease in neural variability in corticostriatal circuits correlates specifically with the decrease in variability of a particular behavior domain .\",\n \"These data suggest that the neural patterns in motor cortex and sensorimotor striatum that give rise to the behavioral patterns that are reinforced are progressively selected .\",\n \"Provocatively , it also suggests that changes in motor variability that are not specifically reinforced but are part of a strategy or driven by effort reduction may be encoded somewhere else .\",\n \"Finally , we also show that corticostriatal plasticity is important for the refinement of specific behavior features .\",\n \"Our data therefore suggests an important role for corticostriatal plasticity in selecting the appropriate implicit neural and behavioral patterns that are reinforced ( Costa , 2011 ) .\",\n \"However corticostriatal plasticity did not seem to be necessary for the increase in behavioral variability in other domains ( Goldberg and Fee , 2011 ) .\",\n \"Although in this study we don't investigate the mechanisms underlying the generation of variability , several studies have suggested that the basal ganglia , dopaminergic system , specific cortical circuits , or cerebellar circuits could subserve this function ( Olveczky et al . , 2005; Costa et al . , 2006; Leblois et al . , 2010; Costa , 2011; Fee and Goldberg , 2011; Shmuelof and Krakauer , 2011; Woolley et al . , 2014 ) .\",\n \"Taken together , our findings suggest that corticostriatal plasticity is important to select the neural patterns that lead to the movement patterns that are reinforced .\",\n \"They highlight that corticostriatal plasticity is not only important for choosing which action to do , but also to shape how to do it to obtain a desired outcome .\"\n ],\n [\n \"All experiments were carried in accordance to the ethics committee guidelines of the Champalimaud Foundation and Instituto Gulbenkian de Ciência , and with approval of the Portuguese DGAV ( Ref . 0421 ) .\",\n \"Experiments were carried out using 20 male , 3 to 5 month old C57BL6/J mice .\",\n \"From these , 13 animals were used exclusively for behavioral training while the remaining seven underwent microelectrode array implantation for neuronal data recordings .\",\n \"Animals were maintained on a light–dark cycle of 12 hr:12 hr starting at 7 AM .\",\n \"All experiments were done during the light cycle .\",\n \"Mice were housed in groups of four animals prior to surgery and individually after the electrodes were implanted .\",\n \"3 to 6 months old RGS9-LCre::Grin1tm1Yql homozygous mice ( N = 7 ) and Cre negative littermate controls ( N = 5 ) were used for the mutant mouse behavioral experiments .\",\n \"Seven C57Bl6/J mice were implanted bilaterally with two micro-electrode arrays ( 2 × 8 ) , 35–50 µm tungsten electrodes with micro-polished tips .\",\n \"One array targeted the primary motor cortex ( M1 , layer 5 ) while the second was targeting the ( DS , sensorimotor area that receives projections from the same area in M1 ) .\",\n \"Craniotomies and electrode array positioning were done according to coordinates from the Mouse Brain Atlas ( Paxinos and Franklin , 2008 ) .\",\n \"M1 array was placed 1 mm rostral and 1 . 6 mm lateral from bregma , and lowered ∼1 mm from the surface of the brain .\",\n \"DS array was placed 0 . 5 mm rostral and 2 . 1 mm lateral from bregma , and lowered ∼2 . 3 mm from the surface of the brain .\",\n \"Electrodes were manually lowered at slow rates while constantly monitoring neural activity in all the channels in order to control for proper electrode function and correct positioning .\",\n \"Final verification of electrode position was done after all the experiments were finished , by perfusing animals with PFA and histological confirmation of Nissl stained 70 µm brain slices ( Figure 4—figure supplement 1A , B ) .\",\n \"After surgery animals were allowed to recover for at least 2 weeks before starting any other experimental procedure .\",\n \"Single and multi unit activity was recorded using Blackrock Microsystems Neural Signal Processor , allowing for online sorting of identified units .\",\n \"Further offline sorting of selected units was done using Plexon Offline Sorter v3 ( Plexon Inc , Dallas , TX , United States ) , based on waveform characteristics , ISI and PCA clustering .\",\n \"Units stability was assessed from waveforms and PCA cluster proprieties .\",\n \"For PCA cluster comparison data from all the training sessions was pooled together to calculate common eigen vectors .\",\n \"Data from individual sessions was then projected into this common PC space , allowing us to determine cluster centroids and dispersion for each session .\",\n \"Clusters were considered stable whenever the centroid in a given session was comprised within the interval of the centroid of the previous session ±1 . 96 * standard deviation of the cluster , in the first two principal components ( Figure 4—figure supplement 1C for a graphical representation of this criteria ) .\",\n \"Animals were trained using operant chambers ( MedAssociates Inc , St . Albans , VT , United States ) placed inside sound attenuating boxes .\",\n \"A retractable lever was extruded in the beginning of each session , simultaneous to the onset of a light .\",\n \"Animals were required to perform a sequence of presses at a minimum frequency in order to obtain a 20 mg food pellet ( Bio-Serv , Flemington , NJ , United States ) .\",\n \"24 hr before the first training session animals were placed under a food restriction schedule .\",\n \"Body weight was constantly monitored in order to be kept above 85% of the initial weight .\",\n \"In order to facilitate learning , animals were initially exposed to one session of magazine training were food pellets would be available on a random time schedule , and to three sessions of continuous reinforcement schedule ( CRF ) 1 day before training , where single lever presses would be reinforced .\",\n \"On the following training sessions animals were reinforced if they performed a sequence of consecutive presses at a minimum frequency ( covert target ) , defined by the inverse of three consecutive inter-press intervals ( IPIs ) , which increased with training .\",\n \"On the first session there was no minimum frequency target , meaning that any consecutive 3 IPIs would lead to reinforcement .\",\n \"In consecutive sessions the minimum frequency that would lead to reinforcement was increased or maintained in the following order: 0 . 375 Hz , 0 . 75 Hz , 0 . 75 Hz , 1 . 5 Hz , 3 Hz , 3 Hz , 4 . 5 Hz and 4 . 5 Hz .\",\n \"This constant increase in the minimum frequency of the covert target forced the animals to systematically adapt to the task requirements and perform faster sequences of presses from session to session .\",\n \"The training protocol for mutant animals and littermate controls was adapted due to difficulties learning the task , to one daily session and using automatic progressive schedules once a minimum number of reinforcements ( 30 or 10 ) was achieved .\",\n \"( Table 1 for performance summary . )\",\n \"Sequences of presses were differentiated based on IPI and occurrence of a magazine head entry .\",\n \"An IPI >2 s ( determined based on the distribution of IPIs ) or a head-entry were used to define the bouts or sequences of presses .\",\n \"The 2 s cutoff was determined from the joint distribution of the instantaneous IPIs ( and the corresponding log distribution ) from all the animals , by determining the valley between the two main peaks of IPIs ( Figure 1—figure supplement 1C , D ) .\",\n \"Frequency of each sequence was defined as the inverse of the average IPI of each sequence .\",\n \"Duration of each sequence was defined as the time between the first and the last press event .\",\n \"Length of each sequence was defined as the number of press events in each sequence .\",\n \"For the matched sequences analysis , sequences with a duration of 0 . 2–2 s and a frequency higher than 2 Hz were selected .\",\n \"Neural activity was averaged in 20-ms bins , shifted by 1 ms , and averaged across trials to construct the peri-event histogram ( PETH ) .\",\n \"Data from the PETH from 5000 to 2000 ms before lever press were considered as baseline activity .\",\n \"A positive modulation in firing rate was defined if at least 20 consecutive bins had firing rate larger than a threshold of 99% above baseline activity , and a negative modulation of firing rate was defined if at least 20 consecutive bins had a firing rate smaller than a threshold of 95% below baseline activity ( Belova et al . , 2007 ) .\",\n \"Paired t-tests between baseline firing rate and sequence firing rate were used to classify individual neurons as sequence-related .\",\n \"The programs to run the tasks presented in this study can be found at http://tinyurl . com/or7ug72 .\",\n \"Analyses were done in Matlab ( MathWorks , Natick , MA , United States ) or GraphPad Prism ( GraphPad Software Inc , La Jolla , CA , United States ) .\",\n \"Normality was verified for all tests using the D'Agostino-Pearson omnibus normality test , or the Kolmogorov–Smirnov test when sample size was too small .\",\n \"Repeated measures ANOVA were used to evaluate changes in behavior and neuronal features .\",\n \"Probability of a magazine check after lever-press was evaluated using one-way ANOVA and post hoc comparisons using Fisher's LSD test , but one subject was excluded from these analysis due to a lack of recorded timestamps for magazine head-entries .\",\n \"Paired t-tests were used to evaluate differences in percentage of lever-presses .\",\n \"Increases in FF modulation were assessed by the Wilcoxon Rank Signed test .\",\n \"Repeated measures two-way ANOVA was used to verify the general effect of the RGS9-NR1 mutants experiment .\",\n \"Bootstrapping statistics were used on the data from the RGS9-NR1 mutants and littermate controls to validate the results from the post hoc tests .\",\n \"Histograms were built from 100000 randomized samples with replacement .\",\n \"Sample sizes were calculated based on α = 0 . 05 and power of 0 . 7 .\",\n \"Trial to trial variability of neuronal and behavior data was assessed using Fano factor .\",\n \"We calculate the Fano factor of individual units by dividing the variance of firing rates across all the trials of a session by the mean over those trials .\",\n \"Fano factor and firing rate modulations for individual stable cells were calculated as the ratio between the difference of values for sequence and baseline and the values during baseline ( Fano factor: [FFsequence − FFbaseline]/FFbaseline; firing rate: [FRsequence − FRbaseline]/FRbaseline ) .\",\n \"Fano factor of the behavioral features was calculated by dividing the variance in the individual features by the mean of the feature for all the trials .\",\n \"To establish correlations between the variability of the neuronal data and the variability of the behavior , Fano factors were calculated using three , five or seven consecutive trials , allowing us to increase the resolution of the variability measures .\",\n \"Correlations between neuronal and behavior data were evaluated using Pearson's linear correlations .\",\n \"To avoid correlations bias due to sample size , statistical significance of all the correlations was assessed using the significance criteria for the session with smaller size .\",\n \"Within animal correlations averaged using Fisher's z transformation ( Silver and Dunlap , 1987 ) returned similar results to grouped correlations for all the tested conditions ( data not shown ) .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Learning to perform a complex motor task requires the optimization of specific behavioral features to cope with task constraints .","We show that when mice learn a novel motor paradigm they differentially refine specific behavioral features .","Animals trained to perform progressively faster sequences of lever presses to obtain reinforcement reduced variability in sequence frequency , but increased variability in an orthogonal feature ( sequence duration ) .","Trial-to-trial variability of the activity of motor cortex and striatal projection neurons was higher early in training and subsequently decreased with learning , without changes in average firing rate .","As training progressed , variability in corticostriatal activity became progressively more correlated with behavioral variability , but specifically with variability in frequency .","Corticostriatal plasticity was required for the reduction in frequency variability , but not for variability in sequence duration .","These data suggest that during motor learning corticostriatal dynamics encode the refinement of specific behavioral features that change the probability of obtaining outcomes ."],"string":"[\n \"Learning to perform a complex motor task requires the optimization of specific behavioral features to cope with task constraints .\",\n \"We show that when mice learn a novel motor paradigm they differentially refine specific behavioral features .\",\n \"Animals trained to perform progressively faster sequences of lever presses to obtain reinforcement reduced variability in sequence frequency , but increased variability in an orthogonal feature ( sequence duration ) .\",\n \"Trial-to-trial variability of the activity of motor cortex and striatal projection neurons was higher early in training and subsequently decreased with learning , without changes in average firing rate .\",\n \"As training progressed , variability in corticostriatal activity became progressively more correlated with behavioral variability , but specifically with variability in frequency .\",\n \"Corticostriatal plasticity was required for the reduction in frequency variability , but not for variability in sequence duration .\",\n \"These data suggest that during motor learning corticostriatal dynamics encode the refinement of specific behavioral features that change the probability of obtaining outcomes .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["Learning a new motor skill typically involves a degree of trial and error .","Movements that achieve the desired outcome—from catching a ball to playing scales—are repeated and refined until they can be produced on demand .","This process is made more difficult as the activity of individual neurons and muscle fibers can vary at random , and this reduces the ability to reproduce a given movement precisely and reliably .","It has been suggested that the motor system overcomes this problem by identifying those parts of a task that are essential for achieving the end goal , and then focusing resources on reducing the variability in the performance of those parts alone .","Santos et al . now provide direct evidence in support of this proposal by recording the activity of neurons in motor regions of the mouse brain as the animals learn a lever pressing task .","By giving mice a food reward each time they pressed the lever four times in a row , Santos et al . trained the animals to press the lever in bouts .","The experiment was then slightly modified , so that the mice had to perform the four lever presses more rapidly in order to earn their reward .","Consistent with predictions , the average speed of lever pressing initially varied greatly , but this variability decreased as the animals learned the task .","By contrast , the total duration of individual bouts of lever pressing—which depends largely on the number of times the mice press the lever—was just as variable after training as before .","A similar pattern emerged for the activity of individual motor neurons in the mouse brain .","Whereas their activity initially varied greatly , this variability decreased over training .","Moreover , it became increasingly linked to the variability in the speed of lever pressing , but not with the variability in the duration of individual bouts .","The work of Santos et al . has thus shown in real time how the motor system focuses its efforts on reducing variability in those specific parts of a task that are essential for achieving a goal .","Without a process called corticostriatal plasticity , by which the motor system adapts , mice could not refine this variability ."],"string":"[\n \"Learning a new motor skill typically involves a degree of trial and error .\",\n \"Movements that achieve the desired outcome—from catching a ball to playing scales—are repeated and refined until they can be produced on demand .\",\n \"This process is made more difficult as the activity of individual neurons and muscle fibers can vary at random , and this reduces the ability to reproduce a given movement precisely and reliably .\",\n \"It has been suggested that the motor system overcomes this problem by identifying those parts of a task that are essential for achieving the end goal , and then focusing resources on reducing the variability in the performance of those parts alone .\",\n \"Santos et al . now provide direct evidence in support of this proposal by recording the activity of neurons in motor regions of the mouse brain as the animals learn a lever pressing task .\",\n \"By giving mice a food reward each time they pressed the lever four times in a row , Santos et al . trained the animals to press the lever in bouts .\",\n \"The experiment was then slightly modified , so that the mice had to perform the four lever presses more rapidly in order to earn their reward .\",\n \"Consistent with predictions , the average speed of lever pressing initially varied greatly , but this variability decreased as the animals learned the task .\",\n \"By contrast , the total duration of individual bouts of lever pressing—which depends largely on the number of times the mice press the lever—was just as variable after training as before .\",\n \"A similar pattern emerged for the activity of individual motor neurons in the mouse brain .\",\n \"Whereas their activity initially varied greatly , this variability decreased over training .\",\n \"Moreover , it became increasingly linked to the variability in the speed of lever pressing , but not with the variability in the duration of individual bouts .\",\n \"The work of Santos et al . has thus shown in real time how the motor system focuses its efforts on reducing variability in those specific parts of a task that are essential for achieving a goal .\",\n \"Without a process called corticostriatal plasticity , by which the motor system adapts , mice could not refine this variability .\"\n]","isConversational":false},"year":{"kind":"string","value":"2015"}}},{"rowIdx":298,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]","isConversational":false},"title":{"kind":"string","value":"Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans"},"id":{"kind":"string","value":"elife-21166-v1"},"sections":{"kind":"list like","value":[["In order to effectively adapt to an environment , organisms need to be able to analyze both their internal state and external surroundings to make behavioral decisions that will maximize their chances of survival .","It has been hypothesized that decision making in animals is controlled primarily at the level of command interneurons or decision centers in the central nervous system , which receive input from an array of sensory neurons and integrate the information conveyed to inform behavioral decisions ( Kristan , 2008 ) .","However , recent work in a number of animal species has demonstrated that modulation of gene expression and neuronal activity by both environmental cues and internal state can occur at the level of sensory neurons , suggesting that aspects of behavioral decision making may also occur in the peripheral nervous system ( Dey et al . , 2015; Farhadian et al . , 2012; Ryan et al . , 2014 ) .","Two pervasive internally derived modifiers of decision making in animals are the nutritional state of the animal and biological sex .","Feeding status and satiety levels have long been known to have dramatic effects on the behaviors and decisions of species across the animal kingdom ( Pool and Scott , 2014; Sengupta , 2013 ) .","In many cases , the nutritional state of the animal will serve as a source of regulation directly on feeding and other food-related behaviors .","For instance , in the medicinal leech , periods of food deprivation can lead to a heightened response to appetitive stimuli in its environment , a response that has been shown to be carried out via a correlation between serotonin production and time of last meal ( Gaudry and Kristan , 2012; Groome et al . , 1993; Lent et al . , 1991 ) .","Starvation or nutritional deprivation can also have less direct effects on feeding behaviors as in Drosophila , where periods of starvation have been demonstrated to both enhance odor sensitivity and attraction as well as abrogate avoidance responses to normally aversive stimuli ( Bräcker et al . , 2013; Inagaki et al . , 2014; Ko et al . , 2015; Root et al . , 2011 ) .","Similarly , the biological sex of an animal is a potent modifier of decision-making .","Not only does the sex of the animal lead to differences in mating behaviors and pheromone attraction , but studies in several systems have suggested that biological sex can also alter olfactory and gustatory preferences , perhaps to guide decision-making in the context of mating and reproduction ( Dey et al . , 2015; Kimchi et al . , 2007; Kurtovic et al . , 2007; Lebreton et al . , 2015; Lee and Portman , 2007; Meunier et al . , 2000; Nakagawa et al . , 2005; Ribeiro and Dickson , 2010; Ryan et al . , 2014; Stowers and Logan , 2010; Stowers et al . , 2002; Vargas et al . , 2010 ) .","The roundworm , Caenorhabditis elegans , and its well-defined nervous system provide a simple experimental system well suited to investigate how internal states and environmental cues can be integrated to modulate animal behavior .","C . elegans exist as one of two sexes: hermaphrodite or male .","Hermaphrodites produce a limited amount of sperm during larval development making them capable of self-fertilization in addition to cross-fertilization .","In contrast , males are only cross-fertile and must find mating partners for reproduction .","As such , the male displays a large behavioral repertoire primarily aimed at facilitating mate finding and successful mating encounters .","These behaviors include complex mating rituals ( Liu and Sternberg , 1995 ) , differential sensitivity to hermaphrodite pheromone ( Srinivasan et al . , 2008 ) , and a mate-searching behavior in adult male animals that is driven by the prioritization of mating over feeding ( Lipton et al . , 2004 ) .","Mate-searching behavior of C . elegans has thus emerged as an interesting case study of decision making in a simple animal , which is subject to regulation by multiple features of internal state—sex , age , and nutritional status—as well as by external sensory experiences such as the presence of food or hermaphrodites in the environment ( Barrios et al . , 2008; Lipton et al . , 2004; Ryan et al . , 2014 ) .","Here , we focus on the DAF-7/TGF-β signaling pathway , which plays a pivotal role in the regulation of diverse aspects of C . elegans physiology and behavior including the dauer developmental decision , reproductive egg laying , feeding and foraging , fat storage , satiety quiescence , longevity , aggregation , aerotaxis and avoidance of pathogenic bacteria ( Chang et al . , 2006; Gallagher et al . , 2013; Greer et al . , 2008; Meisel et al . , 2014; Milward et al . , 2011; Ren et al . , 1996; Shaw et al . , 2007; White and Jorgensen , 2012; You et al . , 2008 ) .","The expression pattern of daf-7 was originally identified as being restricted to a single bilaterally symmetric pair of neurons in the head of the worm , the ASI chemosensory neurons , but it is also expressed in the ADE and OLQ neurons under normal laboratory growth conditions ( Meisel et al . , 2014; Ren et al . , 1996; Schackwitz et al . , 1996 ) .","We recently defined an altered expression pattern for daf-7 in hermaphrodites in response to exposure to bacterial metabolites produced by the pathogenic bacteria Pseudomonas aeruginosa ( Meisel et al . , 2014 ) .","Within minutes of being exposed to P . aeruginosa , daf-7 expression is induced in an additional pair of chemosensory neurons , the ASJ neurons , which in turn promotes behavioral avoidance of the pathogenic bacterial lawn ( Meisel et al . , 2014 ) .","Here , we show that in male C . elegans , daf-7 expression in the ASJ neuron pair is not only induced in response to environmental cues such as Pseudomonas aeruginosa , but can be switched ‘on’ or ‘off’ depending on multiple internal states such as sex , age , and experience .","The differential effects of internal states and environmental experience on the DAF-7 transcriptional switch in the two ASJ sensory neurons reveal a hierarchy among different inputs that can function to modulate behavior ."],["While expression of the DAF-7/TGF-β ligand was long thought to be confined to the ASI chemosensory neurons ( Ren et al . , 1996; Schackwitz et al . , 1996 ) , we recently reported that a change in the microbial environment of the worm—namely , the introduction of the pathogen P . aeruginosa—can alter the neuronal expression pattern of daf-7 , inducing expression in the ASJ neuron pair of hermaphrodite animals ( Meisel et al . , 2014 ) .","In the current study , we observed that adult males exhibit daf-7 expression in both the ASI and the ASJ neuron pairs in the absence of P . aeruginosa ( Figure 1A and B ) .","This expression pattern difference in adult C . elegans , can be observed both through the use of a pdaf-7::gfp transcriptional reporter ( Figure 1A and B ) as well as by fluorescence in situ hybridization ( FISH ) that directly probes for endogenous daf-7 mRNA ( Figure 1—figure supplement 1A–C ) . 10 . 7554/eLife . 21166 . 003Figure 1 . Sex-specific expression of daf-7 in the ASJ neurons of adult males .","( A ) pdaf-7::gfp expression pattern in hermaphrodites ( left ) and males ( right ) .","Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .","Scale bar indicates 50 µm .","( B ) Maximum fluorescence values of pdaf-7::gfp in the ASI ( left ) and ASJ ( right ) neurons of age-matched adult hermaphrodites ( grey ) and males ( black ) .","*p<0 . 05 as determined by an unpaired t-test with Welch’s correction .","Error bars indicate standard deviation ( SD ) .","n . s . , not significant .","( C ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of males through larval development and early adulthood .","The developmental timeline of C . elegans is shown to scale on top .","Time values indicate hours after an egg is laid .","Error bars indicate SD .","( D ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of both males and hermaphrodites on E . coli ( yellow ) or P . aeruginosa ( green ) .","***p<0 . 001 as determined by unpaired t-tests with Welch’s correction .","Error bars indicate SD . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00310 . 7554/eLife . 21166 . 004Figure 1—figure supplement 1 . daf-7 expression in the ASJ neuron pair is specific to adult males .","( A–B )","FISH images of endogenous daf-7 mRNA in adult hermaphrodite ( A ) and adult male ( B ) .","White dotted lines indicate outline of the ASJ neuron ( s ) .","Both images are a single slice through the animal where ptrx-1::gfp was visible and in focus in one or both ASJ neurons .","Other neurons visible anterior to the ASI neurons are likely the OLQ neurons , and the cells posterior to the ASJ neurons that express daf-7 mRNA are likely the ADE neurons .","Scale bar represents 25 µm .","Orientation provided in ( A ) applies to both images .","( C ) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons ( as localized by ptrx-1::gfp ) in males and hermaphrodites .","***p<0 . 001 as determined by unpaired t-test with Welch’s correction .","Error bars represent SD .","( D ) pdaf-7::gfp expression pattern in an L4 larval male .","Filled triangles indicate the ASI neurons .","Scale bar ( shown in C ) indicates 50 µm .","( E–F )","Maximum fluorescence values of pdaf-7::gfp in the ASI ( E ) and ASJ ( F ) neurons of developing males .","**p<0 . 01 , ***p<0 . 001 , as determined by unpaired t-test with Welch’s correction .","Error bars represent SD .","n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 004 Recent studies have suggested that , in addition to the generation of sex-specific neurons late in larval development , both male-specific and sex-shared neurons exhibit changes in gene expression as well as synaptic remodeling as the male undergoes the sexual maturation process ( Oren-Suissa et al . , 2016; Pereira et al . , 2015; Ryan et al . , 2014; Sammut et al . , 2015 ) .","This predicts that a number of additional unidentified changes may occur in the anatomy , connectivity and gene expression profiles of the male nervous system as the animal transitions from larva to adult .","Given this , we asked if daf-7 expression in males was correlated with the developmental stage of the animal .","We observed that in populations of early larval animals carrying the pdaf-7::gfp transcriptional reporter , males and hermaphrodites were indistinguishable , with fluorescence observed only in the ASI neurons ( Figure 1—figure supplement 1D ) .","To determine the onset of daf-7 expression in males , we imaged animals carrying the fluorescent reporter at multiple time points during development beginning in the L4 larval stage and extending into reproductive maturity .","We found that daf-7 expression remains off in the ASJ of males through the end of the last ( L4 ) larval stage , but is switched on during young adulthood as the animals reach reproductive maturity ( Figure 1C ) .","In addition , we examined expression of daf-7 in the ASI neuron pair at several matched timepoints during development and observed a significant drop in the expression of daf-7 in the ASI neurons from L4 to young adulthood ( Figure 1—figure supplements 1E and F , 52 hr and 64 . 5 hr timepoints ) .","At subsequent timepoints during adulthood , however , expression levels in the ASI neurons remained constant as expression in the ASJ neurons increased ( Figure 1—figure supplements 1E , 3F 64 . 5 hr and 71 . 5 hr timepoints ) .","We asked what the role of the genetic sex of the animal is in regulating daf-7 expression in the ASJ neurons .","The C . elegans sex determination pathway terminates on the master regulator GLI-family transcription factor , TRA-1 ( Zarkower and Hodgkin , 1992; Zarkower , 2006 ) .","TRA-1 activity functions to promote hermaphrodite cell fates and inhibit male development .","We first looked at tra-1 ( e1099 ) mutants which are karyotypically XX , but develop as fertile pseudomales due to the lack of active TRA-1 ( Hodgkin and Brenner , 1977 ) .","In these mutant XX males , daf-7 expression could be seen in both the ASI and ASJ neurons , suggesting that daf-7 is regulated by the genetic sex determination pathway either directly or indirectly ( Figure 2—figure supplement 1A ) .","We next looked at the role of genetic sex specifically in the nervous system using transgenic sexual mosaics .","TRA-1 is negatively regulated by the FEM proteins—FEM-1 , FEM-2 , and FEM-3—and it has been demonstrated by many groups that overexpression of FEM-3 cDNA is sufficient to lead to ‘masculinization’ in otherwise hermaphrodite animals ( Mehra et al . , 1999; Oren-Suissa et al . , 2016; Pereira et al . , 2015; Sammut et al . , 2015; White et al . , 2007 ) .","We expressed the FEM-3 cDNA under the control of the pan-neural promoter , rab-3 , and found that daf-7 expression was up-regulated in the ASJ neurons of adult hermaphrodites , suggesting that a genetically ‘male’ nervous system is sufficient for daf-7 expression in ASJ ( Figure 2A , C and E ) .","Although we observed an increase in daf-7 expression in these genetically masculinized animals , the overall expression of daf-7 in the ASJ of masculinized hermaphrodites was still notably lower than what we observe in wild-type males ( e . g . Figure 1B ) .","This could possibly be explained by the lack of male-specific neurons in these transgenic animals .","While genetic masculinization is effective in sexualizing neurons of the sex-shared nervous system , this technique fails to reliably retain the male-specific CEM neurons and produces hermaphrodites which also lack other male-specific neurons found in the tail ( White et al . , 2007 ) .","To assess the importance of these male-specific neurons for daf-7 expression in ASJ , we examined pdaf-7::gfp fluorescence in ceh-30 ( n4289 ) , mab-3 ( e1240 ) , and pkd-2 ( sy606 ) ;lov-1 ( sy582 ) mutant males .","While ceh-30 ( n4289 ) males lack the male-specific CEM neurons in the head ( Schwartz and Horvitz , 2007 ) , mab-3 ( e1240 ) mutants display marked defects in male-tail formation including ray differentiation as well as in male specific circuitry in the head of the animal ( Yi et al . , 2000 ) .","Similarly , PKD-2 and LOV-1 constitute a TRP channel necessary for the function of the B-type ray neurons—another set of male-specific neurons in the tail of the animal ( Barr et al . , 2001; Barr and Sternberg , 1999; Barrios et al . , 2008 ) .","Males of all mutant strains retained wild-type expression levels of daf-7 in ASJ , suggesting that the male-specific neural circuitry is not required for daf-7 expression and is unlikely to underlie the lower daf-7 expression we see in genetically masculinized hermaphrodites ( Figure 2—figure supplement 1B ) . 10 . 7554/eLife . 21166 . 005Figure 2 . Genetic sex regulates male-specific expression of daf-7 in the ASJ neuron pair .","( A–D )","Representative images of pdaf-7::gfp expression in: wild-type ( WT ) hermaphrodites ( A ) and males ( B ) , nervous-system-masculinized hermaphrodites ( C ) and nervous-system-feminized males ( D ) .","Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .","Scale bar indicates 50 µm .","( E ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of control ( black ) and partially masculinized hermaphrodites ( purple ) .","Masculinization was effected by driving expression of fem-3 cDNA under pan-neural ( left ) and ASJ-specific ( right ) promoters .","***p<0 . 001 as determined by unpaired t-test with Welch’s correction .","n . s . , not significant .","Error bars represent SD .","( F ) Maximum fluorescence values of pdaf-7::gfp in ASJ of control ( black ) and partially feminized males ( orange ) .","Feminization was effected by driving expression of tra-2IC under pan-neural ( left ) , ciliated neuron ( middle ) , and ASJ-specific ( right ) promoters .","***p<0 . 001 as determined by unpaired t-test with Welch’s correction .","Error bars indicate SD . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00510 . 7554/eLife . 21166 . 006Figure 2—figure supplement 1 . daf-7 expression in the ASJ neuron pair is regulated by TRA-1 but not by male-specific neurons .","( A ) Representative image of pdaf-7::gfp expression pattern in tra-1 ( e1099 ) pseudomale .","Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .","Scale bar represents 50 µm .","( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of WT , ceh-30 ( n4289 ) , mab-3 ( e1240 ) , and pkd-2 ( sy606 ) ;lov-1 ( sy582 ) mutant animals .","Significance determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","Error bars represent SD .","n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 006 Complementary to masculinization , genetic ‘feminization’ of XO male animals can be achieved via the overexpression of the intracellular domain of the TRA-2 protein , which negatively interacts with FEM-3 to prevent TRA-1 degradation ( Mehra et al . , 1999 ) .","Overexpression of TRA-2IC under the control of the rab-3 promoter led to complete loss of daf-7 expression in the ASJ of otherwise male animals ( Figure 2B , D and F ) .","These results suggest that a genetically male nervous system is both necessary and sufficient for the sex-specific expression pattern of daf-7 .","We next asked whether the sex determination pathway acts cell-autonomously in ASJ to regulate daf-7 expression .","Using the trx-1 ASJ-specific promoter , we masculinized the ASJ neuron pair alone and saw no daf-7 expression in the resulting adult hermaphrodites ( Figure 2E ) .","Similarly , no daf-7 expression could be observed when only the ciliated neurons ( of which ASJ is a member ) were masculinized ( data not shown ) .","These data suggest the requirement that an additional neuron ( or neurons ) or other tissues have a male cell identity for daf-7 to be expressed in the ASJ neurons , although differences in the strength of the heterologous promoters used in the masculinization constructs may also account for these observations .","In contrast , both ciliated neuron-specific and ASJ-specific feminization in males led to down-regulation of daf-7 expression , confirming that a male cell identity in ASJ is necessary , though not sufficient , for proper daf-7 expression ( Figure 2F ) .","We next sought to determine the functional significance of the sex-specific changes in daf-7 expression that we observe in males .","Mate-searching behavior is a behavioral program specific to adult male C . elegans , which features the prioritization of mating over feeding in these animals .","While hermaphrodite animals prefer to remain in a patch of bacterial food indefinitely and exhibit limited exploratory behavior , males will wander away from a food source at a predictable and constant rate ( Lipton et al . , 2004 ) .","This can be examined in an assay in which the movement of the animal is tracked over time to determine the leaving probability of any given genotype ( Figure 3A; Lipton et al . , 2004 ) .","We observed that mutants in both the DAF-7/TGF-β ligand and its Type I receptor , DAF-1 , showed defects in the mate-searching assay ( Figure 3A and B , and Figure 3—figure supplement 1 ) .","This behavioral defect in the daf-7 mutant can be fully suppressed by mutation of the downstream antagonistic co-SMAD , DAF-3 , suggesting that DAF-7 functions through its canonical TGF-β signaling pathway to regulate mate-searching behavior ( Figure 3B , and Figure 3—figure supplement 1B ) .","Given the change in expression of daf-7 in the ASJ neurons of males at the transition to adulthood when this behavior is first displayed , we used genetic ablations of the ASJ neuron pair to ask what their contribution to this behavior might be .","We observed that the ASJ-ablated animals were defective in mate-searching behavior , establishing a role for the ASJ neuron pair in promoting this behavior ( Figure 3C and Figure 3—figure supplement 1A ) .","Importantly , the defects that we see in daf-7 mutant and ASJ-ablated male behavior are not accounted for by global defects in locomotion .","When mutant animals are tested on leaving assay plates on which the bacterial food has been fully spread , these males reach the scoring distance at equivalent rates to WT controls ( Figure 3—figure supplement 2; Barrios et al . , 2012 ) .","This suggests that the reduced leaving behavior we observe in these animals isspecific to the decision to leave the food source in search of a mate . 10 . 7554/eLife . 21166 . 007Figure 3 . DAF-7/TGF-β is required for male mate-searching behavior .","( A ) Schematic of mate-searching assay ( top ) .","Animals are placed individually into the center of a lawn of bacteria .","The tracks of the animal are followed over time and scored for movement beyond 3 cm away from the food source .","A representative data curve is shown on the bottom depicting hermaphrodites ( solid black ) , males ( dashed black ) , and daf-7 ( e1372 ) mutant males ( blue ) .","( B ) Probability of leaving values for WT , daf-7 mutant , and daf-7;daf-3 double mutant males .","Values plotted are the average + SEM for two independent experiments , n = 40 animals for all strains except daf-7 ( ok3125 ) where n = 29 .","*p<0 . 05 , **p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","n . s . , not significant .","( C ) Probability of leaving values for two independent lines of male animals with genetic ablation of the ASJ neurons , compared with corresponding values for WT and daf-7 mutant males .","Values plotted are the average + SEM for two independent experiments for daf-7 mutant animals and three independent experiments for WT control and ASJ ablation strains .","n = 60 animals for all strains except daf-7 ( ok3125 ) where n = 29 animals .","**p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","One replicate was performed with the daf-7;daf-3 double mutant strains in 1B and controls ( WT and daf-7 ) are identical , so probability values for that experiment were used in the averages shown in both B and C of this figure .","( D ) Probability of leaving values for daf-7 ( ok3125 ) mutant animals carrying transgenes expressing daf-7 cDNA specifically under the control of the indicated promoters .","Values on graph are the average + SEM of two independent experiments with an n = 40 total animals for each strain .","**p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00710 . 7554/eLife . 21166 . 008Figure 3—figure supplement 1 . daf-7 regulates mate-searching behavior in males .","( A ) Representative mate-searching data for strains with genetic ablation of the ASJ neurons ( shown in shades of orange ) .","WT hermaphrodites ( solid black ) , WT males ( dashed black ) , and daf-7 mutant ( blue ) animals are included in each assay as controls .","( B ) Representative data for daf-7;daf-3 double mutant ( green ) in the mate-searching assay .","( C ) Representative data for daf-7 rescue strains ( shown in shades of red ) .","( D ) Representative mate-searching curves for daf-1 mutants ( purple ) .","( E ) Probability of leaving values for WT , daf-7 , and daf-1 mutant animals .","Values shown are the average + SEM of two independent experiments .","n = 40 for WT and daf-7 mutants , n = 32 for daf-1 ( e1287 ) and n = 36 for daf-1 ( m402 ) .","*p<0 . 05 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00810 . 7554/eLife . 21166 . 009Figure 3—figure supplement 2 . daf-7 mutant animals and strains carrying genetic ablation of the ASJ neurons exhibit no locomotion defect .","( A ) Schematic of locomotion assay .","E . coli food ( shown in yellow ) was spread to the edges of the plate and a standard mate searching assay performed to determine animal movement on a full lawn of bacteria .","( B–C )","Raw data curves for daf-7 ( blue ) and ASJ-ablation ( orange ) males in locomotion assay .","WT males shown in black for reference . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 009 Since DAF-7 is a secreted ligand , we anticipated that overexpression of DAF-7 from either the ASI or ASJ neurons would be sufficient to rescue daf-7-associated mutant phenotypes .","As expected , we saw that transgenic rescue from the endogenous promoter as well as from ASI- and ASJ-specific promoters were sufficient to restore mate-searching behavior in the daf-7 ( ok3125 ) mutant background ( Figure 3D and Figure 3—figure supplement 1C ) .","Taken together , these data imply that the dynamic expression of daf-7 in the ASJ neurons of adult males regulates mate-searching behavior .","Mate-searching behavior has been demonstrated to be dependent on the feeding state of the animal—starved male animals will re-prioritize feeding over mate searching for a limited period of time until the animal has had sufficient opportunity to re-feed ( Figure 4C; Lipton et al . , 2004 ) .","We asked whether daf-7 expression in the male ASJ neurons might be dependent on the nutritional state of the animal or the bacterial availability in its environment .","We found that males subjected to starvation beginning early in the L4 larval stage failed to express daf-7 in the ASJ neurons as adults ( Figure 4A , conditions i and ii , and Figure 4B ) .","This lack of daf-7 in the ASJ neuron pair was apparent both by GFP visualization as well as by FISH , with daf-7 mRNA entirely absent from the ASJ neurons ( Figure 4A and B ) .","We noticed that daf-7 mRNA was still apparent in the ASI , OLQ , and ADE neurons of these starved animals , suggesting that the loss of daf-7 expression in the ASJ neurons is specific and not representative of a global down-regulation of transcription in these animals ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 21166 . 010Figure 4 . Nutritional state regulates daf-7 expression in the ASJ neurons of males .","( A ) Schematic of experiment design indicating timing and duration of starvation periods ( left ) .","FISH images of endogenous daf-7 mRNA in the ASJ neurons for each experimental condition ( right ) .","White dotted lines indicate outline of ASJ cell body as localized by ptrx-1::gfp .","Scale bar represents 5 µm .","( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons during re-feeding experiment .","Re-fed animals were starved for a period of 24 hr before being reintroduced to E . coli for the indicated amount of time .","***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","Error bars indicate SD .","n . s . , not significant .","( C ) Representative curves of mate-searching data in fed and post-starvation WT males .","Animals were starved for a period of 24 hr as in previous experiments before being assayed for mate-searching behavior . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 01010 . 7554/eLife . 21166 . 011Figure 4—figure supplement 1 . daf-7 expression is down-regulated by starvation specifically in the ASJ neurons .","( A ) Representative FISH image of endogenous daf-7 mRNA in a starved adult male .","White dotted line indicates outline of an ASJ neuron .","Closed triangle indicates an ASI neuron .","Other neurons visible anterior to the ASI neuron are likely the OLQ neurons , and posterior to the ASJ neuron are likely the ADE neurons .","Scale bar represents 25 µm .","( B ) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons ( as localized by ptrx-1::gfp ) of males fed and starved at various different points in their lives .","***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","Error bars represent SD .","n . s . , not significant .","( C ) Fluorescence quantification for daf-7-Cy5 probe in the ASI neurons of starved and fed males .","Significance determined by unpaired t-test with Welch’s correction .","Error bars represent SD .","n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 011 We next asked if daf-7 expression could be dynamically modulated by removal and re-introduction of food at various points during the life of the male .","We first looked at daf-7 expression in animals that had experienced starvation during the L4-to-adulthood period before being returned to an E . coli food source as adults .","In these animals , daf-7 expression could be detected both by the GFP reporter as well as by FISH ( Figure 4A , condition iii , and 4B ) .","We asked whether fully mature adult animals would turn off daf-7 expression in response to starvation after having already induced expression of daf-7 in the ASJ neuron pair .","For these experiments , we utilized FISH of the endogenous daf-7 mRNA because perdurance of the GFP reporter might complicate analysis .","When males were allowed to develop into adulthood unperturbed and then subjected to a period of food deprivation , we observed a complete loss of daf-7 mRNA in the ASJ neurons ( Figure 4A , condition iv ) .","Together this data demonstrates that daf-7 expression in the ASJ neurons is subject to dynamic regulation by periods of starvation and re-feeding .","These data suggest two distinct possibilities for how bacterial food levels in the environment modulate daf-7 transcriptional regulation in the ASJ neuron pair of males .","First , daf-7 transcription could be regulated by the internal nutritional state of the animal ( starved or fed ) .","Alternatively , daf-7 transcription could be activated by the chemosensation ( smell or taste ) of bacterial food .","To distinguish between these two possibilities , we measured the kinetics of daf-7 expression in response to re-feeding in starved animals .","We observed that daf-7 expression in the ASJ neuron pair is not significantly increased compared to starved controls until 9 hr after the re-introduction of an E . coli food source ( Figure 4B ) .","Although some animals could be observed expressing daf-7 weakly in the ASJ neuron pair at 4 hr of re-feeding , the majority of animals still exhibited no pdaf-7::gfp expression in the ASJ neuron pair until later time points .","The extended period of time required to restore daf-7 expression in the ASJ neuron pair suggests that internal nutritional state , not the chemosensation of bacterial food , exerts control over daf-7 transcription in the ASJ neuron pair .","Moreover , we observed that the onset of daf-7 expression in the ASJ neurons of males following the re-introduction of food are consistent with the previously noted kinetics of resumption of mate-searching behavior after males have been starved ( Figure 4C; Lipton et al . , 2004 ) .","We recently showed that the microbial environment is an important regulator of daf-7 expression in the ASJ neurons in hermaphrodite animals ( Figure 1D; Meisel et al . , 2014 ) .","Given that males induce expression of daf-7 in the same ASJ neuron pair independently of the bacterial food source , we sought to determine if they would be capable of responding to exposure to P . aeruginosa in the same way as their hermaphrodite siblings—namely , through additional up-regulation of daf-7 expression in the ASJ neurons .","We observe that , when transferred to P . aeruginosa , males carrying the pdaf-7::gfp transcriptional reporter exhibited an increase in daf-7 expression in the ASJ neurons compared to E . coli-fed controls ( Figure 1D ) .","The additive nature of DAF-7 levels in males suggests that the ASJ neuron pair of males retain the ability to respond to P . aeruginosa that we observe in hermaphrodites .","Our data suggest that daf-7 expression in the ASJ neurons is subject to regulation by both sensory cues from the microbial environment as well as by internal state sensing mechanisms .","Because daf-7 expression in the ASJ neuron pair and mate-searching behavior in males are dependent on nutritional state during feeding on the non-pathogenic bacterial food , E . coli , we sought to determine if expression of daf-7 in response to P . aeruginosa might also be dependent on the nutritional state in males .","We tested this by challenging starved animals with E . coli or P . aeruginosa to see how daf-7 expression would be affected and to observe any behavioral differences .","Looking at animals carrying the pdaf-7::gfp reporter during post-starvation re-feeding , we observed a rapid up-regulation of daf-7 expression in the ASJ neurons of starved males returned to P . aeruginosa as a food source ( Figure 5A and B , and Figure 5—figure supplement 1E ) .","This increase in daf-7 expression in the ASJ neuron pair could be seen as early as 2 hr after first exposure to the pathogenic bacteria ( Figure 5—figure supplement 1E ) .","In contrast , starved males that were returned to the non-pathogenic E . coli showed little to no daf-7 expression in the ASJ neurons until much later time points ( Figure 5A , B and 4B ) .","The kinetics of the increase in fluorescence of the pdaf-7::gfp reporter in response to P . aeruginosa secondary metabolites , which lags behind the immediate response observed by FISH is consistent with what we observed previously for hermaphrodite animals ( Meisel et al . , 2014 ) , and notably faster than the slower increase in pdaf-7::gfp reporter fluorescence in response to repletion of the non-pathogenic E . coli bacterial food . 10 . 7554/eLife . 21166 . 012Figure 5 . Prioritization of multiple inputs through the regulation of daf-7 expression in the ASJ neurons of males .","( A ) Schematic of refeeding experimental design indicating timing and duration of each starving and feeding period ( left ) .","On right , representative images of daf-7 expression following 4 hr of post-starvation refeeding on E . coli ( left ) or P . aeruginosa ( right ) .","Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .","Scale bar represents 50 µm .","( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced either to the normal E . coli food source or to pathogenic P . aeruginosa .","Images were taken for quantification after 4 hr of re-feeding .","***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","Error bars indicate SD .","n . s . , not significant .","( C ) Representative curve of food-leaving data in starved animals on E . coli ( yellow ) and P . aeruginosa ( green ) .","Animals were starved as in previous experiments before being run in the assay . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 01210 . 7554/eLife . 21166 . 013Figure 5—figure supplement 1 . P .","aeruginosa influences exploratory behavior and daf-7 expression in both males and hermaphrodites .","( A ) Representative leaving curves for males ( dashed ) and hermaphrodites ( solid ) on E . coli ( shades of grey ) and P . aeruginosa ( shades of green ) .","All strains were fed in this experiment .","( B ) Probability of leaving values for males and hermaphrodites on E . coli and P . aeruginosa .","All strains were fed in these experiments .","Values plotted are the mean + SEM for two independent experiments , n = 40 animals for all conditions .","*p<0 . 05 as determined by unpaired t-test with Welch’s correction .","n . s . , not significant .","( C ) Representative raw data curves for starved hermaphrodites on E . coli and P . aeruginosa .","Fed hermaphrodites shown as control .","( D ) Maximum fluorescence of pdaf-7::gfp in the ASJ neurons of starved hermaphrodites re-fed E . coli or P . aeruginosa for four hours ( compare to Figure 5B ) .","Error bars indicate SD .","( E ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced to P . aeruginosa for the indicated amount of time .","The experiment in Figure 4B was run in parallel , controls ( fed and starved ) were shared between conditions .","These controls are shown in both figures for comparison purposes .","*p<0 . 05 and ***p<0 . 001 , as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .","Error bars indicate S . D . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 013 We followed up by asking if this difference in expression after starvation is also reflected in behavioral differences of post-starvation animals experiencing different microbial environments .","Normally , following a period of starvation , a male prefers feeding over leaving a lawn of bacteria in search of a mate ( Figure 4C; Lipton et al . , 2004 ) , but we reasoned that these priorities might be re-set if the only available food is pathogenic to the starved animal .","Indeed , whereas starved males will remain on the E . coli lawn for several hours , starved males who are instead returned to a lawn of P . aeruginosa will readily leave the bacterial lawn at a rate similar to non-starved animals on the normal E . coli food source ( Figures 4C and 5C ) .","This lawn-leaving behavior is not male-specific , as both fed and starved hermaphrodites will also readily leave a lawn of P . aeruginosa and starved hermaphrodites are also capable of responding to P . aeruginosa through an upregulation of daf-7 in the ASJ neurons ( Figure 5—figure supplement 1A–1D ) .","In this regard , one might consider that lawn-leaving under these conditions is driven by the presence of pathogenic P . aeruginosa , and not mate-searching .","Our data suggest that daf-7 expression in the ASJ neurons is one way through which animals are both able to interpret internal and external cues and prioritize their behaviors accordingly ."],["We have presented a set of experiments that suggest that behavioral prioritization and decision-making may be effected through the transcriptional control of a key neuroendocrine regulator , the TGF-β ligand , DAF-7 , in a pair of chemosensory neurons , the ASJ neurons .","Our data suggest that daf-7 expression in the ASJ neuron pair is utilized to promote exploratory behaviors in different physiological and ecological contexts .","In hermaphrodites , expression of daf-7 is induced by the detection of secondary metabolites and functions to allow animals to discriminate between microbial species and promote avoidance of pathogenic bacteria ( Meisel et al . , 2014 ) .","In essence , pathogen avoidance represents the behavioral choice an animal makes when confronted with two competing needs—the need to eat and remain well fed and the need to avoid danger and infection .","Mate searching presents a paralagous but sex-specific behavior , where the decision instead is between food and mating .","Our data suggest that modulation of daf-7 expression provides a striking example of how a neuroendocrine signaling pathway may be coopted to guide behavioral decisions in entirely different contexts .","Our study suggests that internal states and environmental cues that converge on the transcriptional control of DAF-7 in the ASJ neuron pair are processed in a hierarchical manner that , in turn , serves to modulate the exploratory mate-searching behavior in the male ( Figure 6 ) .","The work presented here suggests that the transcriptional regulation of daf-7 in the ASJ neurons functions as an ON/OFF switch , where each input holds the switch—and thus , daf-7 expression—in one of two states .","However , the various regulatory inputs into this switch exert differential control , with environmental cues being prioritized over all others .","This differential control of the transcriptional switch , in turn allows for re-adjustments of behavioral priorities in the worm based on its current and past experience . 10 . 7554/eLife . 21166 . 014Figure 6 . A hierarchy of inputs regulates daf-7 expression in the ASJ neurons to modulate exploratory behaviors . We show that the information from many different sources both internal and external is integrated hierarchically in the regulation of daf-7 expression in the ASJ neuron pair .","This hierarchical regulation of daf-7 in turn leads to a distinct prioritization of exploratory behaviors in the male worm . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 014 Interestingly , dynamic transcriptional regulation has previously been implicated in the regulation of sex-specific decision-making behavior in C . elegans .","Expression of the chemoreceptor , ODR-10 , in the sex-shared sensory AWA neurons has also been shown to be dependent on both internal and external cues to regulate food-related decision making in male C . elegans ( Ryan et al . , 2014 ) .","Given the similarities in the regulation of these two genes by both sex and satiety , it is possible that DAF-7 and ODR-10 function in a single pathway to regulate decision-making behaviors in the male .","Previous studies of daf-7 have suggested that it is involved in regulating the expression of numerous chemoreceptors in many different neuron types ( Nolan et al . , 2002 ) .","While no interaction has been identified between daf-7 and odr-10 in the hermaphrodite nervous system , there has been no examination as yet of the effects of daf-7 mutation on odr-10 in the relevant cell types , sex , or under different food conditions , which may be an interesting line of future research .","DAF-7/TGF-β has previously been found to be involved in the regulation of other satiety-regulated behaviors such as quiescence and foraging ( Gallagher et al . , 2013; Milward et al . , 2011; You et al . , 2008 ) .","daf-7 mutants show marked defects in the ability to respond to fasting through an increase in quiescence , suggesting that DAF-7 may serve as a critical signal of food availability in the worm ( You et al . , 2008 ) .","Similarly , DAF-7 signaling has also been implicated in promoting food-leaving ( or foraging ) in the context of a rapidly depleting food source , further underscoring the importance of this pathway for communicating nutritional availability ( Milward et al . , 2011 ) .","While DAF-7 from the ASI neurons , and not the ASJ neurons , contributes to these other possibly related phenotypes , similar mechanisms may be involved in regulating daf-7 expression and release in these three distinct food/satiety-dependent assay contexts .","Taken together with our data , the important role of daf-7 in these different behavioral paradigms suggests that the daf-7 pathway may act as a more global food signal that is critical for allowing the animal to sense food and satiety and modulate its behaviors accordingly .","Finally , our results support a model where integration of internal and external sensory information can occur at the level of the sensory neuron itself to regulate behavioral prioritization .","Our data on the role of the ASJ sensory neurons in male mate-searching behavior are consistent with emerging evidence from studies in several species to suggest that the regulatory control of sensory neurons contributes to information processing and decision making behavior ( Dey et al . , 2015; Lebreton et al . , 2015; Peckol et al . , 2001; Ryan et al . , 2014 ) ."],["C . elegans were grown on E . coli OP50 as a food source as previously described ( Brenner , 1974 ) .","Strains were grown at 20°C except for assays containing dauer-constitutive mutants , in which case , all strains were grown at 16°C until they passed the dauer developmental decision .","Unless otherwise indicated , all strains carry the him-5 ( e1490 ) mutation to increase the number of spontaneous males available .","For a complete list of strains used in this study , please see Supplementary file","1 . For quantification of daf-7 expression in mutants and developing animals , animals were egg laid for 2 hr on OP50 to obtain synchronized populations of animals .","The animals were then mounted and anesthetized in 50 mM sodium azide at the indicated hour of development or at 72 hr if not indicated .","The animals were imaged at 40x using a Zeiss Axioimager Z1 microscope .","15–20 animals were imaged for each condition or strain .","For quantification , maximum intensity values of GFP within the ASJ neurons was calculated using FIJI software ( Schindelin et al . , 2012 ) ; Fiji , RRID:SCR_002285 ) .","For P . aeruginosa experiments , the P . aeruginosa PA14 strain was grown overnight at 37°C in 3 mL of LB as previously described and the next day 7 µL of culture was seeded onto SKA plates ( Tan et al . , 1999 ) .","Plates were incubated at 37°C overnight and then allowed to grow for an additional two days at room temperature before being used for experiments .","Animals for P . aeruginosa experiments were synchronized by treatment with bleach and then allowed to hatch and arrest as L1 larvae in M9 media overnight .","This provided synchronized populations free from any possible bacterial contamination .","Animals were transferred to PA14 plates or OP50 controls at the L4 larval stage .","Imaging and quantification were performed as described above at 16 hr after transfer to PA14 plates .","To generate the neural feminization and masculinization transgenes , the pGH8 plasmid containing the pan-neural rab-3 promoter along with mCherry::unc-54 3’UTR was linearized by PCR .","The fem-3 and intracellular tra-2 cDNAs were amplified from cDNA generated with an Ambion RETROScript kit .","The unc-54 3’UTR was amplified from the pPD95 . 75 vector and the SL2 trans-splicing sequence leader ( Huang et al . , 2001; Spieth et al . , 1993 ) was amplified from genomic DNA .","The fem-3 or tra-2 cDNA along with the unc-54 3’UTR and SL2 sequence were cloned into the linearized pGH8 vector by Gibson assembly ( Gibson et al . , 2009 ) .","For cell specific manipulations , the ciliated neuron bbs-1 promoter ( 1 . 9 kb ) and ASJ specific trx-1 promoter ( 1 . 1 kb; Fierro-González et al . , 2011 ) were amplified from genomic DNA and inserted in the place of the rab-3 promoter by Gibson assembly .","All plasmids were sequenced to confirm identity and injected into ksIs2;him-5 ( e1490 ) animals at a concentration of 50 ng/µL , along with a plasmid carrying ofm-1::gfp ( 50 ng/µL ) .","At least three independent transgenic lines were obtained and analyzed for each construct and one representative line is shown .","For a complete list of primers used in this paper , please see Supplementary file","2 . Mate-searching behavior was assessed in animals through a food leaving assay as previously described ( See diagram in Figure 3A; Lipton et al . , 2004 ) .","Briefly , animals were placed individually onto a 1 cm lawn ( ~20 µL of culture ) of E . coli OP50 in the center of a 10 cm plate .","Movement of each animal was tracked at hourly time points .","When an animal’s tracks extended a distance of 3 cm away from the starting lawn , the animal was scored as a ‘leaver’ .","It has been previously demonstrated that male leaving behavior can be modeled by the single exponential decay function N ( t ) /N ( 0 ) =exp ( -λt ) , where N ( t ) refers to the number of non-leavers at a given time and the hazard value ( λ ) gives an estimate for the probability of leaving , which remains constant over the course of an assay ( Lipton et al . , 2004 ) .","Hazard values were calculated by fitting data using the R survival package with an exponential parametric survival model using maximum likelihood ( R Project for Statistical Computing , RRID:SCR_001905 ) .","These values were pooled across replicates .","At least two independent experiments were performed for each genotype ( ~40 animals per genotype ) .","For leaving assays with P . aeruginosa , plates were supplemented with additional peptone and were seeded with 15 µL of bacterial culture .","In addition , all plates for PA14 assays were incubated overnight at 37°C after being seeded with bacteria and then for an additional 2 days at room temperature before being used for the assay .","For locomotion assays , 10 cm mate-searching plates were seeded with ~400 µL of E . coli OP50 culture spread to the edge of the plate .","Plates were allowed to dry and remained at room temperature for ~16 hr before being used in the assay .","Males were placed individually on these plates and their movement tracked over time as in the standard mate-searching assay .","The same scoring distance was used for locomotion assays as for the mate-searching assay .","FISH was performed as described previously ( Raj et al . , 2008 ) .","Animals were fixed in 4% formaldehyde for 45 min at 20°C .","Following washing in PBS , animals were resuspended in 70% ethanol and incubated overnight at 4°C .","Samples were then hybridized with probes to the endogenous daf-7 mRNA as previously published ( Meisel et al . , 2014 ) .","Hybridization was carried out at 30°C overnight .","Imaging was performed using a Nikon Eclipse Ti Inverted microscope with a Princeton Instruments PIXIS 1024 camera .","All data were analyzed in FIJI .","For quantifications of FISH data , fluorescence values were calculated within the ASJ ( or ASI ) neurons for the indicated conditions .","All samples were hybridized using daf-7-Cy5 probes and imaged at an exposure time of 1500 ms . For all FISH experiments , animals carrying the ofEx4[ptrx-1::gfp] transgene were used in order to localize the ASJ neurons .","For all starvation experiments , animals were synchronized by treatment with bleach and then were allowed to hatch and arrest as L1s in M9 media for 14 hr .","After 14 hr , animals were dropped onto plates containing abundant OP50 E . coli and were allowed to develop for 34–36 hr at 20°C .","At this time point , animals had already entered the L4 larval stage .","All animals were washed from the plates and subjected to an additional 4–5 washes with M9 media to remove any remaining bacteria .","Animals were placed either back onto plates seeded with E . coli or were dropped onto plates lacking peptone to inhibit any bacterial growth .","For re-feeding experiments , animals were starved on peptone free plates for 24 hr at 20°C .","After 24 hr of starvation , nearly all of the male animals had molted into adults as evident by tail and gonadal morphology .","Only animals with adult morphology were used for experiments and imaging .","They were subsequently collected and placed onto plates seeded with either E . coli or P . aeruginosa for the designated amount of time .","For the FISH experiment shown in Figure 3 , animals were re-fed on E . coli OP50 for 16 hr at 20°C before being fixed for imaging .","For the late starve experiments , animals dropped back onto E . coli after the washing step were allowed to grow for an additional 24 hr at 20°C before being collected and washed 4–5 times in M9 media .","These animals were then placed onto peptone-free plates and starved for 16 hr at 20°C before being fixed for imaging .","All statistical analysis was performed using the GraphPad Prism software ( Graphpad Prism , RRID:SCR_002798 ) .","Statistical tests used are indicated in each figure legend ."]],"string":"[\n [\n \"In order to effectively adapt to an environment , organisms need to be able to analyze both their internal state and external surroundings to make behavioral decisions that will maximize their chances of survival .\",\n \"It has been hypothesized that decision making in animals is controlled primarily at the level of command interneurons or decision centers in the central nervous system , which receive input from an array of sensory neurons and integrate the information conveyed to inform behavioral decisions ( Kristan , 2008 ) .\",\n \"However , recent work in a number of animal species has demonstrated that modulation of gene expression and neuronal activity by both environmental cues and internal state can occur at the level of sensory neurons , suggesting that aspects of behavioral decision making may also occur in the peripheral nervous system ( Dey et al . , 2015; Farhadian et al . , 2012; Ryan et al . , 2014 ) .\",\n \"Two pervasive internally derived modifiers of decision making in animals are the nutritional state of the animal and biological sex .\",\n \"Feeding status and satiety levels have long been known to have dramatic effects on the behaviors and decisions of species across the animal kingdom ( Pool and Scott , 2014; Sengupta , 2013 ) .\",\n \"In many cases , the nutritional state of the animal will serve as a source of regulation directly on feeding and other food-related behaviors .\",\n \"For instance , in the medicinal leech , periods of food deprivation can lead to a heightened response to appetitive stimuli in its environment , a response that has been shown to be carried out via a correlation between serotonin production and time of last meal ( Gaudry and Kristan , 2012; Groome et al . , 1993; Lent et al . , 1991 ) .\",\n \"Starvation or nutritional deprivation can also have less direct effects on feeding behaviors as in Drosophila , where periods of starvation have been demonstrated to both enhance odor sensitivity and attraction as well as abrogate avoidance responses to normally aversive stimuli ( Bräcker et al . , 2013; Inagaki et al . , 2014; Ko et al . , 2015; Root et al . , 2011 ) .\",\n \"Similarly , the biological sex of an animal is a potent modifier of decision-making .\",\n \"Not only does the sex of the animal lead to differences in mating behaviors and pheromone attraction , but studies in several systems have suggested that biological sex can also alter olfactory and gustatory preferences , perhaps to guide decision-making in the context of mating and reproduction ( Dey et al . , 2015; Kimchi et al . , 2007; Kurtovic et al . , 2007; Lebreton et al . , 2015; Lee and Portman , 2007; Meunier et al . , 2000; Nakagawa et al . , 2005; Ribeiro and Dickson , 2010; Ryan et al . , 2014; Stowers and Logan , 2010; Stowers et al . , 2002; Vargas et al . , 2010 ) .\",\n \"The roundworm , Caenorhabditis elegans , and its well-defined nervous system provide a simple experimental system well suited to investigate how internal states and environmental cues can be integrated to modulate animal behavior .\",\n \"C . elegans exist as one of two sexes: hermaphrodite or male .\",\n \"Hermaphrodites produce a limited amount of sperm during larval development making them capable of self-fertilization in addition to cross-fertilization .\",\n \"In contrast , males are only cross-fertile and must find mating partners for reproduction .\",\n \"As such , the male displays a large behavioral repertoire primarily aimed at facilitating mate finding and successful mating encounters .\",\n \"These behaviors include complex mating rituals ( Liu and Sternberg , 1995 ) , differential sensitivity to hermaphrodite pheromone ( Srinivasan et al . , 2008 ) , and a mate-searching behavior in adult male animals that is driven by the prioritization of mating over feeding ( Lipton et al . , 2004 ) .\",\n \"Mate-searching behavior of C . elegans has thus emerged as an interesting case study of decision making in a simple animal , which is subject to regulation by multiple features of internal state—sex , age , and nutritional status—as well as by external sensory experiences such as the presence of food or hermaphrodites in the environment ( Barrios et al . , 2008; Lipton et al . , 2004; Ryan et al . , 2014 ) .\",\n \"Here , we focus on the DAF-7/TGF-β signaling pathway , which plays a pivotal role in the regulation of diverse aspects of C . elegans physiology and behavior including the dauer developmental decision , reproductive egg laying , feeding and foraging , fat storage , satiety quiescence , longevity , aggregation , aerotaxis and avoidance of pathogenic bacteria ( Chang et al . , 2006; Gallagher et al . , 2013; Greer et al . , 2008; Meisel et al . , 2014; Milward et al . , 2011; Ren et al . , 1996; Shaw et al . , 2007; White and Jorgensen , 2012; You et al . , 2008 ) .\",\n \"The expression pattern of daf-7 was originally identified as being restricted to a single bilaterally symmetric pair of neurons in the head of the worm , the ASI chemosensory neurons , but it is also expressed in the ADE and OLQ neurons under normal laboratory growth conditions ( Meisel et al . , 2014; Ren et al . , 1996; Schackwitz et al . , 1996 ) .\",\n \"We recently defined an altered expression pattern for daf-7 in hermaphrodites in response to exposure to bacterial metabolites produced by the pathogenic bacteria Pseudomonas aeruginosa ( Meisel et al . , 2014 ) .\",\n \"Within minutes of being exposed to P . aeruginosa , daf-7 expression is induced in an additional pair of chemosensory neurons , the ASJ neurons , which in turn promotes behavioral avoidance of the pathogenic bacterial lawn ( Meisel et al . , 2014 ) .\",\n \"Here , we show that in male C . elegans , daf-7 expression in the ASJ neuron pair is not only induced in response to environmental cues such as Pseudomonas aeruginosa , but can be switched ‘on’ or ‘off’ depending on multiple internal states such as sex , age , and experience .\",\n \"The differential effects of internal states and environmental experience on the DAF-7 transcriptional switch in the two ASJ sensory neurons reveal a hierarchy among different inputs that can function to modulate behavior .\"\n ],\n [\n \"While expression of the DAF-7/TGF-β ligand was long thought to be confined to the ASI chemosensory neurons ( Ren et al . , 1996; Schackwitz et al . , 1996 ) , we recently reported that a change in the microbial environment of the worm—namely , the introduction of the pathogen P . aeruginosa—can alter the neuronal expression pattern of daf-7 , inducing expression in the ASJ neuron pair of hermaphrodite animals ( Meisel et al . , 2014 ) .\",\n \"In the current study , we observed that adult males exhibit daf-7 expression in both the ASI and the ASJ neuron pairs in the absence of P . aeruginosa ( Figure 1A and B ) .\",\n \"This expression pattern difference in adult C . elegans , can be observed both through the use of a pdaf-7::gfp transcriptional reporter ( Figure 1A and B ) as well as by fluorescence in situ hybridization ( FISH ) that directly probes for endogenous daf-7 mRNA ( Figure 1—figure supplement 1A–C ) . 10 . 7554/eLife . 21166 . 003Figure 1 . Sex-specific expression of daf-7 in the ASJ neurons of adult males .\",\n \"( A ) pdaf-7::gfp expression pattern in hermaphrodites ( left ) and males ( right ) .\",\n \"Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .\",\n \"Scale bar indicates 50 µm .\",\n \"( B ) Maximum fluorescence values of pdaf-7::gfp in the ASI ( left ) and ASJ ( right ) neurons of age-matched adult hermaphrodites ( grey ) and males ( black ) .\",\n \"*p<0 . 05 as determined by an unpaired t-test with Welch’s correction .\",\n \"Error bars indicate standard deviation ( SD ) .\",\n \"n . s . , not significant .\",\n \"( C ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of males through larval development and early adulthood .\",\n \"The developmental timeline of C . elegans is shown to scale on top .\",\n \"Time values indicate hours after an egg is laid .\",\n \"Error bars indicate SD .\",\n \"( D ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of both males and hermaphrodites on E . coli ( yellow ) or P . aeruginosa ( green ) .\",\n \"***p<0 . 001 as determined by unpaired t-tests with Welch’s correction .\",\n \"Error bars indicate SD . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00310 . 7554/eLife . 21166 . 004Figure 1—figure supplement 1 . daf-7 expression in the ASJ neuron pair is specific to adult males .\",\n \"( A–B )\",\n \"FISH images of endogenous daf-7 mRNA in adult hermaphrodite ( A ) and adult male ( B ) .\",\n \"White dotted lines indicate outline of the ASJ neuron ( s ) .\",\n \"Both images are a single slice through the animal where ptrx-1::gfp was visible and in focus in one or both ASJ neurons .\",\n \"Other neurons visible anterior to the ASI neurons are likely the OLQ neurons , and the cells posterior to the ASJ neurons that express daf-7 mRNA are likely the ADE neurons .\",\n \"Scale bar represents 25 µm .\",\n \"Orientation provided in ( A ) applies to both images .\",\n \"( C ) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons ( as localized by ptrx-1::gfp ) in males and hermaphrodites .\",\n \"***p<0 . 001 as determined by unpaired t-test with Welch’s correction .\",\n \"Error bars represent SD .\",\n \"( D ) pdaf-7::gfp expression pattern in an L4 larval male .\",\n \"Filled triangles indicate the ASI neurons .\",\n \"Scale bar ( shown in C ) indicates 50 µm .\",\n \"( E–F )\",\n \"Maximum fluorescence values of pdaf-7::gfp in the ASI ( E ) and ASJ ( F ) neurons of developing males .\",\n \"**p<0 . 01 , ***p<0 . 001 , as determined by unpaired t-test with Welch’s correction .\",\n \"Error bars represent SD .\",\n \"n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 004 Recent studies have suggested that , in addition to the generation of sex-specific neurons late in larval development , both male-specific and sex-shared neurons exhibit changes in gene expression as well as synaptic remodeling as the male undergoes the sexual maturation process ( Oren-Suissa et al . , 2016; Pereira et al . , 2015; Ryan et al . , 2014; Sammut et al . , 2015 ) .\",\n \"This predicts that a number of additional unidentified changes may occur in the anatomy , connectivity and gene expression profiles of the male nervous system as the animal transitions from larva to adult .\",\n \"Given this , we asked if daf-7 expression in males was correlated with the developmental stage of the animal .\",\n \"We observed that in populations of early larval animals carrying the pdaf-7::gfp transcriptional reporter , males and hermaphrodites were indistinguishable , with fluorescence observed only in the ASI neurons ( Figure 1—figure supplement 1D ) .\",\n \"To determine the onset of daf-7 expression in males , we imaged animals carrying the fluorescent reporter at multiple time points during development beginning in the L4 larval stage and extending into reproductive maturity .\",\n \"We found that daf-7 expression remains off in the ASJ of males through the end of the last ( L4 ) larval stage , but is switched on during young adulthood as the animals reach reproductive maturity ( Figure 1C ) .\",\n \"In addition , we examined expression of daf-7 in the ASI neuron pair at several matched timepoints during development and observed a significant drop in the expression of daf-7 in the ASI neurons from L4 to young adulthood ( Figure 1—figure supplements 1E and F , 52 hr and 64 . 5 hr timepoints ) .\",\n \"At subsequent timepoints during adulthood , however , expression levels in the ASI neurons remained constant as expression in the ASJ neurons increased ( Figure 1—figure supplements 1E , 3F 64 . 5 hr and 71 . 5 hr timepoints ) .\",\n \"We asked what the role of the genetic sex of the animal is in regulating daf-7 expression in the ASJ neurons .\",\n \"The C . elegans sex determination pathway terminates on the master regulator GLI-family transcription factor , TRA-1 ( Zarkower and Hodgkin , 1992; Zarkower , 2006 ) .\",\n \"TRA-1 activity functions to promote hermaphrodite cell fates and inhibit male development .\",\n \"We first looked at tra-1 ( e1099 ) mutants which are karyotypically XX , but develop as fertile pseudomales due to the lack of active TRA-1 ( Hodgkin and Brenner , 1977 ) .\",\n \"In these mutant XX males , daf-7 expression could be seen in both the ASI and ASJ neurons , suggesting that daf-7 is regulated by the genetic sex determination pathway either directly or indirectly ( Figure 2—figure supplement 1A ) .\",\n \"We next looked at the role of genetic sex specifically in the nervous system using transgenic sexual mosaics .\",\n \"TRA-1 is negatively regulated by the FEM proteins—FEM-1 , FEM-2 , and FEM-3—and it has been demonstrated by many groups that overexpression of FEM-3 cDNA is sufficient to lead to ‘masculinization’ in otherwise hermaphrodite animals ( Mehra et al . , 1999; Oren-Suissa et al . , 2016; Pereira et al . , 2015; Sammut et al . , 2015; White et al . , 2007 ) .\",\n \"We expressed the FEM-3 cDNA under the control of the pan-neural promoter , rab-3 , and found that daf-7 expression was up-regulated in the ASJ neurons of adult hermaphrodites , suggesting that a genetically ‘male’ nervous system is sufficient for daf-7 expression in ASJ ( Figure 2A , C and E ) .\",\n \"Although we observed an increase in daf-7 expression in these genetically masculinized animals , the overall expression of daf-7 in the ASJ of masculinized hermaphrodites was still notably lower than what we observe in wild-type males ( e . g . Figure 1B ) .\",\n \"This could possibly be explained by the lack of male-specific neurons in these transgenic animals .\",\n \"While genetic masculinization is effective in sexualizing neurons of the sex-shared nervous system , this technique fails to reliably retain the male-specific CEM neurons and produces hermaphrodites which also lack other male-specific neurons found in the tail ( White et al . , 2007 ) .\",\n \"To assess the importance of these male-specific neurons for daf-7 expression in ASJ , we examined pdaf-7::gfp fluorescence in ceh-30 ( n4289 ) , mab-3 ( e1240 ) , and pkd-2 ( sy606 ) ;lov-1 ( sy582 ) mutant males .\",\n \"While ceh-30 ( n4289 ) males lack the male-specific CEM neurons in the head ( Schwartz and Horvitz , 2007 ) , mab-3 ( e1240 ) mutants display marked defects in male-tail formation including ray differentiation as well as in male specific circuitry in the head of the animal ( Yi et al . , 2000 ) .\",\n \"Similarly , PKD-2 and LOV-1 constitute a TRP channel necessary for the function of the B-type ray neurons—another set of male-specific neurons in the tail of the animal ( Barr et al . , 2001; Barr and Sternberg , 1999; Barrios et al . , 2008 ) .\",\n \"Males of all mutant strains retained wild-type expression levels of daf-7 in ASJ , suggesting that the male-specific neural circuitry is not required for daf-7 expression and is unlikely to underlie the lower daf-7 expression we see in genetically masculinized hermaphrodites ( Figure 2—figure supplement 1B ) . 10 . 7554/eLife . 21166 . 005Figure 2 . Genetic sex regulates male-specific expression of daf-7 in the ASJ neuron pair .\",\n \"( A–D )\",\n \"Representative images of pdaf-7::gfp expression in: wild-type ( WT ) hermaphrodites ( A ) and males ( B ) , nervous-system-masculinized hermaphrodites ( C ) and nervous-system-feminized males ( D ) .\",\n \"Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .\",\n \"Scale bar indicates 50 µm .\",\n \"( E ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of control ( black ) and partially masculinized hermaphrodites ( purple ) .\",\n \"Masculinization was effected by driving expression of fem-3 cDNA under pan-neural ( left ) and ASJ-specific ( right ) promoters .\",\n \"***p<0 . 001 as determined by unpaired t-test with Welch’s correction .\",\n \"n . s . , not significant .\",\n \"Error bars represent SD .\",\n \"( F ) Maximum fluorescence values of pdaf-7::gfp in ASJ of control ( black ) and partially feminized males ( orange ) .\",\n \"Feminization was effected by driving expression of tra-2IC under pan-neural ( left ) , ciliated neuron ( middle ) , and ASJ-specific ( right ) promoters .\",\n \"***p<0 . 001 as determined by unpaired t-test with Welch’s correction .\",\n \"Error bars indicate SD . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00510 . 7554/eLife . 21166 . 006Figure 2—figure supplement 1 . daf-7 expression in the ASJ neuron pair is regulated by TRA-1 but not by male-specific neurons .\",\n \"( A ) Representative image of pdaf-7::gfp expression pattern in tra-1 ( e1099 ) pseudomale .\",\n \"Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .\",\n \"Scale bar represents 50 µm .\",\n \"( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of WT , ceh-30 ( n4289 ) , mab-3 ( e1240 ) , and pkd-2 ( sy606 ) ;lov-1 ( sy582 ) mutant animals .\",\n \"Significance determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"Error bars represent SD .\",\n \"n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 006 Complementary to masculinization , genetic ‘feminization’ of XO male animals can be achieved via the overexpression of the intracellular domain of the TRA-2 protein , which negatively interacts with FEM-3 to prevent TRA-1 degradation ( Mehra et al . , 1999 ) .\",\n \"Overexpression of TRA-2IC under the control of the rab-3 promoter led to complete loss of daf-7 expression in the ASJ of otherwise male animals ( Figure 2B , D and F ) .\",\n \"These results suggest that a genetically male nervous system is both necessary and sufficient for the sex-specific expression pattern of daf-7 .\",\n \"We next asked whether the sex determination pathway acts cell-autonomously in ASJ to regulate daf-7 expression .\",\n \"Using the trx-1 ASJ-specific promoter , we masculinized the ASJ neuron pair alone and saw no daf-7 expression in the resulting adult hermaphrodites ( Figure 2E ) .\",\n \"Similarly , no daf-7 expression could be observed when only the ciliated neurons ( of which ASJ is a member ) were masculinized ( data not shown ) .\",\n \"These data suggest the requirement that an additional neuron ( or neurons ) or other tissues have a male cell identity for daf-7 to be expressed in the ASJ neurons , although differences in the strength of the heterologous promoters used in the masculinization constructs may also account for these observations .\",\n \"In contrast , both ciliated neuron-specific and ASJ-specific feminization in males led to down-regulation of daf-7 expression , confirming that a male cell identity in ASJ is necessary , though not sufficient , for proper daf-7 expression ( Figure 2F ) .\",\n \"We next sought to determine the functional significance of the sex-specific changes in daf-7 expression that we observe in males .\",\n \"Mate-searching behavior is a behavioral program specific to adult male C . elegans , which features the prioritization of mating over feeding in these animals .\",\n \"While hermaphrodite animals prefer to remain in a patch of bacterial food indefinitely and exhibit limited exploratory behavior , males will wander away from a food source at a predictable and constant rate ( Lipton et al . , 2004 ) .\",\n \"This can be examined in an assay in which the movement of the animal is tracked over time to determine the leaving probability of any given genotype ( Figure 3A; Lipton et al . , 2004 ) .\",\n \"We observed that mutants in both the DAF-7/TGF-β ligand and its Type I receptor , DAF-1 , showed defects in the mate-searching assay ( Figure 3A and B , and Figure 3—figure supplement 1 ) .\",\n \"This behavioral defect in the daf-7 mutant can be fully suppressed by mutation of the downstream antagonistic co-SMAD , DAF-3 , suggesting that DAF-7 functions through its canonical TGF-β signaling pathway to regulate mate-searching behavior ( Figure 3B , and Figure 3—figure supplement 1B ) .\",\n \"Given the change in expression of daf-7 in the ASJ neurons of males at the transition to adulthood when this behavior is first displayed , we used genetic ablations of the ASJ neuron pair to ask what their contribution to this behavior might be .\",\n \"We observed that the ASJ-ablated animals were defective in mate-searching behavior , establishing a role for the ASJ neuron pair in promoting this behavior ( Figure 3C and Figure 3—figure supplement 1A ) .\",\n \"Importantly , the defects that we see in daf-7 mutant and ASJ-ablated male behavior are not accounted for by global defects in locomotion .\",\n \"When mutant animals are tested on leaving assay plates on which the bacterial food has been fully spread , these males reach the scoring distance at equivalent rates to WT controls ( Figure 3—figure supplement 2; Barrios et al . , 2012 ) .\",\n \"This suggests that the reduced leaving behavior we observe in these animals isspecific to the decision to leave the food source in search of a mate . 10 . 7554/eLife . 21166 . 007Figure 3 . DAF-7/TGF-β is required for male mate-searching behavior .\",\n \"( A ) Schematic of mate-searching assay ( top ) .\",\n \"Animals are placed individually into the center of a lawn of bacteria .\",\n \"The tracks of the animal are followed over time and scored for movement beyond 3 cm away from the food source .\",\n \"A representative data curve is shown on the bottom depicting hermaphrodites ( solid black ) , males ( dashed black ) , and daf-7 ( e1372 ) mutant males ( blue ) .\",\n \"( B ) Probability of leaving values for WT , daf-7 mutant , and daf-7;daf-3 double mutant males .\",\n \"Values plotted are the average + SEM for two independent experiments , n = 40 animals for all strains except daf-7 ( ok3125 ) where n = 29 .\",\n \"*p<0 . 05 , **p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"n . s . , not significant .\",\n \"( C ) Probability of leaving values for two independent lines of male animals with genetic ablation of the ASJ neurons , compared with corresponding values for WT and daf-7 mutant males .\",\n \"Values plotted are the average + SEM for two independent experiments for daf-7 mutant animals and three independent experiments for WT control and ASJ ablation strains .\",\n \"n = 60 animals for all strains except daf-7 ( ok3125 ) where n = 29 animals .\",\n \"**p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"One replicate was performed with the daf-7;daf-3 double mutant strains in 1B and controls ( WT and daf-7 ) are identical , so probability values for that experiment were used in the averages shown in both B and C of this figure .\",\n \"( D ) Probability of leaving values for daf-7 ( ok3125 ) mutant animals carrying transgenes expressing daf-7 cDNA specifically under the control of the indicated promoters .\",\n \"Values on graph are the average + SEM of two independent experiments with an n = 40 total animals for each strain .\",\n \"**p<0 . 01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00710 . 7554/eLife . 21166 . 008Figure 3—figure supplement 1 . daf-7 regulates mate-searching behavior in males .\",\n \"( A ) Representative mate-searching data for strains with genetic ablation of the ASJ neurons ( shown in shades of orange ) .\",\n \"WT hermaphrodites ( solid black ) , WT males ( dashed black ) , and daf-7 mutant ( blue ) animals are included in each assay as controls .\",\n \"( B ) Representative data for daf-7;daf-3 double mutant ( green ) in the mate-searching assay .\",\n \"( C ) Representative data for daf-7 rescue strains ( shown in shades of red ) .\",\n \"( D ) Representative mate-searching curves for daf-1 mutants ( purple ) .\",\n \"( E ) Probability of leaving values for WT , daf-7 , and daf-1 mutant animals .\",\n \"Values shown are the average + SEM of two independent experiments .\",\n \"n = 40 for WT and daf-7 mutants , n = 32 for daf-1 ( e1287 ) and n = 36 for daf-1 ( m402 ) .\",\n \"*p<0 . 05 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 00810 . 7554/eLife . 21166 . 009Figure 3—figure supplement 2 . daf-7 mutant animals and strains carrying genetic ablation of the ASJ neurons exhibit no locomotion defect .\",\n \"( A ) Schematic of locomotion assay .\",\n \"E . coli food ( shown in yellow ) was spread to the edges of the plate and a standard mate searching assay performed to determine animal movement on a full lawn of bacteria .\",\n \"( B–C )\",\n \"Raw data curves for daf-7 ( blue ) and ASJ-ablation ( orange ) males in locomotion assay .\",\n \"WT males shown in black for reference . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 009 Since DAF-7 is a secreted ligand , we anticipated that overexpression of DAF-7 from either the ASI or ASJ neurons would be sufficient to rescue daf-7-associated mutant phenotypes .\",\n \"As expected , we saw that transgenic rescue from the endogenous promoter as well as from ASI- and ASJ-specific promoters were sufficient to restore mate-searching behavior in the daf-7 ( ok3125 ) mutant background ( Figure 3D and Figure 3—figure supplement 1C ) .\",\n \"Taken together , these data imply that the dynamic expression of daf-7 in the ASJ neurons of adult males regulates mate-searching behavior .\",\n \"Mate-searching behavior has been demonstrated to be dependent on the feeding state of the animal—starved male animals will re-prioritize feeding over mate searching for a limited period of time until the animal has had sufficient opportunity to re-feed ( Figure 4C; Lipton et al . , 2004 ) .\",\n \"We asked whether daf-7 expression in the male ASJ neurons might be dependent on the nutritional state of the animal or the bacterial availability in its environment .\",\n \"We found that males subjected to starvation beginning early in the L4 larval stage failed to express daf-7 in the ASJ neurons as adults ( Figure 4A , conditions i and ii , and Figure 4B ) .\",\n \"This lack of daf-7 in the ASJ neuron pair was apparent both by GFP visualization as well as by FISH , with daf-7 mRNA entirely absent from the ASJ neurons ( Figure 4A and B ) .\",\n \"We noticed that daf-7 mRNA was still apparent in the ASI , OLQ , and ADE neurons of these starved animals , suggesting that the loss of daf-7 expression in the ASJ neurons is specific and not representative of a global down-regulation of transcription in these animals ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 21166 . 010Figure 4 . Nutritional state regulates daf-7 expression in the ASJ neurons of males .\",\n \"( A ) Schematic of experiment design indicating timing and duration of starvation periods ( left ) .\",\n \"FISH images of endogenous daf-7 mRNA in the ASJ neurons for each experimental condition ( right ) .\",\n \"White dotted lines indicate outline of ASJ cell body as localized by ptrx-1::gfp .\",\n \"Scale bar represents 5 µm .\",\n \"( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons during re-feeding experiment .\",\n \"Re-fed animals were starved for a period of 24 hr before being reintroduced to E . coli for the indicated amount of time .\",\n \"***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"Error bars indicate SD .\",\n \"n . s . , not significant .\",\n \"( C ) Representative curves of mate-searching data in fed and post-starvation WT males .\",\n \"Animals were starved for a period of 24 hr as in previous experiments before being assayed for mate-searching behavior . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 01010 . 7554/eLife . 21166 . 011Figure 4—figure supplement 1 . daf-7 expression is down-regulated by starvation specifically in the ASJ neurons .\",\n \"( A ) Representative FISH image of endogenous daf-7 mRNA in a starved adult male .\",\n \"White dotted line indicates outline of an ASJ neuron .\",\n \"Closed triangle indicates an ASI neuron .\",\n \"Other neurons visible anterior to the ASI neuron are likely the OLQ neurons , and posterior to the ASJ neuron are likely the ADE neurons .\",\n \"Scale bar represents 25 µm .\",\n \"( B ) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons ( as localized by ptrx-1::gfp ) of males fed and starved at various different points in their lives .\",\n \"***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"Error bars represent SD .\",\n \"n . s . , not significant .\",\n \"( C ) Fluorescence quantification for daf-7-Cy5 probe in the ASI neurons of starved and fed males .\",\n \"Significance determined by unpaired t-test with Welch’s correction .\",\n \"Error bars represent SD .\",\n \"n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 011 We next asked if daf-7 expression could be dynamically modulated by removal and re-introduction of food at various points during the life of the male .\",\n \"We first looked at daf-7 expression in animals that had experienced starvation during the L4-to-adulthood period before being returned to an E . coli food source as adults .\",\n \"In these animals , daf-7 expression could be detected both by the GFP reporter as well as by FISH ( Figure 4A , condition iii , and 4B ) .\",\n \"We asked whether fully mature adult animals would turn off daf-7 expression in response to starvation after having already induced expression of daf-7 in the ASJ neuron pair .\",\n \"For these experiments , we utilized FISH of the endogenous daf-7 mRNA because perdurance of the GFP reporter might complicate analysis .\",\n \"When males were allowed to develop into adulthood unperturbed and then subjected to a period of food deprivation , we observed a complete loss of daf-7 mRNA in the ASJ neurons ( Figure 4A , condition iv ) .\",\n \"Together this data demonstrates that daf-7 expression in the ASJ neurons is subject to dynamic regulation by periods of starvation and re-feeding .\",\n \"These data suggest two distinct possibilities for how bacterial food levels in the environment modulate daf-7 transcriptional regulation in the ASJ neuron pair of males .\",\n \"First , daf-7 transcription could be regulated by the internal nutritional state of the animal ( starved or fed ) .\",\n \"Alternatively , daf-7 transcription could be activated by the chemosensation ( smell or taste ) of bacterial food .\",\n \"To distinguish between these two possibilities , we measured the kinetics of daf-7 expression in response to re-feeding in starved animals .\",\n \"We observed that daf-7 expression in the ASJ neuron pair is not significantly increased compared to starved controls until 9 hr after the re-introduction of an E . coli food source ( Figure 4B ) .\",\n \"Although some animals could be observed expressing daf-7 weakly in the ASJ neuron pair at 4 hr of re-feeding , the majority of animals still exhibited no pdaf-7::gfp expression in the ASJ neuron pair until later time points .\",\n \"The extended period of time required to restore daf-7 expression in the ASJ neuron pair suggests that internal nutritional state , not the chemosensation of bacterial food , exerts control over daf-7 transcription in the ASJ neuron pair .\",\n \"Moreover , we observed that the onset of daf-7 expression in the ASJ neurons of males following the re-introduction of food are consistent with the previously noted kinetics of resumption of mate-searching behavior after males have been starved ( Figure 4C; Lipton et al . , 2004 ) .\",\n \"We recently showed that the microbial environment is an important regulator of daf-7 expression in the ASJ neurons in hermaphrodite animals ( Figure 1D; Meisel et al . , 2014 ) .\",\n \"Given that males induce expression of daf-7 in the same ASJ neuron pair independently of the bacterial food source , we sought to determine if they would be capable of responding to exposure to P . aeruginosa in the same way as their hermaphrodite siblings—namely , through additional up-regulation of daf-7 expression in the ASJ neurons .\",\n \"We observe that , when transferred to P . aeruginosa , males carrying the pdaf-7::gfp transcriptional reporter exhibited an increase in daf-7 expression in the ASJ neurons compared to E . coli-fed controls ( Figure 1D ) .\",\n \"The additive nature of DAF-7 levels in males suggests that the ASJ neuron pair of males retain the ability to respond to P . aeruginosa that we observe in hermaphrodites .\",\n \"Our data suggest that daf-7 expression in the ASJ neurons is subject to regulation by both sensory cues from the microbial environment as well as by internal state sensing mechanisms .\",\n \"Because daf-7 expression in the ASJ neuron pair and mate-searching behavior in males are dependent on nutritional state during feeding on the non-pathogenic bacterial food , E . coli , we sought to determine if expression of daf-7 in response to P . aeruginosa might also be dependent on the nutritional state in males .\",\n \"We tested this by challenging starved animals with E . coli or P . aeruginosa to see how daf-7 expression would be affected and to observe any behavioral differences .\",\n \"Looking at animals carrying the pdaf-7::gfp reporter during post-starvation re-feeding , we observed a rapid up-regulation of daf-7 expression in the ASJ neurons of starved males returned to P . aeruginosa as a food source ( Figure 5A and B , and Figure 5—figure supplement 1E ) .\",\n \"This increase in daf-7 expression in the ASJ neuron pair could be seen as early as 2 hr after first exposure to the pathogenic bacteria ( Figure 5—figure supplement 1E ) .\",\n \"In contrast , starved males that were returned to the non-pathogenic E . coli showed little to no daf-7 expression in the ASJ neurons until much later time points ( Figure 5A , B and 4B ) .\",\n \"The kinetics of the increase in fluorescence of the pdaf-7::gfp reporter in response to P . aeruginosa secondary metabolites , which lags behind the immediate response observed by FISH is consistent with what we observed previously for hermaphrodite animals ( Meisel et al . , 2014 ) , and notably faster than the slower increase in pdaf-7::gfp reporter fluorescence in response to repletion of the non-pathogenic E . coli bacterial food . 10 . 7554/eLife . 21166 . 012Figure 5 . Prioritization of multiple inputs through the regulation of daf-7 expression in the ASJ neurons of males .\",\n \"( A ) Schematic of refeeding experimental design indicating timing and duration of each starving and feeding period ( left ) .\",\n \"On right , representative images of daf-7 expression following 4 hr of post-starvation refeeding on E . coli ( left ) or P . aeruginosa ( right ) .\",\n \"Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons .\",\n \"Scale bar represents 50 µm .\",\n \"( B ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced either to the normal E . coli food source or to pathogenic P . aeruginosa .\",\n \"Images were taken for quantification after 4 hr of re-feeding .\",\n \"***p<0 . 001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"Error bars indicate SD .\",\n \"n . s . , not significant .\",\n \"( C ) Representative curve of food-leaving data in starved animals on E . coli ( yellow ) and P . aeruginosa ( green ) .\",\n \"Animals were starved as in previous experiments before being run in the assay . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 01210 . 7554/eLife . 21166 . 013Figure 5—figure supplement 1 . P .\",\n \"aeruginosa influences exploratory behavior and daf-7 expression in both males and hermaphrodites .\",\n \"( A ) Representative leaving curves for males ( dashed ) and hermaphrodites ( solid ) on E . coli ( shades of grey ) and P . aeruginosa ( shades of green ) .\",\n \"All strains were fed in this experiment .\",\n \"( B ) Probability of leaving values for males and hermaphrodites on E . coli and P . aeruginosa .\",\n \"All strains were fed in these experiments .\",\n \"Values plotted are the mean + SEM for two independent experiments , n = 40 animals for all conditions .\",\n \"*p<0 . 05 as determined by unpaired t-test with Welch’s correction .\",\n \"n . s . , not significant .\",\n \"( C ) Representative raw data curves for starved hermaphrodites on E . coli and P . aeruginosa .\",\n \"Fed hermaphrodites shown as control .\",\n \"( D ) Maximum fluorescence of pdaf-7::gfp in the ASJ neurons of starved hermaphrodites re-fed E . coli or P . aeruginosa for four hours ( compare to Figure 5B ) .\",\n \"Error bars indicate SD .\",\n \"( E ) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced to P . aeruginosa for the indicated amount of time .\",\n \"The experiment in Figure 4B was run in parallel , controls ( fed and starved ) were shared between conditions .\",\n \"These controls are shown in both figures for comparison purposes .\",\n \"*p<0 . 05 and ***p<0 . 001 , as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test .\",\n \"Error bars indicate S . D . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 013 We followed up by asking if this difference in expression after starvation is also reflected in behavioral differences of post-starvation animals experiencing different microbial environments .\",\n \"Normally , following a period of starvation , a male prefers feeding over leaving a lawn of bacteria in search of a mate ( Figure 4C; Lipton et al . , 2004 ) , but we reasoned that these priorities might be re-set if the only available food is pathogenic to the starved animal .\",\n \"Indeed , whereas starved males will remain on the E . coli lawn for several hours , starved males who are instead returned to a lawn of P . aeruginosa will readily leave the bacterial lawn at a rate similar to non-starved animals on the normal E . coli food source ( Figures 4C and 5C ) .\",\n \"This lawn-leaving behavior is not male-specific , as both fed and starved hermaphrodites will also readily leave a lawn of P . aeruginosa and starved hermaphrodites are also capable of responding to P . aeruginosa through an upregulation of daf-7 in the ASJ neurons ( Figure 5—figure supplement 1A–1D ) .\",\n \"In this regard , one might consider that lawn-leaving under these conditions is driven by the presence of pathogenic P . aeruginosa , and not mate-searching .\",\n \"Our data suggest that daf-7 expression in the ASJ neurons is one way through which animals are both able to interpret internal and external cues and prioritize their behaviors accordingly .\"\n ],\n [\n \"We have presented a set of experiments that suggest that behavioral prioritization and decision-making may be effected through the transcriptional control of a key neuroendocrine regulator , the TGF-β ligand , DAF-7 , in a pair of chemosensory neurons , the ASJ neurons .\",\n \"Our data suggest that daf-7 expression in the ASJ neuron pair is utilized to promote exploratory behaviors in different physiological and ecological contexts .\",\n \"In hermaphrodites , expression of daf-7 is induced by the detection of secondary metabolites and functions to allow animals to discriminate between microbial species and promote avoidance of pathogenic bacteria ( Meisel et al . , 2014 ) .\",\n \"In essence , pathogen avoidance represents the behavioral choice an animal makes when confronted with two competing needs—the need to eat and remain well fed and the need to avoid danger and infection .\",\n \"Mate searching presents a paralagous but sex-specific behavior , where the decision instead is between food and mating .\",\n \"Our data suggest that modulation of daf-7 expression provides a striking example of how a neuroendocrine signaling pathway may be coopted to guide behavioral decisions in entirely different contexts .\",\n \"Our study suggests that internal states and environmental cues that converge on the transcriptional control of DAF-7 in the ASJ neuron pair are processed in a hierarchical manner that , in turn , serves to modulate the exploratory mate-searching behavior in the male ( Figure 6 ) .\",\n \"The work presented here suggests that the transcriptional regulation of daf-7 in the ASJ neurons functions as an ON/OFF switch , where each input holds the switch—and thus , daf-7 expression—in one of two states .\",\n \"However , the various regulatory inputs into this switch exert differential control , with environmental cues being prioritized over all others .\",\n \"This differential control of the transcriptional switch , in turn allows for re-adjustments of behavioral priorities in the worm based on its current and past experience . 10 . 7554/eLife . 21166 . 014Figure 6 . A hierarchy of inputs regulates daf-7 expression in the ASJ neurons to modulate exploratory behaviors . We show that the information from many different sources both internal and external is integrated hierarchically in the regulation of daf-7 expression in the ASJ neuron pair .\",\n \"This hierarchical regulation of daf-7 in turn leads to a distinct prioritization of exploratory behaviors in the male worm . DOI: http://dx . doi . org/10 . 7554/eLife . 21166 . 014 Interestingly , dynamic transcriptional regulation has previously been implicated in the regulation of sex-specific decision-making behavior in C . elegans .\",\n \"Expression of the chemoreceptor , ODR-10 , in the sex-shared sensory AWA neurons has also been shown to be dependent on both internal and external cues to regulate food-related decision making in male C . elegans ( Ryan et al . , 2014 ) .\",\n \"Given the similarities in the regulation of these two genes by both sex and satiety , it is possible that DAF-7 and ODR-10 function in a single pathway to regulate decision-making behaviors in the male .\",\n \"Previous studies of daf-7 have suggested that it is involved in regulating the expression of numerous chemoreceptors in many different neuron types ( Nolan et al . , 2002 ) .\",\n \"While no interaction has been identified between daf-7 and odr-10 in the hermaphrodite nervous system , there has been no examination as yet of the effects of daf-7 mutation on odr-10 in the relevant cell types , sex , or under different food conditions , which may be an interesting line of future research .\",\n \"DAF-7/TGF-β has previously been found to be involved in the regulation of other satiety-regulated behaviors such as quiescence and foraging ( Gallagher et al . , 2013; Milward et al . , 2011; You et al . , 2008 ) .\",\n \"daf-7 mutants show marked defects in the ability to respond to fasting through an increase in quiescence , suggesting that DAF-7 may serve as a critical signal of food availability in the worm ( You et al . , 2008 ) .\",\n \"Similarly , DAF-7 signaling has also been implicated in promoting food-leaving ( or foraging ) in the context of a rapidly depleting food source , further underscoring the importance of this pathway for communicating nutritional availability ( Milward et al . , 2011 ) .\",\n \"While DAF-7 from the ASI neurons , and not the ASJ neurons , contributes to these other possibly related phenotypes , similar mechanisms may be involved in regulating daf-7 expression and release in these three distinct food/satiety-dependent assay contexts .\",\n \"Taken together with our data , the important role of daf-7 in these different behavioral paradigms suggests that the daf-7 pathway may act as a more global food signal that is critical for allowing the animal to sense food and satiety and modulate its behaviors accordingly .\",\n \"Finally , our results support a model where integration of internal and external sensory information can occur at the level of the sensory neuron itself to regulate behavioral prioritization .\",\n \"Our data on the role of the ASJ sensory neurons in male mate-searching behavior are consistent with emerging evidence from studies in several species to suggest that the regulatory control of sensory neurons contributes to information processing and decision making behavior ( Dey et al . , 2015; Lebreton et al . , 2015; Peckol et al . , 2001; Ryan et al . , 2014 ) .\"\n ],\n [\n \"C . elegans were grown on E . coli OP50 as a food source as previously described ( Brenner , 1974 ) .\",\n \"Strains were grown at 20°C except for assays containing dauer-constitutive mutants , in which case , all strains were grown at 16°C until they passed the dauer developmental decision .\",\n \"Unless otherwise indicated , all strains carry the him-5 ( e1490 ) mutation to increase the number of spontaneous males available .\",\n \"For a complete list of strains used in this study , please see Supplementary file\",\n \"1 . For quantification of daf-7 expression in mutants and developing animals , animals were egg laid for 2 hr on OP50 to obtain synchronized populations of animals .\",\n \"The animals were then mounted and anesthetized in 50 mM sodium azide at the indicated hour of development or at 72 hr if not indicated .\",\n \"The animals were imaged at 40x using a Zeiss Axioimager Z1 microscope .\",\n \"15–20 animals were imaged for each condition or strain .\",\n \"For quantification , maximum intensity values of GFP within the ASJ neurons was calculated using FIJI software ( Schindelin et al . , 2012 ) ; Fiji , RRID:SCR_002285 ) .\",\n \"For P . aeruginosa experiments , the P . aeruginosa PA14 strain was grown overnight at 37°C in 3 mL of LB as previously described and the next day 7 µL of culture was seeded onto SKA plates ( Tan et al . , 1999 ) .\",\n \"Plates were incubated at 37°C overnight and then allowed to grow for an additional two days at room temperature before being used for experiments .\",\n \"Animals for P . aeruginosa experiments were synchronized by treatment with bleach and then allowed to hatch and arrest as L1 larvae in M9 media overnight .\",\n \"This provided synchronized populations free from any possible bacterial contamination .\",\n \"Animals were transferred to PA14 plates or OP50 controls at the L4 larval stage .\",\n \"Imaging and quantification were performed as described above at 16 hr after transfer to PA14 plates .\",\n \"To generate the neural feminization and masculinization transgenes , the pGH8 plasmid containing the pan-neural rab-3 promoter along with mCherry::unc-54 3’UTR was linearized by PCR .\",\n \"The fem-3 and intracellular tra-2 cDNAs were amplified from cDNA generated with an Ambion RETROScript kit .\",\n \"The unc-54 3’UTR was amplified from the pPD95 . 75 vector and the SL2 trans-splicing sequence leader ( Huang et al . , 2001; Spieth et al . , 1993 ) was amplified from genomic DNA .\",\n \"The fem-3 or tra-2 cDNA along with the unc-54 3’UTR and SL2 sequence were cloned into the linearized pGH8 vector by Gibson assembly ( Gibson et al . , 2009 ) .\",\n \"For cell specific manipulations , the ciliated neuron bbs-1 promoter ( 1 . 9 kb ) and ASJ specific trx-1 promoter ( 1 . 1 kb; Fierro-González et al . , 2011 ) were amplified from genomic DNA and inserted in the place of the rab-3 promoter by Gibson assembly .\",\n \"All plasmids were sequenced to confirm identity and injected into ksIs2;him-5 ( e1490 ) animals at a concentration of 50 ng/µL , along with a plasmid carrying ofm-1::gfp ( 50 ng/µL ) .\",\n \"At least three independent transgenic lines were obtained and analyzed for each construct and one representative line is shown .\",\n \"For a complete list of primers used in this paper , please see Supplementary file\",\n \"2 . Mate-searching behavior was assessed in animals through a food leaving assay as previously described ( See diagram in Figure 3A; Lipton et al . , 2004 ) .\",\n \"Briefly , animals were placed individually onto a 1 cm lawn ( ~20 µL of culture ) of E . coli OP50 in the center of a 10 cm plate .\",\n \"Movement of each animal was tracked at hourly time points .\",\n \"When an animal’s tracks extended a distance of 3 cm away from the starting lawn , the animal was scored as a ‘leaver’ .\",\n \"It has been previously demonstrated that male leaving behavior can be modeled by the single exponential decay function N ( t ) /N ( 0 ) =exp ( -λt ) , where N ( t ) refers to the number of non-leavers at a given time and the hazard value ( λ ) gives an estimate for the probability of leaving , which remains constant over the course of an assay ( Lipton et al . , 2004 ) .\",\n \"Hazard values were calculated by fitting data using the R survival package with an exponential parametric survival model using maximum likelihood ( R Project for Statistical Computing , RRID:SCR_001905 ) .\",\n \"These values were pooled across replicates .\",\n \"At least two independent experiments were performed for each genotype ( ~40 animals per genotype ) .\",\n \"For leaving assays with P . aeruginosa , plates were supplemented with additional peptone and were seeded with 15 µL of bacterial culture .\",\n \"In addition , all plates for PA14 assays were incubated overnight at 37°C after being seeded with bacteria and then for an additional 2 days at room temperature before being used for the assay .\",\n \"For locomotion assays , 10 cm mate-searching plates were seeded with ~400 µL of E . coli OP50 culture spread to the edge of the plate .\",\n \"Plates were allowed to dry and remained at room temperature for ~16 hr before being used in the assay .\",\n \"Males were placed individually on these plates and their movement tracked over time as in the standard mate-searching assay .\",\n \"The same scoring distance was used for locomotion assays as for the mate-searching assay .\",\n \"FISH was performed as described previously ( Raj et al . , 2008 ) .\",\n \"Animals were fixed in 4% formaldehyde for 45 min at 20°C .\",\n \"Following washing in PBS , animals were resuspended in 70% ethanol and incubated overnight at 4°C .\",\n \"Samples were then hybridized with probes to the endogenous daf-7 mRNA as previously published ( Meisel et al . , 2014 ) .\",\n \"Hybridization was carried out at 30°C overnight .\",\n \"Imaging was performed using a Nikon Eclipse Ti Inverted microscope with a Princeton Instruments PIXIS 1024 camera .\",\n \"All data were analyzed in FIJI .\",\n \"For quantifications of FISH data , fluorescence values were calculated within the ASJ ( or ASI ) neurons for the indicated conditions .\",\n \"All samples were hybridized using daf-7-Cy5 probes and imaged at an exposure time of 1500 ms . For all FISH experiments , animals carrying the ofEx4[ptrx-1::gfp] transgene were used in order to localize the ASJ neurons .\",\n \"For all starvation experiments , animals were synchronized by treatment with bleach and then were allowed to hatch and arrest as L1s in M9 media for 14 hr .\",\n \"After 14 hr , animals were dropped onto plates containing abundant OP50 E . coli and were allowed to develop for 34–36 hr at 20°C .\",\n \"At this time point , animals had already entered the L4 larval stage .\",\n \"All animals were washed from the plates and subjected to an additional 4–5 washes with M9 media to remove any remaining bacteria .\",\n \"Animals were placed either back onto plates seeded with E . coli or were dropped onto plates lacking peptone to inhibit any bacterial growth .\",\n \"For re-feeding experiments , animals were starved on peptone free plates for 24 hr at 20°C .\",\n \"After 24 hr of starvation , nearly all of the male animals had molted into adults as evident by tail and gonadal morphology .\",\n \"Only animals with adult morphology were used for experiments and imaging .\",\n \"They were subsequently collected and placed onto plates seeded with either E . coli or P . aeruginosa for the designated amount of time .\",\n \"For the FISH experiment shown in Figure 3 , animals were re-fed on E . coli OP50 for 16 hr at 20°C before being fixed for imaging .\",\n \"For the late starve experiments , animals dropped back onto E . coli after the washing step were allowed to grow for an additional 24 hr at 20°C before being collected and washed 4–5 times in M9 media .\",\n \"These animals were then placed onto peptone-free plates and starved for 16 hr at 20°C before being fixed for imaging .\",\n \"All statistical analysis was performed using the GraphPad Prism software ( Graphpad Prism , RRID:SCR_002798 ) .\",\n \"Statistical tests used are indicated in each figure legend .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["Animal behavior is directed by the integration of sensory information from internal states and the environment .","Neuroendocrine regulation of diverse behaviors of Caenorhabditis elegans is under the control of the DAF-7/TGF-β ligand that is secreted from sensory neurons .","Here , we show that C . elegans males exhibit an altered , male-specific expression pattern of daf-7 in the ASJ sensory neuron pair with the onset of reproductive maturity , which functions to promote male-specific mate-searching behavior .","Molecular genetic analysis of the switch-like regulation of daf-7 expression in the ASJ neuron pair reveals a hierarchy of regulation among multiple inputs—sex , age , nutritional status , and microbial environment—which function in the modulation of behavior .","Our results suggest that regulation of gene expression in sensory neurons can function in the integration of a wide array of sensory information and facilitate decision-making behaviors in C . elegans ."],"string":"[\n \"Animal behavior is directed by the integration of sensory information from internal states and the environment .\",\n \"Neuroendocrine regulation of diverse behaviors of Caenorhabditis elegans is under the control of the DAF-7/TGF-β ligand that is secreted from sensory neurons .\",\n \"Here , we show that C . elegans males exhibit an altered , male-specific expression pattern of daf-7 in the ASJ sensory neuron pair with the onset of reproductive maturity , which functions to promote male-specific mate-searching behavior .\",\n \"Molecular genetic analysis of the switch-like regulation of daf-7 expression in the ASJ neuron pair reveals a hierarchy of regulation among multiple inputs—sex , age , nutritional status , and microbial environment—which function in the modulation of behavior .\",\n \"Our results suggest that regulation of gene expression in sensory neurons can function in the integration of a wide array of sensory information and facilitate decision-making behaviors in C . elegans .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["For almost all species of animal , males and females will often behave differently in similar situations .","Little is known about how these sex-specific differences are generated or , for example , how different the nervous system of a male is to that of a female .","Moreover , it is also poorly understood how these underlying differences based on the biological sex of an animal are integrated with and influenced by its experiences and environment .","The roundworm Caenorhabditis elegans has two sexes , hermaphrodites and males .","The male worms behave differently to the hermaphrodites in a number of situations .","This means that these animals offer the opportunity to explore and understand sex-specific differences in behavior .","It is also possible to analyze the underlying factors that contribute to behavior in C . elegans , because it has a relatively simple and well-defined nervous system .","Now , Hilbert and Kim show that a signal that influences how C . elegans explores in response to chemicals in its environment is expressed differently in male and hermaphrodite worms .","The signal in question is molecule called DAF-7 , which is released by several sensory neurons—nerve cells that are used for detecting cues from the environment .","The sensory neurons that release DAF-7 are found in both sexes of C . elegans but the specific way that the male worms express this signal encourages them to search for mates .","Hermaphrodites , on the other hand , do not need to search for mates because they can fertilize their own eggs .","Hilbert and Kim showed that the biological sex in combination with multiple other inputs – including the animal’s past diet and age – regulate how the DAF-7 signal is expressed in C . elegans .","These inputs all converge onto a single pair of sensory neurons , which integrate the inputs and enable the worm to assess its current and past experiences and alter its behavior accordingly .","Moving forward the next challenge is to understand how information about both external environment and internal states , such as hunger , are communicated to and integrated by these sensory neurons .","Decoding the signals behind this process may illuminate how biological sex and internal states influence behavior in other species of animals ."],"string":"[\n \"For almost all species of animal , males and females will often behave differently in similar situations .\",\n \"Little is known about how these sex-specific differences are generated or , for example , how different the nervous system of a male is to that of a female .\",\n \"Moreover , it is also poorly understood how these underlying differences based on the biological sex of an animal are integrated with and influenced by its experiences and environment .\",\n \"The roundworm Caenorhabditis elegans has two sexes , hermaphrodites and males .\",\n \"The male worms behave differently to the hermaphrodites in a number of situations .\",\n \"This means that these animals offer the opportunity to explore and understand sex-specific differences in behavior .\",\n \"It is also possible to analyze the underlying factors that contribute to behavior in C . elegans , because it has a relatively simple and well-defined nervous system .\",\n \"Now , Hilbert and Kim show that a signal that influences how C . elegans explores in response to chemicals in its environment is expressed differently in male and hermaphrodite worms .\",\n \"The signal in question is molecule called DAF-7 , which is released by several sensory neurons—nerve cells that are used for detecting cues from the environment .\",\n \"The sensory neurons that release DAF-7 are found in both sexes of C . elegans but the specific way that the male worms express this signal encourages them to search for mates .\",\n \"Hermaphrodites , on the other hand , do not need to search for mates because they can fertilize their own eggs .\",\n \"Hilbert and Kim showed that the biological sex in combination with multiple other inputs – including the animal’s past diet and age – regulate how the DAF-7 signal is expressed in C . elegans .\",\n \"These inputs all converge onto a single pair of sensory neurons , which integrate the inputs and enable the worm to assess its current and past experiences and alter its behavior accordingly .\",\n \"Moving forward the next challenge is to understand how information about both external environment and internal states , such as hunger , are communicated to and integrated by these sensory neurons .\",\n \"Decoding the signals behind this process may illuminate how biological sex and internal states influence behavior in other species of animals .\"\n]","isConversational":false},"year":{"kind":"string","value":"2017"}}},{"rowIdx":299,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["developmental biology","cell biology"],"string":"[\n \"developmental biology\",\n \"cell biology\"\n]","isConversational":false},"title":{"kind":"string","value":"TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice"},"id":{"kind":"string","value":"elife-03464-v1"},"sections":{"kind":"list like","value":[["Chronic proliferative dermatitis mutation ( cpdm ) mice are deficient in SHARPIN ( Sharpincpdm/cpdm: henceforth referred to as Shpnm/m; protein: SHARPIN ) and develop dermatitis , multi-organ pathology and an immunological phenotype including disrupted lymphoid architecture , splenomegaly , liver inflammation and a loss of Peyer's patches in the gut ( HogenEsch et al . , 1993 , 1999; Seymour et al . , 2007 ) .","SHARPIN is required for normal tumour necrosis factor ( TNF ) receptor 1 ( TNFR1 ) -mediated gene induction and prevention of TNF-mediated death of various cells , including epidermal keratinocytes , in vitro ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) .","The dermatitis is characterized by epidermal cell death marked by cleaved caspase-3- , -8- and -9-positive cells ( Ikeda et al . , 2011; Liang and Sundberg , 2011; Potter et al . , 2014 ) .","Since the dermatitis and inflammatory phenotype were shown to be TNF dependent , and because the only TNF signaling output that was aberrantly increased in the absence of SHARPIN was cell death , we previously proposed TNF/TNFR1-mediated cell death to be causative of the cpdm phenotype ( Gerlach et al . , 2011 ) .","The role of neither TNFR1 nor cell death has been confirmed in vivo , however .","TNFR1 signaling typically involves the intracellular recruitment of TNFR1-associated death domain protein ( TRADD ) , TNF receptor-associated factor 2 ( TRAF2 ) , cellular inhibitor of apoptosis ( cIAPs ) , and receptor interacting protein kinase 1 ( RIPK1 ) ( Silke , 2011 ) .","The heterotrimeric linear ubiquitin chain assembly complex ( LUBAC ) of SHARPIN ( also known as SIPL ) , HOIL-1 ( RBCK1/RNF54 ) and HOIL-1L-interacting protein ( HOIP; RNF31 ) ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) is also recruited to the TNFR1 signaling complex .","Here , it assembles a linear ubiquitin scaffold needed for full recruitment of the NF-κB essential modulator ( NEMO ) /NF-κB kinase subunit gamma ( IKKγ ) -containing IKK complex , which activates pro-survival NF-κB signaling .","TNFR1-induced c-Jun N-terminal protein kinase ( JNK ) and p38 signaling is also regulated by LUBAC .","SHARPIN deficiency blunts the TNFR1 pro-survival transcriptional signal and sensitizes cells to TNF-induced cell death .","The E3 ligase activity of HOIP catalyzes the addition of linear ubiquitin to target proteins , and SHARPIN and HOIL-1 are key regulators of the stability and activity of HOIP ( Gerlach et al . , 2011 ) .","In addition to TNFR1 , LUBAC has also been shown to regulate the transcriptional response from the interleukin-1 receptor ( IL-1R ) , CD40 , lymphotoxin beta receptor ( LTβR ) , toll-like-receptor 4 ( TLR4 ) , and nucleotide-binding oligomerization domain-containing protein 2 ( NOD2 ) receptor signaling complexes ( Schmukle and Walczak , 2012 ) .","Deletion of Il1rap , needed for IL-1 signaling , has been reported to almost completely prevent the development of cpdm dermatitis ( Liang et al . , 2010 ) .","This suggests that IL-1R signaling is a significant driver of disease , but the effect of Il1rap deficiency on the rest of the Shpnm/m phenotype was not reported .","Cpdm mice have prominent eosinophil infiltration into the skin; however , deletion of Il5 , which dramatically reduces the number of cutaneous and circulating eosinophils , fails to ameliorate disease ( Renninger et al . , 2010 ) .","Shpnm/mRag1−/− mice lacking functional lymphocytes develop dermatitis , indicating that T and B cell cells are not required for the skin phenotype ( Potter et al . , 2014 ) .","Furthermore , hematopoietic cell transfer with bone marrow and spleen cells from cpdm mice to syngeneic wild-type C57BL/Ka mice failed to transfer disease in mice 2 months post reconstitution .","Finally , cpdm skin transplanted onto nude mice retained the donor dermatitis phenotype 3 months post transplant , while syngeneic healthy skin transplanted onto cpdm mice did not acquire the disease over the same time ( HogenEsch et al . , 1993; Gijbels et al . , 1995 ) .","Together these studies indicate that a skin-intrinsic defect in cpdm mice drives the inflammatory disease , however they do not rule out a role for the hematopoietic system in amplifying it .","Impaired pro-survival TNFR1 signaling can induce both caspase-8-dependent apoptotic and RIPK3- and mixed lineage kinase domain-like protein ( MLKL ) -dependent necroptotic cell death via a cytosolic death platform ( Micheau and Tschopp , 2003; He et al . , 2009; Sun et al . , 2012; Zhao et al . , 2012; Murphy et al . , 2013 ) .","Necroptosis involves the release of cellular contents including potential damage-associated molecular patterns ( DAMPs ) such as mitochondrial DNA , high mobility group box 1 protein ( HMGB1 ) , IL-33 , and IL-1α ( Kaczmarek et al . , 2013 ) .","By contrast , apoptosis is considered to be immunologically silent , although this is clearly context dependent because excessive apoptosis resulting from conditional epidermal deletion of the caspase inhibitor cFLIP can cause severe skin inflammation ( Panayotova-Dimitrova et al . , 2013 ) .","Caspase-8 can cleave both RIPK1 and RIPK3 and is needed to keep the necroptotic pathway in check ( Vandenabeele et al . , 2010; Kaiser et al . , 2011; Oberst et al . , 2011 ) .","Regulation of necroptotic signaling is crucial for skin homeostasis because deletion of either caspase-8 , the caspase-8 adaptor protein FADD ( Fas-associated protein with death domain ) , or RIPK1 , leads to RIPK3- and MLKL-dependent epidermal hyperplasia and inflammation ( Kovalenko et al . , 2009; Lee et al . , 2009; Bonnet et al . , 2011; Kaiser et al . , 2011; Oberst et al . , 2011; Dannappel et al . , 2014; Dillon et al . , 2014; Rickard et al . , 2014 ) .","Although the precise factors that determine whether TNFR1 mediates apoptosis or necroptosis are unclear , high levels of RIPK3 , loss of cIAPs , and CYLD-mediated deubiquitylation of RIPK1 appear conducive to necroptosis ( Silke and Vaux , 2014 ) .","In addition to a crucial role in necroptosis , RIPK3 may also regulate inflammasome-induced IL-1ß production in the absence of IAPs or caspase-8 ( Vince et al . , 2012; Kang et al . , 2013 ) .","Thus the effects of loss of RIPK3 on an inflammatory phenotype may not be due to loss of necroptotic cell death but to a less well-defined role in IL-1ß production .","This complicates interpretation of the role of RIPK3 in a disease , particularly when IL-1 is pathogenic such as in cpdm dermatitis .","MLKL is downstream of RIPK3 in necroptosis and appears not to be required for cytokine production in the same situations as RIPK3 ( Wong et al . , 2014 ) .","Thus Mlkl−/− mice may provide an opportunity to disentangle the relative contribution of necroptosis and deregulated cytokine production in disease .","Here we provide genetic evidence in support of our hypothesis that TNFR1-induced cell death is a driver of the inflammatory disease in cpdm mice ( Gerlach et al . , 2011 ) .","We show that the cpdm phenotype is TNFR1 and cell-death dependent .","Ripk3 or Mlkl deficiency largely prevented cpdm liver inflammation and ameliorated the splenic phenotype and leukocytosis .","Remarkably , given the skin-inflammation phenotype of skin-specific Casp8 knock-out mice ( Kovalenko et al . , 2009; Lee et al . , 2009 ) , Casp8 heterozygosity potently suppressed the inflammatory skin phenotype while leaving the systemic inflammation unaffected .","Strikingly , combined Ripk3 deficiency and Casp8 heterozygosity completely prevented the cpdm dermatitis in all but one of the mice analyzed at 42 to 45 weeks of age , prevented liver inflammation and grossly restored splenic architecture .","Surprisingly , given the importance of LTßR ( also known as TNFRSF3 ) signaling in the formation of Peyer's patches ( De Togni et al . , 1994; Koni et al . , 1997 ) and the role of SHARPIN in this pathway ( Tokunaga et al . , 2011 ) , apparently normal Peyer's patches were also present in Shpnm/mCasp8+/-Ripk3−/− mice ."],["The dermatitis in Shpnm/m mice has previously been shown to be driven by TNF ( Gerlach et al . , 2011 ) and IL-1 signaling ( Liang et al . , 2010 ) .","Because the environment may influence the onset of the disease we wished to test the importance of TNF and IL-1 signaling in a head-to-head manner .","Furthermore the relative contribution of TNFR1 and TNFR2 in cpdm dermatitis has not been determined .","We therefore generated Shpnm/m mice deficient in Tnfr1 , Tnfr2 or Il1r1 ( Figure 1A ) .","All the knock-out mouse strains used in this study have been backcrossed at least ten times onto C57BL/6 , or were generated on the C57BL/6 background ( Mlkl−/− mice ) .","However , the cpdm mutation arose on a C57BL/Ka background ( HogenEsch et al . , 1993 ) .","To control for background modifier effects , we backcrossed C57BL/Ka Shpnm/m mice once or twice onto the C57BL/6 background , equivalent to the strategy we used in generating all our Shpnm/m compound knock-out strains to generate Shpnm/m C57BL/6 control mice .","These control mice developed the cpdm phenotype indistinguishably from the C57BL/Ka Shpnm/m mice , typically presenting with a visible skin phenotype by 5 to 6 weeks and invariably requiring euthanasia due to the dermatitis before 14 weeks of age .","Tnfr2 deletion did not ameliorate the dermatitis but Shpnm/mTnfr1−/− mice showed no outward signs of disease even after 35 weeks ( Figure 1A–C ) .","Il1r1 deletion significantly delayed the appearance of dermatitis , with markedly reduced epidermal hyperplasia in 13-week-old Shpnm/mIl1r1−/− mice compared with 12-week-old Shpnm/m mice ( Figure 1B , C ) .","However , by 19–20 weeks , Shpnm/mIl1r1−/− mice typically developed disease and required euthanasia .","Tnfr1 deletion reduced periportal liver inflammation and partially ameliorated the splenic phenotype , but did not restore Peyer's patches , whilst Tnfr2 and Il1r1 deletion did not prevent pathology in any of these organs ( Figure 1B , D ) . 10 . 7554/eLife . 03464 . 003Figure 1 . Cpdm dermatitis is mediated by TNFR1 , IL-1R to a lesser extent , and not TNFR2 . ( A ) Representative photos of mice of indicated genotypes and age .","( B ) Histological analysis of mice of genotype and age as indicated; representative of n ≥ 3 mice for each genotype or group .","Black arrows in liver images point to areas of periportal inflammation .","Black arrows in small intestine image point to Peyer's patches .","Shpnm/m: Sharpincpdm/cpdm .","Control mice are Shpn+/+ or +/m , Tnfr1−/− control mice are Shpn+/+ or +/mTnfr1−/− .","Scale bars: Skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .","H&E: hematoxylin and eosin .","( C ) Epidermal thickness of mice of indicated age and genotypes .","Each point represents the average of at least 14 measurements from multiple fields of view per mouse that were taken by a researcher who was blind to the genotype .","Dotted lines are drawn at 30 µm and 14 weeks .","Red numbers ( and black for middle graph ) correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .","Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mTnfr1−/− and Shpn+/+ or +/mIl1r1−/− in upper , middle and lower graphs respectively .","*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .","( D ) Average spleen weights of mice of indicated genotypes .","Spleen weights were taken from 12-week-old mice ( except Tnfr1−/− mice that were 15 or 35 weeks old ) , or younger mice if they required euthanasia due to their dermatitis .","Data are represented as mean + SEM , *p ≤ 0 . 05 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 003 To gain insight into the pathogenesis of cpdm dermatitis we assessed cytokine levels in skin lysates from 6-week-old cpdm and control mice using a BioPlex kit ( BioRad ) ( Figure 2A ) to determine which cytokines were elevated early in the disease process .","TNF levels were slightly elevated ( log scale , Figure 2A ) and , consistent with reports of eosinophilia in cpdm mice ( HogenEsch et al . , 1993; Gijbels et al . , 1996 ) , IL-5 ( a key inducer of eosinophil maturation ) was also elevated .","The monocyte and macrophage chemoattractant protein MCP-1 was significantly elevated in cpdm skin , as was the IL-12 p40 subunit .","There was also a trend for macrophage inflammatory protein 1α ( MIP-1α ) levels to be elevated .","Consistent with this , there was an increase in F4/80+ cells in the Shpnm/m dermis , and this was evident in patches at 3 weeks , before significant epidermal hyperplasia was present ( Figure 2B ) .","Ikeda et al . reported cleaved caspase-3-positive keratinocytes in 10-week-old Shpnm/m mice ( Ikeda et al . , 2011 ) , and we found cleaved caspase-3-positive cells were already present in the epidermis of 3-week-old Shpnm/m mice , indicating that apoptosis is an early event in the dermatitis and occurs before significant hyperplasia ( Figure 2C ) .","There were slightly more cleaved caspase-3-positive cells in Shpnm/m livers than controls ( almost exclusively in the infiltrating cells ) , however they were infrequent .","An increased number of cleaved caspase-3-positive cells were detected in Shpnm/m spleens , but , again , were much less appreciable than in the epidermis ( Figure 2C ) . 10 . 7554/eLife . 03464 . 004Figure 2 . Keratinocyte cell death and dermal macrophage infiltration are early events in cpdm dermatitis .","( A ) BioPlex cytokine analysis of skin lysates from mice of indicated genotypes .","Data are represented as mean +S . E . M . *p ≤ 0 . 05 .","( B ) F4/80 staining ( brown ) of skin sections counterstained with hematoxylin ( blue ) .","Control: Shpn+/+ or +/m .","( C ) Cleaved caspase-3 staining ( brown ) of skin , liver and spleen sections counterstained with hematoxylin ( blue ) .","Black arrows show examples of cleaved-caspase-3 positive cells .","Control: Shpn+/+ or +/m .","( A–C )","Three mice were analyzed for each genotype or group .","Scale bars: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 004 Reconstitution of wild-type mice with Shpnm/m bone marrow and/or spleen cells fails to transfer the disease ( HogenEsch et al . , 1993 ) , suggesting a skin-intrinsic defect underlies cpdm dermatitis .","These mice , however , were only followed for 8 weeks post reconstitution and this may not have allowed sufficient time for the dermatitis to develop .","To test this possibility , we reconstituted wild-type mice with Shpnm/m bone marrow cells and followed them for 12 months .","Reconstitution efficiency was high but the mice did not demonstrate any skin , liver , or spleen phenotype during this time ( Figure 3 ) .","Collectively these results suggest that the hematopoietic system in isolation cannot cause Shpnm/m dermatitis , and that macrophages may play a role in the amplification of the disease , particularly given they can be a prominent source of TNF . 10 . 7554/eLife . 03464 . 005Figure 3 . No dermatitis in wild-type mice reconstituted with Shpnm/m bone marrow after 12 months .","( A ) No dermatitis was observed in wild-type mice 12 months after reconstitution with Shpnm/m bone marrow .","( B ) Flow cytometry analysis showing percentage contribution of Ly5 . 1 ( recipient ) vs Ly5 . 2 ( donor ) white blood cells in peripheral blood 12 months after reconstitution .","( C ) Histological and immunofluorescence analysis of dorsal skin from mice 12 months after reconstitution .","( D ) Histological analysis of the spleen and liver from mice 12 months after reconstitution .","Scale bars: Skin 100 µm , liver 200 µm and spleen 500 µm .","H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 005 Tnf deletion prevents cpdm dermatitis and TNF kills Shpnm/m keratinocytes in vitro , suggesting that TNF-induced cell death drives the Shpnm/m skin phenotype ( Gerlach et al . , 2011 ) .","Consistent with this , TNF-induced cell death in Shpnm/m keratinocytes that was partially blocked by Q-VD-OPh or Nec-1 treatment ( Figure 4A ) .","Shpnm/m mouse dermal fibroblasts ( MDFs ) were also sensitive to TNF-induced cell death , and this death was almost completely blocked by the RIPK1 kinase inhibitor Necrostatin-1 ( Nec-1 ) , but not by the pan-caspase inhibitor Q-VD-OPh ( Figure 4B ) .","To test whether TNF could induce caspase cleavage we treated primary Shpnm/m keratinocytes and MDFs with TNF for up to 4 hr ( Figure 4C , D , Figure 4—figure supplement 1 ) .","TNF treatment led to caspase-3 and -8 cleavage at 4 hr in Shpnm/m , but not wild-type , keratinocytes .","In Shpnm/m MDFs there was also caspase-3 and -8 cleavage , and , surprisingly , this was reduced by Nec-1 , but not Q-VD , treatment ( Figure 4D ) .","In Shpnm/m keratinocytes and MDFs , the increased caspase-8 activity coincided with a marked processing of cFLIP , but other outcomes of the signaling pathway were less obviously affected .","Together with the early presence of cleaved caspase-3 staining and the elevation of TNF in Shpnm/m skin , these results indicate that TNF-induced apoptosis may play a pathogenic role in the skin phenotype . 10 . 7554/eLife . 03464 . 006Figure 4 . TNF induces death in multiple primary cell types , and is marked by caspase-3 and -8 cleavage in primary keratinocytes and mouse dermal fibroblasts in vitro .","( A and B )","Primary keratinocytes ( A ) and mouse dermal fibroblasts ( MDFs ) ( B ) were treated with 100 ng/ml human Fc-TNF , 50 µM Nec-1 or 10 µM Q-VD-OPh for 24 hr as indicated , then viability was assessed by propidium iodide ( PI ) uptake and flow cytometry .","Cells were generated from three different mice for each group .","Control: Shpn+/+ or +/m .","( C and D )","Western blot analysis of primary keratinocytes ( C ) and MDFs ( D ) treated as indicated ( concentrations as in A and B ) then lysed and lysates separated on SDS/PAGE and western blotted with indicated antibodies .","Shpn+/m: n = 1; Shpnm/m: n = 2 ( n = 1 for MDFs ) mice analyzed shown above .","* Indicates non-specific band .","Data from additional mice is shown in Figure 4—figure supplement 1 .","( E ) Neutrophils , monocytes and bone-marrow-derived macrophages were treated with 100 ng/ml human Fc-TNF , 50 µM Nec-1 , 20 µM Q-VD-OPh ( 10 µM for macrophages ) or 500 nM Compound A ( CpdA ) for 20 hr ( 24 hr for macrophages ) as indicated .","Viability was assessed by PI uptake and flow cytometry .","The Smac mimetic CpdA sensitizes cells to TNF-induced cell death and serves as a control .","Cells were generated from three different mice for each group except for macrophages , where six to eight mice were analyzed .","( A , B , E )","Data are represented as mean + SEM , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 00610 . 7554/eLife . 03464 . 007Figure 4—figure supplement 1 . TNF induces caspase-3 and -8 cleavage in primary keratinocytes and mouse dermal fibroblasts in vitro . Western blot analysis of primary keratinocytes and mouse dermal fibroblasts ( MDFs ) treated as indicated ( reagent concentrations as per Figure 4A , B ) then lysed and lysates separated on SDS/PAGE and western blotted with indicated antibodies .","* Indicates non-specific band .","Lysates generated from different mice to those shown in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 007 Given the appearance of cleaved caspase-3-positive cells in the Shpnm/m dermis ( Figure 2C ) , we sought to determine whether some of these might be non-fibroblast cells such as immune cells .","Since lymphoid cells are not required for cpdm dermatitis ( Potter et al . , 2014 ) and macrophage infiltration appears early , we tested whether myeloid cells were also sensitive to TNF-induced death .","Shpnm/m neutrophils , monocytes and bone-marrow-derived macrophages ( BMDMs ) were all sensitive to killing by TNF , whereas the wild-type cells were not ( Figure 4E ) .","Neutrophil and monocyte cell death was more efficiently blocked by a combination of Nec-1 and Q-VD , whereas in macrophages Nec-1 was sufficient to block cell death .","These data suggest that myeloid cell death may contribute to the inflammatory skin phenotype .","We did not detect a defect in p38 , JNK or NF-κB pro-survival signaling in SHARPIN-deficient keratinocytes and MDFs , as has been shown for other cell types ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) .","Consistent with earlier reports ( Gerlach et al . , 2011; Peltzer et al . , 2014 ) , however , using an antibody that specifically recognises linear ubiquitin we detected substantially reduced linear ubiquitylation in the native TNFR1 signaling complex obtained from Shpnm/m vs wild-type mouse embryonic fibroblasts ( MEFs ) ( Figure 5A ) .","We also found that HaCaT keratinocytes ( a human immortalized cell line ) stably expressing catalytically inactive HOIPC885S were sensitive to TNF-induced cell death ( Figure 5B ) , indicating a requirement for LUBAC and its linear-ubiquitin-chain-forming activity in preventing TNF-induced keratinocyte death . 10 . 7554/eLife . 03464 . 008Figure 5 . Sharpin is required for normal linear ubiquitylation of the TNF-R1 signaling complex , and HOIP protects keratinocytes from TNF-induced cell death .","( A ) Anti Flag Immuno-Precipitation ( IP ) of the TNF receptor signaling complex in immortalised mouse embryonic fibroblasts ( MEFs ) treated with Flag-TNF for the times indicated .","( B ) HaCaT human keratinocytes stably expressing HOIP , the catalytically inactive HOIPC885S , or an empty vector ( control ) were treated with 100 ng/ml TNF for 24 hr .","Viability was assessed by propidium iodide ( PI ) uptake and flow cytometry .","Data are presented as mean + SEM , n = 3 , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 008 To investigate the importance of the caspase-8-mediated apoptotic pathway in vivo we generated Shpnm/mCasp8+/− mice .","We did not attempt to generate Shpnm/mCasp8−/− mice because Casp8 deletion results in embryonic lethality at around E10 . 5 ( Varfolomeev et al . , 1998 ) .","In contrast to Shpnm/m mice , 12-week-old Shpnm/mCasp8+/− mice had almost no epidermal hyperplasia and largely normal keratin 6 and 14 expression ( Figure 6A , B ) .","By 15 weeks of age , however , significant hyperplasia was observed in one of these mice ( Figure 6B ) .","Shpnm/mCasp8+/− mice retained other aspects of the phenotype: splenomegaly with disrupted splenic architecture , liver inflammation , and a lack of intestinal Peyer's patches ( Figure 6C ) .","Some Shpnm/mCasp8+/− mice succumbed to a pulmonary infection with Pasteurella and required euthanasia while littermates were unaffected , suggesting that Shpnm/mCasp8+/− mice are partially immunocompromised .","Mice with obvious signs of infection were not used for the spleen , blood , or liver analyses but were included in the skin analysis .","Occult infection , however , in Shpnm/mCasp8+/− mice used for hematopoietic , liver , and spleen analyses cannot be completely excluded . 10 . 7554/eLife . 03464 . 009Figure 6 . Protection from cpdm dermatitis with Casp8 heterozygosity but not Bid deletion .","( A ) Histological and immunofluorescence skin analysis .","( B ) Epidermal thickness of mice of indicated age and genotypes determined as in Figure 1B , by an investigator blinded to genotype .","Top panel is a repeat of data in 1B for reference purposes only .","Dotted lines are drawn at 30 µm and 14 weeks .","Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .","Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mBid−/− , and Shpn+/+ or +/mCasp8+/− in upper , middle and lower graphs , respectively .","*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .","( C ) Histological analysis of spleen , liver , and small intestine .","Black arrows in liver images point to areas of periportal inflammation .","Black arrow in small intestine image points to Peyer's patch .","( D ) Average spleen weights of mice of indicated genotypes .","Spleen weights were taken from 12–14-week-old mice , or younger mice if they required euthanasia due to their dermatitis .","Data are represented as mean + SEM , ***p ≤ 0 . 005 .","( A and C )","Control mice are Shpn+/+ or +/m , n ≥ 3 mice analyzed each genotype or group .","Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .","H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 009 In certain cell types such as hepatocytes , BID can be cleaved by caspase-8 to generate truncated BID ( tBID ) that , in turn , mediates caspase-9- and mitochondria-dependent apoptosis ( Czabotar et al . , 2014 ) .","BID is a key regulator of UV-induced apoptosis in keratinocytes , indicating that this pathway is of importance in the skin ( Pradhan et al . , 2008 ) .","Because cleaved caspase-9 is found in the Shpnm/m epidermis ( Ikeda et al . , 2011 ) and Shpnm/m keratinocyte extracts contained caspase-9-substrate-cleaving activity ( Liang and Sundberg , 2011 ) , we hypothesized that TNF-induced BID-dependent apoptosis may be a driver of the cpdm phenotype and , hence , generated Shpnm/mBid−/− mice .","We observed no protection from any aspects of the cpdm phenotype ( Figure 6A–D ) , however , indicating that caspase-8 , but not BID , is an important driver of this disease .","Caspase-8 heterozygosity significantly delayed epidermal hyperplasia , but other aspects of the cpdm phenotype ( e . g . splenomegaly and liver inflammation ) remained .","We therefore sought to test the role of necroptosis in the inflammatory disease by generating Shpnm/mRipk3−/− mice .","Whereas control mice invariably developed severe dermatitis by 12 weeks of age , roughly half of the 12-week-old Shpnm/mRipk3−/− mice had a less severe epidermal phenotype at this point ( Figure 7A , B ) .","When aged over 12 weeks , all Shpnm/mRipk3−/− animals went on to develop severe disease and were euthanized due to their skin phenotype before 18 weeks of age .","The remainder of Shpnm/mRipk3−/− mice developed skin disease at the same rate as control mice .","12 week-old Shpnm/mRipk3−/− mice had no signs of liver inflammation and significantly less splenomegaly , although they still lacked Peyer's patches ( Figure 7C , D ) .","We also generated Shpnm/mMlkl−/− mice and found they had a similar epidermal phenotype to Shpnm/m mice ( Figure 7B ) .","Like Shpnm/mRipk3−/− mice , however , 12-week-old Shpnm/mMlkl−/− animals had reduced splenomegaly and only one out of 12 mice showed signs of liver inflammation ( Figure 7C , D ) .","Collectively these results indicate that RIPK3 and MLKL are important drivers of the liver and splenic cpdm phenotype , and that RIPK3 contributes to the epidermal phenotype , importantly in a non-MLKL-dependent ( hence , most likely , necroptosis-independent ) manner . 10 . 7554/eLife . 03464 . 010Figure 7 . Ripk3 deficiency slightly delays cpdm dermatitis onset , and Ripk3 and Mlkl deficiency partially protects against the cpdm splenic phenotype and markedly attenuates liver inflammation .","( A ) Histological and immunofluorescence skin analysis .","( B ) Epidermal thickness of mice of indicated age and genotypes determined as in Figure 1B , by an investigator blinded to genotype .","Top panel is a repeat of data in 1B for reference purposes only .","Dotted lines are drawn at 30 µm and 14 weeks .","Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .","Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mRipk3−/− , and Shpn+/+ or +/mMlkl−/− in upper , middle and lower graphs , respectively .","*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 , # significantly different to Shpnm/m mice ( Fisher's exact test ) , p ≤ 0 . 05 .","( C ) Histological analysis of spleen , liver , and small intestine .","Black arrows in liver image points to areas of periportal inflammation .","Black arrows in small intestine image points to Peyer's patches .","( D ) Average spleen weights of mice of indicated genotypes .","Spleen weights were taken from 12-week-old mice , or younger mice if they required euthanasia due to their dermatitis .","Data are represented as mean + SEM , ***p ≤ 0 . 005 .","( A and C )","Control mice are Shpn+/+ or +/m , n ≥ 3 mice analyzed each genotype or group .","Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .","H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 010 Since deletion neither of one allele of Casp8 nor of two alleles of Ripk3 was able to fully rescue all the multi-organ pathology in cpdm mice , we sought to generate Shpnm/mCasp8−/−Ripk3−/− mice , taking advantage of the fact that Ripk3 deletion prevents Casp8−/−-mediated embryonic lethality ( Kaiser et al . , 2011; Oberst et al . , 2011 ) .","We generated these mice independently at two separate facilities .","At one facility , most of these triple-deficient mice died perinatally , typically in a window between E17 and 1 to 2 days after birth , and no Shpnm/mCasp8-/-Ripk3−/− animals were obtained at weaning ( Figure 8A , B; numbers in A refer to mice analyzed at first facility only ) .","Most Shpnm/mCasp8−/−Ripk3−/− embryos obtained by Caesarean section at E19 appeared edematous or were dead .","Some were able to breathe; however , these died within the first 2 days after birth .","Hematopoietic analysis at E19 did not reveal any consistent differences ( Figure 8C ) .","At a separate facility , two viable Shpnm/mCasp8−/−Ripk3−/− mice were obtained from a limited number of matings .","By roughly 3 months of age these mice appeared runted and were euthanized .","Histologically these mice had no epidermal phenotype , but disrupted splenic architecture was apparent ( Figure 8D ) . 10 . 7554/eLife . 03464 . 011Figure 8 . Shpnm/mCasp8−/−Ripk3−/− mice are prone to perinatal lethality .","( A ) Table of segregation of expected and observed genotypes from Shpn+/mCasp8-/-Ripk3−/− or Shpnm/mCasp8+/-Ripk3−/− intercrosses at various developmental stages .","E10 . 5 controls: Shpn+/+ or +/mCasp8-/-Ripk3−/−; E19 controls: Shpnm/mCasp8+/+ or +/−Ripk3−/−; weaning controls: Shpn+/+ or +/mCasp8−/−Ripk3−/− or Shpnm/mCasp8+/+ or +/−Ripk3−/− .","*Significantly different to expected value , Fisher's exact test p < 0 . 0005 .","( B ) Photos of E19 embryos obtained by Caesarian section .","The Shpnm/mCasp8−/−Ripk3−/− mouse on the right was recovered dead , all others were alive .","Other embryos are control mice: Shpnm/mCasp8+/+ or +/−Ripk3−/− .","( C ) ADVIA blood analysis from E19 embryos .","RBC: red blood cells; WBC: white blood cells; MCV: mean cell volume .","Horizontal lines depict data means .","Control mice: Shpnm/mCasp8+/+ or +/−Ripk3−/− .","( D ) Histological analysis of tissue from 12-week-old mice of indicated genotypes .","Mice were from a separate facility to those in A–C and are not included in the table in A . Two mice were analyzed for each genotype .","Scale bars: skin 100 µm , spleen 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 011 While the reason for the lethality of the triple-deficient animals is unknown , we were readily able to obtain viable Shpnm/mCasp8+/−Ripk3−/− mice .","These mice were indistinguishable from control mice at 12 weeks of age and had no skin , liver , or spleen pathology ( Figure 9A–E ) .","Remarkably , Peyer's patches were present ( Figure 9C , Figure 9—figure supplement 1 ) .","One 67-week-old and three 45-week-old Shpnm/mCasp8+/−Ripk3−/− mice had no signs of any skin phenotype , however another mouse developed dermatitis at 42 weeks ( Figure 9A , B , D; note that epidermal thickness was not quantified for the 42-week-old mouse ) . 10 . 7554/eLife . 03464 . 012Figure 9 . Ripk3 deletion and Casp8 heterozygosity markedly delays emergence of cpdm dermatitis and liver inflammation and restores Peyer’s patches .","( A ) Representative photos of mice of indicated genotypes and age .","Three Shpnm/mCasp8+/-Ripk3−/− mice were analyzed at 45 weeks and one at 67 weeks with no detectable dermatitis , however another mouse developed dermatitis by 42 weeks of age .","( B ) Histological and immunofluorescence skin analysis .","( C ) Histological analysis of spleen , liver , and small intestine .","Black arrows in liver image point to areas of periportal inflammation .","Black arrows in small intestine images point to Peyer's patches .","( D ) Epidermal thickness of mice of indicated age and genotypes measured as described in Figure 1B .","Top panel is a repeat of data in 1B for reference purposes only .","Dotted lines are drawn at 30 µm and 14 weeks .","Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .","Control mice are Shpn+/+ or +/m and Shpn+/+ or +/mCasp8+/−Ripk3−/− in upper and lower graphs , respectively .","*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .","( E ) Average spleen weights of mice of indicated genotypes .","Spleen weights were taken from 12-week-old mice , or younger mice if they required euthanasia due to their dermatitis .","Data are represented as mean + SEM , ***p ≤ 0 . 005 .","( B and C )","Control mice are Shpn+/+ or +/m , n ≥ 3 mice analysed each genotype or group .","Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .","H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 01210 . 7554/eLife . 03464 . 013Figure 9—figure supplement 1 . Restoration of Peyer's patches in 8-week-old Shpnm/mCasp8+/−Ripk3−/− mice . Numbers of Peyer's patches per small intestine counted from 8-week-old mice of indicated genotypes .","Horizontal lines depict data means . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 013 To investigate the effect of the various genetic crosses on Shpnm/m leukocytosis , we analyzed white blood cell levels in peripheral blood using an ADVIA hematological analyzer ( Figure 10 ) .","Tnfr1 deletion was more effective at reducing leukocyte numbers than Tnfr2 or Il1r1 deletion , although all of these compound knock-out mice had leukocyte subsets that were elevated compared to controls .","Caspase-8 heterozygosity or Bid deletion did not prevent the leukocytosis , whereas Ripk3 and Mlkl deletion markedly reduced it , suggesting the hematopoietic phenotype is driven predominantly by necroptosis .","Shpnm/mRipk3−/− and Shpnm/mMlkl−/− mice still had elevated neutrophils , however Shpnm/mCasp8+/−Ripk3−/− mice had no white blood cell elevation . 10 . 7554/eLife . 03464 . 014Figure 10 . Peripheral blood counts from various crosses . Peripheral blood was collected from 11–14-week-old mice ( reconstituted mice: Shpnm/m → wild type were 12 months old and Tnfr1−/− mice were 35 weeks old ) , or younger if the mice were euthanized due to severe dermatitis .","Shpn+/+ or +/m and Shpnm/m mice are highlighted in blue and red for reference purposes .","Blood was analyzed using an ADVIA 2120 hematological analyzer .","RBC: red blood cells .","Horizontal lines depict data means .","Control mice are Shpn+/+ or +/m , for example Tnfr1−/− control mice are Shpn+/+ or +/mTnfr1−/− .","DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 014"],["LUBAC , composed of three proteins , HOIL-1 , HOIP and SHARPIN , has recently emerged as a regulator of a diverse set of signaling complexes ( Zak et al . , 2011; Schmukle and Walczak , 2012; Tokunaga , 2013 ) .","Lack of HOIP causes mid-gestational embryonic lethality ( embryonic day 10 . 5 ) , and this is prevented by simultaneous loss of Tnfr1 ( Peltzer et al . , 2014 ) .","Together , these data demonstrate that LUBAC is required for full-strength IL-1ß and TNF signaling and deficiency in these signaling pathways would be expected to impair an inflammatory response .","The consequence , however , of Sharpin deficiency in mice is multi-organ inflammation ( HogenEsch et al . , 1993 ) .","Likewise , loss-of-expression and loss-of-function mutations in HOIL-1 result in a fatal , human , inherited disorder characterized by chronic inflammation; consistent with loss of the major defensive inflammatory signaling pathways , affected patients suffer from invasive bacterial infections ( Boisson et al . , 2012 ) .","In vitro experiments suggest that HOIL-1 is an essential component of LUBAC , which is present in many cell types .","With this in mind it is surprising that HOIL-1-deficient mice were reported to be normal ( Tokunaga et al . , 2009 ) .","If , however , we set this observation to one side , it seems that in humans and mice loss of LUBAC can precipitate an inflammatory disease .","This could be because the loss of the homeostatic component of TNFR1 , IL-1R1 , and TLR signaling reduces the ability of an organism to resist infection , which , even in the weakened signaling environment of LUBAC deficiency , drives inflammatory signaling .","Another possibility is that full-strength signaling from TNF , IL-1ß , or TLR ligands is required to upregulate essential negative feedback regulators such as IκBα and A20 .","Signaling , therefore , although reduced , is constitutively active .","This explanation seems less likely because IκBα and A20 transcripts were still upregulated in response to TNF or Pam3CSK4 in HOIL-1/HOIP-deficient and cpdm cells ( Haas et al . , 2009; Tokunaga et al . , 2009; Gerlach et al . , 2011; Zak et al . , 2011 ) .","The fact that the phenotype from the cpdm mice could not be transferred by the hematopoietic compartment , and that the skin phenotype is maintained following skin transplantation onto nude mice , indicates that these animals suffer from an intrinsic skin defect ( HogenEsch et al . , 1993; Gijbels et al . , 1995 ) .","This is not unprecedented; loss of Notch signaling in the skin causes a dermatitis disease in mice that is sufficient to drive systemic inflammation with similar features to the cpdm phenotype ( Dumortier et al . , 2010 ) .","The earlier hematopoietic reconstitution experiments , however , may not have allowed sufficient time for the inflammatory phenotype to develop .","The work described here excludes this caveat because reconstituted mice did not develop signs of the cpdm phenotype even a year after reconstitution; thus , the inflammatory skin phenotype in the Shpnm/m mutant mice is the result of an intrinsic skin defect .","It is , however , noteworthy that loss of Mlkl did not affect the Shpnm/m skin phenotype but significantly reduced or prevented splenomegaly and liver inflammation .","Conversely , reduction in caspase-8 markedly ameliorated the skin phenotype but did not prevent splenomegaly or liver inflammation .","This shows that the skin and systemic phenotype are separable , although the exact mechanism is unclear .","The fact that cpdm keratinocytes are sensitized to TNF-induced apoptosis and necroptosis led us to hypothesize that TNF/TNFR1-dependent cell death was causative for the skin phenotype ( Gerlach et al . , 2011 ) .","Because of the systemic inflammation we suspected that necroptotic cell death and the release of DAMPs was the driver .","Consistent with this hypothesis , we show here that Tnfr1 deficiency ( as with Tnf deficiency ) suppresses the cpdm phenotype , whereas Tnfr2 deficiency had no effect on the phenotype .","Loss of IL-1 signaling has been shown to suppress the cpdm phenotype ( Liang et al . , 2010 ) , but because the environment of the mice likely plays a large part in the onset and severity of the inflammatory disease it was not possible to determine whether loss of IL-1 signaling is as potent as Tnf deficiency at preventing the phenotype .","We therefore generated Shpnm/mIl1r1−/− mice , which succumbed to the inflammatory disease much later than Shpnm/m mice , but were far more inflammation prone than any of the Shpnm/mTnf−/+ , Shpnm/mTnf−/− , or Shpnm/mTnfr1−/− mice .","These data are again consistent with our original hypothesis , suggesting that TNF/TNFR1 signaling is the main driver of inflammation and that IL-1 contributes to exacerbating the disease .","Casp8 deficiency results in early embryonic lethality in mice ( Varfolomeev et al . , 1998 ) , and loss of Casp8 or Fadd in the skin leads to keratinocyte hyperplasia and inflammatory skin disease ( Kovalenko et al . , 2009; Bonnet et al . , 2011 ) .","Therefore , we considered it unlikely that loss of caspase-8 would diminish the chronic proliferative dermatitis of the Shpnm/m mutant mice .","To our surprise , however , loss of a single allele of Casp8 was strikingly effective at reducing the skin phenotype and appearance of cleaved caspase-3 in the skin .","It has been proposed that the intrinsic mitochondrial apoptosis pathway mediated by caspase-9 and caspase-3 plays a role in the keratinocyte hyperplasia observed in Shpnm/m mutant mice based on the presence of disrupted mitochondria in cpdm skin and in vitro experiments ( Liang and Sundberg , 2011 ) .","Given the prominent role that caspase-8 plays in the dermatitis , it would be expected that if the intrinsic apoptosis pathway is engaged it should be downstream of caspase-8 and require cleavage of the BH3 protein , BID ( Czabotar et al . , 2014 ) .","Cells that require BID cleavage by caspase-8 in order to undergo apoptosis are known as type II cells ( Barnhart et al . , 2003 ) , but it is unclear whether keratinocytes are type I or type II ( Pradhan et al . , 2008; Geserick et al . , 2014 ) .","Genetic deletion of Bid did not suppress the dermatitis or any other aspect of the cpdm phenotype , therefore we conclude that the intrinsic mitochondrial pathway is unlikely to play a significant role in the disease .","Because necroptotic cell death can be inflammatory , by provoking the release of DAMPs , we expected that deficiency in RIPK3 or MLKL , essential effectors of the necroptotic cell death pathway , would reduce the severity of the inflammation in Shpnm/m mice .","Whilst there was a modest delay in the appearance of the dermatitis in Shpnm/mRipk3−/− mice , all animals went on to develop severe skin disease .","In contrast to RIPK3 deficiency , deletion of Mlkl did not even delay appearance of the dermatitis .","Together , this suggests that RIPK3 may exacerbate the skin phenotype independently of necroptosis , possibly via a direct role in cytokine production .","Combined with the markedly ameliorated dermatitis seen in Shpnm/mCasp8+/− mice , this indicates that apoptosis is the main driver of Shpnm/m dermatitis .","Although apoptosis is generally regarded as being immunologically inert , when in excess it can result in severe inflammation .","For example , conditional deletion of cFLIP in adult keratinocytes caused severe inflammation in the epidermis ( Panayotova-Dimitrova et al . , 2013 ) .","Like the cpdm phenotype , this inflammation was TNF dependent .","An excess of apoptotic cells may cause disease by overwhelming phagocytosis and clearance of apoptotic bodies , leading to secondary necrosis and DAMP release .","It has also been proposed that in certain contexts , such as viral infections , apoptosis may be inflammatory ( Cullen et al . , 2013 ) .","In other Shpnm/m organs such as the liver , spleen , and hematopoietic system , Ripk3 and Mlkl deletion ameliorated or protected against disease , indicating that Sharpin deficiency triggers apoptosis in some tissues but necroptosis in others .","Because it has been shown that RIPK3 or MLKL can mediate cytokine production induced by the absence of caspase-8 ( Kang et al . , 2013 ) , and potentially other , as yet undescribed , pathways , we cannot completely exclude the possibility that Ripk3 or Mlkl deletion affords protection by blunting cytokine production .","Absence of SHARPIN , however , leads to increased , not decreased , caspase-8 activity , so it is not obvious whether these observations apply in this case .","Furthermore , whereas it is clear that RIPK3 plays a role in promoting cytokine production in response to a number of stimuli ( Vince et al . , 2012 ) , we have so far only observed a defect in necroptosis and not in inflammatory cytokine production in Mlkl−/− cells ( Murphy et al . , 2013; Allam et al . , 2014; Rickard et al . , 2014 ) .","Whilst Shpnm/mCasp8+/− , Shpnm/mRipk3−/− , and Shpnm/mMlkl−/− mice all developed significant disease in either the liver , spleen , hematopoietic compartment , or skin , Shpnm/mCasp8+/−Ripk3−/− mice were almost completely protected from disease for approximately 10–11 months , and in one case 15 months .","One of these mice developed the skin phenotype at 10 months of age , indicating that the single allele of Casp8 is enough to eventually cause dermatitis .","This is supported by the development of epidermal phenotype in 14–15-week-old Shpnm/mCasp8+/− mice .","SHARPIN has been shown to regulate LTß/LTßR signaling , and LTßR is required for the development of Peyer's patches ( De Togni et al . , 1994 ) , therefore we had assumed that the absence of Peyer's patches was due to defective LTßR signaling .","A recent study , however , demonstrated that rudimentary Peyer's patches formed in Shpnm/m embryos , but that these regressed post-natally ( Seymour et al . , 2013 ) .","Our data show that whilst neither Casp8 heterozygosity nor Ripk3 deletion in isolation restored intestinal Peyer's patches , the combination was able to do so .","This suggests that the post-natal regression of these secondary lymphoid organs in Shpnm/m mice is due to deregulated cell death .","In a wider sense , our results are particularly important when interpreting the recent finding that crossing cpdm mice with Ripk1K45A/K45A mice ( which lack RIPK1 kinase activity ) completely suppresses the cpdm phenotype ( Berger et al . , 2014 ) .","RIPK1 has a promiscuous role in immune signaling , regulating pro-survival , necroptotic , and apoptotic pathways .","However , while the RIPK1 kinase domain is well known for its ability to activate the necroptotic pathway , it is not believed to be required to cause apoptosis ( Vandenabeele et al . , 2010 ) .","Yet we show here that the Shpnm/m skin phenotype is associated with the appearance of processed caspase-3 in the skin , and both the epidermal hyperplasia and appearance of processed caspase-3 is markedly reduced by loss of a single allele of Casp8 .","Consistent with the hypothesis that Shpnm/m keratinocyte hyperplasia is due to apoptosis , it is not prevented by loss of the key necroptotic effector MLKL .","Furthermore , purified Shpnm/m keratinocytes and dermal fibroblasts rapidly activate caspase-8 and caspase-3 in response to TNF .","Taken together with the work of Berger et al . our work inescapably suggests that in the context of the Shpnm/m epidermis the dominant role of the RIPK1 kinase domain is to activate apoptosis .","Unexpectedly , but supporting this conclusion , we showed that Nec-1 ( the RIPK1 kinase inhibitor ) blocked TNF-induced caspase-8 and caspase-3 activation in Shpnm/m dermal fibroblasts .","This conclusion is particularly confronting because we and others have shown that loss of RIPK1 in the skin results in a RIPK3/MLKL-dependent hyperplasia that is presumably dependent on necroptosis ( Dannappel et al . , 2014; Rickard et al . , 2014 ) .","Thus RIPK1 is able to both activate and inhibit either apoptosis or necroptosis in a highly context-dependent manner .","Ripk3 deletion prevents the embryonic lethality seen with either Fadd or Casp8 deletion and the mice survive to adulthood ( Kaiser et al . , 2011; Oberst et al . , 2011; Dillon et al . , 2012 ) .","Given this , the perinatal lethality we observed in Shpnm/mCasp8−/−Ripk3−/− mice was completely unexpected .","At E19 some of these mice were recovered alive by Caesarean section and established regular breathing , some failed to establish normal breathing , some appeared edematous and were not recovered alive , whilst others were in the process of being resorbed from earlier embryonic lethality .","To complicate the picture , two viable Shpnm/mCasp8−/−Ripk3−/− mice were obtained at a separate facility .","At 3 months of age these mice had no epidermal phenotype but were runted .","One potential explanation for the different penetrance of the phenotype may be a different genetic background because the mice described in Figure 8A were exon 3-deleted caspase-8 mice ( Beisner et al . , 2005 ) , whereas the mice in Figure 8D were exon 3- and 4-deleted caspase-8 ( Salmena et al . , 2003 ) .","Exon 3- and 4-deleted caspase-8 mice in yet a third facility ( not shown ) , however , did not survive past weaning , indicating that environmental differences undoubtedly also contribute to the variable penetrance .","Future efforts aimed at understanding the lethality of Shpnm/mCasp8−/−Ripk3−/− mice should yield important insights into not only the biology of SHARPIN and linear ubiquitin , but also that of caspase-8 and RIPK3 .","In summary , we provide strong evidence that Sharpin deficiency sensitizes keratinocytes to TNF/TNFR1-induced , caspase-8-mediated apoptosis , and that this defect appears to drive the cpdm dermatitis .","Ripk3 deletion provided only a modest and variable delay in the presentation of dermatitis .","Unlike Casp8 heterozygosity , Ripk3 and Mlkl deletion ameliorated many aspects of the systemic cpdm phenotype .","This indicates a tissue-specific role for Sharpin in regulating cell death pathways .","Only combined Casp8 heterozygosity and Ripk3 deficiency was able to almost completely prevent all aspects of the cpdm systemic phenotype that we evaluated , including the early loss of Peyer's patches .","Whilst inflammation is a known sequelae to necroptotic DAMP release , these findings provide further evidence that excessive apoptosis can also cause inflammatory disease .","Furthermore , these results indicate that the suppression of cpdm dermatitis seen by crossing to mice lacking RIPK1 kinase activity ( Berger et al . , 2014 ) may , surprisingly , be due to RIPK1’s kinase activity being upstream of caspase-8 in mediating TNF-induced apoptosis ."],["Mice were maintained at the Walter and Eliza Hall Institute of Medical Research ( WEHI ) and University College London ( UCL ) .","C57BL/Ka Sharpincpdm/cpdm mice were obtained from Jax ( Bar Harbor , ME ) , and then either backcrossed one to two times onto C57BL/6 or crossed with C57BL/6 Ripk3−/− , Mlkl−/− , Casp8+/− , Casp8+/-Ripk3−/− , Bid−/− , Il1r1−/− , Tnfr1−/− , or Tnfr2−/− mice .","For timed matings , mice were analyzed by Caesarean section .","For E19 timed matings , pregnant females were injected at E17 and E18 with progesterone .","Primary keratinocytes and MDFs were isolated and cultured as described previously ( Gerlach et al . , 2011; Etemadi et al . , 2013 ) .","Cell lysates were prepared using DISC buffer ( 1% NP-40 , 10% glycerol , 150 mM NaCl , 20 mM Tris pH 7 . 5 , 2 mM EDTA , cOmplete protease inhibitor cocktail ( Roche; Penzberg , Germany ) , 2 mM sodium orthovanadate , 10 mM sodium fluoride , β-glycerophosphate , N2O2PO7 ) .","Cell lysates were loaded in NuPAGE Bis-Tris gels ( Life Technologies/Thermo Fisher Scientific; Waltham , MA ) and transferred on to Immobilon-P PVDF membranes ( Millipore; Billerica , MA ) or Hybond-C Extra ( GE Healthcare; Little Chalfont , UK ) .","Membranes were blocked and antibodies diluted in 5% skim milk powder or Bovine Serum Albumin ( BSA ) in 0 . 1% PBS or TBS-Tween20 .","Antibodies used for western blot: cleaved caspase-3 ( 9661 ) and −8 ( 8592 ) , phospho-JNK1/2 ( 4668P ) , phospho-p38 ( 4511 ) , p38 ( 9212 ) , caspase-8 ( 4927 ) , JNK1/2 ( 9252 ) , IκBα ( CN: 9242 ) , and phospho-p65 ( 3033 ) from Cell Signaling Technology ( Danvers , MA ) , β-actin ( A-1978; Sigma Aldrich; St . Louis , MO ) , RIPK1 ( 610458; BD Biosciences; Franklin Lakes , NJ ) , cFLIP ( AG-20B-0005; Adipogen; Liestal , Switzerland ) , FADD ( generated in-house; gift from Lorraine O’Reilly ) and MLKL ( generated in house; Murphy et al . , 2013 ) .","Signals were detected by chemoluminescence ( Millipore ) after incubation with secondary antibodies conjugated to horseradish peroxidase .","For isolation of neutrophils and monocytes , red blood cells were lysed and bone marrow cells were stained with flurochrome-conjugated anti-mouse Ig antibodies ( CD11b [Mac-1] and Ly6G [1A8] ) and sorted using a FACS ARIA instrument ( BD Biosciences ) .","Neutrophils ( CD11b+ Ly6G+ ) and monocytes ( CD11b+Ly6G− ) were cultured in 5% FCS RPMI at 1 × 105 and 0 . 5 × 105 cells per well , respectively , in a 96-well u-bottom tissue culture plate .","BMDMs were isolated and cultured as described previously ( Wong et al . , 2014 ) .","Keratinocytes , MDFs , neutrophils , monocytes , and BMDMs were stimulated with TNF ( 100 ng/ml ) , Nec-1 ( 50 μM ) , QVD-Oph ( 10 μM , 20 μM for neutrophils and monocytes ) and CpdA ( 911 , 500 nM ) .","After 24 hr ( 20 hr for neutrophils and monocytes ) all cells except keratinocytes were stained with propidium iodide ( PI ) and cell death analyzed on a FACScalibur instrument ( BD Biosciences ) .","For the keratinocyte MTS viability assay phenazine methosulfate ( PMS; 0 . 92 mg/ml in PBS; Sigma-Aldrich ) and 3- ( 4 , 5-dimethylthiazol-2-yl ) -5- ( 3-carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) -2H-tetrazolium ( MTS; 2 mg/ml in PBS; Promega; Fitchburg , WI ) were combined in a 1:20 ratio .","The mixture was added to cell culture media in a 1:5 ratio and incubated for 1–4 hr at 37°C in a humidified 5% CO2 incubator .","Media was transferred to a flat-bottom 96-well plate and absorbance was measured at 490 nm .","Viability was calculated relative to the untreated sample .","For HaCaT experiments , cells stably expressing HOIP , HOIPC885S ( catalytically inactive ) , or an empty vector were seeded and incubated the next day with 100 ng/ml histidine-tagged TNF for 24 hr .","Supernatant and adherent cells were harvested and resuspended in PBS containing 5 mg/ml PI .","PI-positive cells were measured by flow cytometry ( BD Accuri; BD Biosciences ) .","Paraffin-embedded tissue was fixed in 10% neutral buffered formalin then processed and stained with hematoxylin and eosin ( H&E ) according to standard practices .","Immunohistochemistry and immunofluorescence analysis was performed as described previously ( Rickard et al . , 2014 ) .","Ly5 . 1 mice were irradiated with 2 × 550 rads spaced 3 hr apart .","Following red blood cell lysis , ∼ 5 × 106 BM cells from Shpnm/m ( Ly5 . 2 ) mice were intravenously injected .","Mice were maintained on 2 mg/ml neomycin in drinking water for 3 weeks post irradiation .","Reconstitution efficiency was assessed 6 weeks and 12 months post reconstitution by staining for Ly5 . 1 and Ly5 . 2 in peripheral blood obtained from retro-orbital bleeding .","Cytokines were analyzed using a BioPlex Pro mouse cytokine 23-plex kit ( Bio-Rad; Hercules , CA ) , or for analysis of TNF levels a mouse TNF ELISA kit ( eBioscience; San Diego , CA ) was used .","Skin lysates were prepared by homogenizing skin in ice cold protein lysis buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 2 mM EDTA , 1% Triton-X100 , 10% glycerol ) using a Tissue Lyser II ( QIAGEN; Hilden , Germany ) for 12 cycles of 30 s at 30 Hz .","A BCA kit ( Thermo Fisher Scientific ) was used to normalise protein levels .","Values below the reference range were assigned the value of the lowest standard .","Peripheral blood was collected into EDTA tubes ( Sarstedt; Nümbrecht , Germany ) and analyzed using an ADVIA 2120 hematological analyzer .","For TNF-RSC isolation , immortalised MEFs were stimulated with 3xFlag-2xStrep-TNF at 0 . 5 μg/ml for the indicated times , or left untreated .","Cells were subsequently solubilized in lysis buffer ( 30 mM Tris–HCl ( pH 7 . 4 ) , 150 mM NaCl , 2 mM EDTA , 2 mM KCl , 10% Glycerol , 1% Triton X100 , EDTA-free proteinase inhibitor cocktail ( Roche ) and 1x phosphatase inhibitor cocktail 2 ( Sigma Aldrich ) ) at 4°C for 30 min .","The lysates were cleared by centrifugation , and 3xFlag-2xStrep-TNF was added to the untreated samples .","Next , lysates were subjected to anti-Flag immunoprecipitation using M2 beads ( Sigma Aldrich ) for 16 hr .","The beads were washed three times with lysis buffer , proteins were eluted in 50 μl of LDS buffer ( Life Technologies/Thermo Fisher Scientific ) containing 50 mM DTT .","Samples were analyzed by western blotting .","Antibodies used were: HOIP ( custom-made , Thermo Fisher Scientific ) , SHARPIN ( 14626-1-AP; ProteinTech; Chicago , IL ) , TNFR1 ( ab19139; Abcam; Cambridge , UK ) , and linear ubiquitin ( Genentech; South San Francisco , CA ) .","Pearson chi-square and Fisher's exact test were used to assess frequencies of observed vs expected genotypes during development , at birth , and at weaning .","Fisher's exact test was used for epidermal thickness statistical calculations .","Student's t test was used to calculate statistical significance shown for all other data ."]],"string":"[\n [\n \"Chronic proliferative dermatitis mutation ( cpdm ) mice are deficient in SHARPIN ( Sharpincpdm/cpdm: henceforth referred to as Shpnm/m; protein: SHARPIN ) and develop dermatitis , multi-organ pathology and an immunological phenotype including disrupted lymphoid architecture , splenomegaly , liver inflammation and a loss of Peyer's patches in the gut ( HogenEsch et al . , 1993 , 1999; Seymour et al . , 2007 ) .\",\n \"SHARPIN is required for normal tumour necrosis factor ( TNF ) receptor 1 ( TNFR1 ) -mediated gene induction and prevention of TNF-mediated death of various cells , including epidermal keratinocytes , in vitro ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) .\",\n \"The dermatitis is characterized by epidermal cell death marked by cleaved caspase-3- , -8- and -9-positive cells ( Ikeda et al . , 2011; Liang and Sundberg , 2011; Potter et al . , 2014 ) .\",\n \"Since the dermatitis and inflammatory phenotype were shown to be TNF dependent , and because the only TNF signaling output that was aberrantly increased in the absence of SHARPIN was cell death , we previously proposed TNF/TNFR1-mediated cell death to be causative of the cpdm phenotype ( Gerlach et al . , 2011 ) .\",\n \"The role of neither TNFR1 nor cell death has been confirmed in vivo , however .\",\n \"TNFR1 signaling typically involves the intracellular recruitment of TNFR1-associated death domain protein ( TRADD ) , TNF receptor-associated factor 2 ( TRAF2 ) , cellular inhibitor of apoptosis ( cIAPs ) , and receptor interacting protein kinase 1 ( RIPK1 ) ( Silke , 2011 ) .\",\n \"The heterotrimeric linear ubiquitin chain assembly complex ( LUBAC ) of SHARPIN ( also known as SIPL ) , HOIL-1 ( RBCK1/RNF54 ) and HOIL-1L-interacting protein ( HOIP; RNF31 ) ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) is also recruited to the TNFR1 signaling complex .\",\n \"Here , it assembles a linear ubiquitin scaffold needed for full recruitment of the NF-κB essential modulator ( NEMO ) /NF-κB kinase subunit gamma ( IKKγ ) -containing IKK complex , which activates pro-survival NF-κB signaling .\",\n \"TNFR1-induced c-Jun N-terminal protein kinase ( JNK ) and p38 signaling is also regulated by LUBAC .\",\n \"SHARPIN deficiency blunts the TNFR1 pro-survival transcriptional signal and sensitizes cells to TNF-induced cell death .\",\n \"The E3 ligase activity of HOIP catalyzes the addition of linear ubiquitin to target proteins , and SHARPIN and HOIL-1 are key regulators of the stability and activity of HOIP ( Gerlach et al . , 2011 ) .\",\n \"In addition to TNFR1 , LUBAC has also been shown to regulate the transcriptional response from the interleukin-1 receptor ( IL-1R ) , CD40 , lymphotoxin beta receptor ( LTβR ) , toll-like-receptor 4 ( TLR4 ) , and nucleotide-binding oligomerization domain-containing protein 2 ( NOD2 ) receptor signaling complexes ( Schmukle and Walczak , 2012 ) .\",\n \"Deletion of Il1rap , needed for IL-1 signaling , has been reported to almost completely prevent the development of cpdm dermatitis ( Liang et al . , 2010 ) .\",\n \"This suggests that IL-1R signaling is a significant driver of disease , but the effect of Il1rap deficiency on the rest of the Shpnm/m phenotype was not reported .\",\n \"Cpdm mice have prominent eosinophil infiltration into the skin; however , deletion of Il5 , which dramatically reduces the number of cutaneous and circulating eosinophils , fails to ameliorate disease ( Renninger et al . , 2010 ) .\",\n \"Shpnm/mRag1−/− mice lacking functional lymphocytes develop dermatitis , indicating that T and B cell cells are not required for the skin phenotype ( Potter et al . , 2014 ) .\",\n \"Furthermore , hematopoietic cell transfer with bone marrow and spleen cells from cpdm mice to syngeneic wild-type C57BL/Ka mice failed to transfer disease in mice 2 months post reconstitution .\",\n \"Finally , cpdm skin transplanted onto nude mice retained the donor dermatitis phenotype 3 months post transplant , while syngeneic healthy skin transplanted onto cpdm mice did not acquire the disease over the same time ( HogenEsch et al . , 1993; Gijbels et al . , 1995 ) .\",\n \"Together these studies indicate that a skin-intrinsic defect in cpdm mice drives the inflammatory disease , however they do not rule out a role for the hematopoietic system in amplifying it .\",\n \"Impaired pro-survival TNFR1 signaling can induce both caspase-8-dependent apoptotic and RIPK3- and mixed lineage kinase domain-like protein ( MLKL ) -dependent necroptotic cell death via a cytosolic death platform ( Micheau and Tschopp , 2003; He et al . , 2009; Sun et al . , 2012; Zhao et al . , 2012; Murphy et al . , 2013 ) .\",\n \"Necroptosis involves the release of cellular contents including potential damage-associated molecular patterns ( DAMPs ) such as mitochondrial DNA , high mobility group box 1 protein ( HMGB1 ) , IL-33 , and IL-1α ( Kaczmarek et al . , 2013 ) .\",\n \"By contrast , apoptosis is considered to be immunologically silent , although this is clearly context dependent because excessive apoptosis resulting from conditional epidermal deletion of the caspase inhibitor cFLIP can cause severe skin inflammation ( Panayotova-Dimitrova et al . , 2013 ) .\",\n \"Caspase-8 can cleave both RIPK1 and RIPK3 and is needed to keep the necroptotic pathway in check ( Vandenabeele et al . , 2010; Kaiser et al . , 2011; Oberst et al . , 2011 ) .\",\n \"Regulation of necroptotic signaling is crucial for skin homeostasis because deletion of either caspase-8 , the caspase-8 adaptor protein FADD ( Fas-associated protein with death domain ) , or RIPK1 , leads to RIPK3- and MLKL-dependent epidermal hyperplasia and inflammation ( Kovalenko et al . , 2009; Lee et al . , 2009; Bonnet et al . , 2011; Kaiser et al . , 2011; Oberst et al . , 2011; Dannappel et al . , 2014; Dillon et al . , 2014; Rickard et al . , 2014 ) .\",\n \"Although the precise factors that determine whether TNFR1 mediates apoptosis or necroptosis are unclear , high levels of RIPK3 , loss of cIAPs , and CYLD-mediated deubiquitylation of RIPK1 appear conducive to necroptosis ( Silke and Vaux , 2014 ) .\",\n \"In addition to a crucial role in necroptosis , RIPK3 may also regulate inflammasome-induced IL-1ß production in the absence of IAPs or caspase-8 ( Vince et al . , 2012; Kang et al . , 2013 ) .\",\n \"Thus the effects of loss of RIPK3 on an inflammatory phenotype may not be due to loss of necroptotic cell death but to a less well-defined role in IL-1ß production .\",\n \"This complicates interpretation of the role of RIPK3 in a disease , particularly when IL-1 is pathogenic such as in cpdm dermatitis .\",\n \"MLKL is downstream of RIPK3 in necroptosis and appears not to be required for cytokine production in the same situations as RIPK3 ( Wong et al . , 2014 ) .\",\n \"Thus Mlkl−/− mice may provide an opportunity to disentangle the relative contribution of necroptosis and deregulated cytokine production in disease .\",\n \"Here we provide genetic evidence in support of our hypothesis that TNFR1-induced cell death is a driver of the inflammatory disease in cpdm mice ( Gerlach et al . , 2011 ) .\",\n \"We show that the cpdm phenotype is TNFR1 and cell-death dependent .\",\n \"Ripk3 or Mlkl deficiency largely prevented cpdm liver inflammation and ameliorated the splenic phenotype and leukocytosis .\",\n \"Remarkably , given the skin-inflammation phenotype of skin-specific Casp8 knock-out mice ( Kovalenko et al . , 2009; Lee et al . , 2009 ) , Casp8 heterozygosity potently suppressed the inflammatory skin phenotype while leaving the systemic inflammation unaffected .\",\n \"Strikingly , combined Ripk3 deficiency and Casp8 heterozygosity completely prevented the cpdm dermatitis in all but one of the mice analyzed at 42 to 45 weeks of age , prevented liver inflammation and grossly restored splenic architecture .\",\n \"Surprisingly , given the importance of LTßR ( also known as TNFRSF3 ) signaling in the formation of Peyer's patches ( De Togni et al . , 1994; Koni et al . , 1997 ) and the role of SHARPIN in this pathway ( Tokunaga et al . , 2011 ) , apparently normal Peyer's patches were also present in Shpnm/mCasp8+/-Ripk3−/− mice .\"\n ],\n [\n \"The dermatitis in Shpnm/m mice has previously been shown to be driven by TNF ( Gerlach et al . , 2011 ) and IL-1 signaling ( Liang et al . , 2010 ) .\",\n \"Because the environment may influence the onset of the disease we wished to test the importance of TNF and IL-1 signaling in a head-to-head manner .\",\n \"Furthermore the relative contribution of TNFR1 and TNFR2 in cpdm dermatitis has not been determined .\",\n \"We therefore generated Shpnm/m mice deficient in Tnfr1 , Tnfr2 or Il1r1 ( Figure 1A ) .\",\n \"All the knock-out mouse strains used in this study have been backcrossed at least ten times onto C57BL/6 , or were generated on the C57BL/6 background ( Mlkl−/− mice ) .\",\n \"However , the cpdm mutation arose on a C57BL/Ka background ( HogenEsch et al . , 1993 ) .\",\n \"To control for background modifier effects , we backcrossed C57BL/Ka Shpnm/m mice once or twice onto the C57BL/6 background , equivalent to the strategy we used in generating all our Shpnm/m compound knock-out strains to generate Shpnm/m C57BL/6 control mice .\",\n \"These control mice developed the cpdm phenotype indistinguishably from the C57BL/Ka Shpnm/m mice , typically presenting with a visible skin phenotype by 5 to 6 weeks and invariably requiring euthanasia due to the dermatitis before 14 weeks of age .\",\n \"Tnfr2 deletion did not ameliorate the dermatitis but Shpnm/mTnfr1−/− mice showed no outward signs of disease even after 35 weeks ( Figure 1A–C ) .\",\n \"Il1r1 deletion significantly delayed the appearance of dermatitis , with markedly reduced epidermal hyperplasia in 13-week-old Shpnm/mIl1r1−/− mice compared with 12-week-old Shpnm/m mice ( Figure 1B , C ) .\",\n \"However , by 19–20 weeks , Shpnm/mIl1r1−/− mice typically developed disease and required euthanasia .\",\n \"Tnfr1 deletion reduced periportal liver inflammation and partially ameliorated the splenic phenotype , but did not restore Peyer's patches , whilst Tnfr2 and Il1r1 deletion did not prevent pathology in any of these organs ( Figure 1B , D ) . 10 . 7554/eLife . 03464 . 003Figure 1 . Cpdm dermatitis is mediated by TNFR1 , IL-1R to a lesser extent , and not TNFR2 . ( A ) Representative photos of mice of indicated genotypes and age .\",\n \"( B ) Histological analysis of mice of genotype and age as indicated; representative of n ≥ 3 mice for each genotype or group .\",\n \"Black arrows in liver images point to areas of periportal inflammation .\",\n \"Black arrows in small intestine image point to Peyer's patches .\",\n \"Shpnm/m: Sharpincpdm/cpdm .\",\n \"Control mice are Shpn+/+ or +/m , Tnfr1−/− control mice are Shpn+/+ or +/mTnfr1−/− .\",\n \"Scale bars: Skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .\",\n \"H&E: hematoxylin and eosin .\",\n \"( C ) Epidermal thickness of mice of indicated age and genotypes .\",\n \"Each point represents the average of at least 14 measurements from multiple fields of view per mouse that were taken by a researcher who was blind to the genotype .\",\n \"Dotted lines are drawn at 30 µm and 14 weeks .\",\n \"Red numbers ( and black for middle graph ) correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .\",\n \"Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mTnfr1−/− and Shpn+/+ or +/mIl1r1−/− in upper , middle and lower graphs respectively .\",\n \"*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .\",\n \"( D ) Average spleen weights of mice of indicated genotypes .\",\n \"Spleen weights were taken from 12-week-old mice ( except Tnfr1−/− mice that were 15 or 35 weeks old ) , or younger mice if they required euthanasia due to their dermatitis .\",\n \"Data are represented as mean + SEM , *p ≤ 0 . 05 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 003 To gain insight into the pathogenesis of cpdm dermatitis we assessed cytokine levels in skin lysates from 6-week-old cpdm and control mice using a BioPlex kit ( BioRad ) ( Figure 2A ) to determine which cytokines were elevated early in the disease process .\",\n \"TNF levels were slightly elevated ( log scale , Figure 2A ) and , consistent with reports of eosinophilia in cpdm mice ( HogenEsch et al . , 1993; Gijbels et al . , 1996 ) , IL-5 ( a key inducer of eosinophil maturation ) was also elevated .\",\n \"The monocyte and macrophage chemoattractant protein MCP-1 was significantly elevated in cpdm skin , as was the IL-12 p40 subunit .\",\n \"There was also a trend for macrophage inflammatory protein 1α ( MIP-1α ) levels to be elevated .\",\n \"Consistent with this , there was an increase in F4/80+ cells in the Shpnm/m dermis , and this was evident in patches at 3 weeks , before significant epidermal hyperplasia was present ( Figure 2B ) .\",\n \"Ikeda et al . reported cleaved caspase-3-positive keratinocytes in 10-week-old Shpnm/m mice ( Ikeda et al . , 2011 ) , and we found cleaved caspase-3-positive cells were already present in the epidermis of 3-week-old Shpnm/m mice , indicating that apoptosis is an early event in the dermatitis and occurs before significant hyperplasia ( Figure 2C ) .\",\n \"There were slightly more cleaved caspase-3-positive cells in Shpnm/m livers than controls ( almost exclusively in the infiltrating cells ) , however they were infrequent .\",\n \"An increased number of cleaved caspase-3-positive cells were detected in Shpnm/m spleens , but , again , were much less appreciable than in the epidermis ( Figure 2C ) . 10 . 7554/eLife . 03464 . 004Figure 2 . Keratinocyte cell death and dermal macrophage infiltration are early events in cpdm dermatitis .\",\n \"( A ) BioPlex cytokine analysis of skin lysates from mice of indicated genotypes .\",\n \"Data are represented as mean +S . E . M . *p ≤ 0 . 05 .\",\n \"( B ) F4/80 staining ( brown ) of skin sections counterstained with hematoxylin ( blue ) .\",\n \"Control: Shpn+/+ or +/m .\",\n \"( C ) Cleaved caspase-3 staining ( brown ) of skin , liver and spleen sections counterstained with hematoxylin ( blue ) .\",\n \"Black arrows show examples of cleaved-caspase-3 positive cells .\",\n \"Control: Shpn+/+ or +/m .\",\n \"( A–C )\",\n \"Three mice were analyzed for each genotype or group .\",\n \"Scale bars: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 004 Reconstitution of wild-type mice with Shpnm/m bone marrow and/or spleen cells fails to transfer the disease ( HogenEsch et al . , 1993 ) , suggesting a skin-intrinsic defect underlies cpdm dermatitis .\",\n \"These mice , however , were only followed for 8 weeks post reconstitution and this may not have allowed sufficient time for the dermatitis to develop .\",\n \"To test this possibility , we reconstituted wild-type mice with Shpnm/m bone marrow cells and followed them for 12 months .\",\n \"Reconstitution efficiency was high but the mice did not demonstrate any skin , liver , or spleen phenotype during this time ( Figure 3 ) .\",\n \"Collectively these results suggest that the hematopoietic system in isolation cannot cause Shpnm/m dermatitis , and that macrophages may play a role in the amplification of the disease , particularly given they can be a prominent source of TNF . 10 . 7554/eLife . 03464 . 005Figure 3 . No dermatitis in wild-type mice reconstituted with Shpnm/m bone marrow after 12 months .\",\n \"( A ) No dermatitis was observed in wild-type mice 12 months after reconstitution with Shpnm/m bone marrow .\",\n \"( B ) Flow cytometry analysis showing percentage contribution of Ly5 . 1 ( recipient ) vs Ly5 . 2 ( donor ) white blood cells in peripheral blood 12 months after reconstitution .\",\n \"( C ) Histological and immunofluorescence analysis of dorsal skin from mice 12 months after reconstitution .\",\n \"( D ) Histological analysis of the spleen and liver from mice 12 months after reconstitution .\",\n \"Scale bars: Skin 100 µm , liver 200 µm and spleen 500 µm .\",\n \"H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 005 Tnf deletion prevents cpdm dermatitis and TNF kills Shpnm/m keratinocytes in vitro , suggesting that TNF-induced cell death drives the Shpnm/m skin phenotype ( Gerlach et al . , 2011 ) .\",\n \"Consistent with this , TNF-induced cell death in Shpnm/m keratinocytes that was partially blocked by Q-VD-OPh or Nec-1 treatment ( Figure 4A ) .\",\n \"Shpnm/m mouse dermal fibroblasts ( MDFs ) were also sensitive to TNF-induced cell death , and this death was almost completely blocked by the RIPK1 kinase inhibitor Necrostatin-1 ( Nec-1 ) , but not by the pan-caspase inhibitor Q-VD-OPh ( Figure 4B ) .\",\n \"To test whether TNF could induce caspase cleavage we treated primary Shpnm/m keratinocytes and MDFs with TNF for up to 4 hr ( Figure 4C , D , Figure 4—figure supplement 1 ) .\",\n \"TNF treatment led to caspase-3 and -8 cleavage at 4 hr in Shpnm/m , but not wild-type , keratinocytes .\",\n \"In Shpnm/m MDFs there was also caspase-3 and -8 cleavage , and , surprisingly , this was reduced by Nec-1 , but not Q-VD , treatment ( Figure 4D ) .\",\n \"In Shpnm/m keratinocytes and MDFs , the increased caspase-8 activity coincided with a marked processing of cFLIP , but other outcomes of the signaling pathway were less obviously affected .\",\n \"Together with the early presence of cleaved caspase-3 staining and the elevation of TNF in Shpnm/m skin , these results indicate that TNF-induced apoptosis may play a pathogenic role in the skin phenotype . 10 . 7554/eLife . 03464 . 006Figure 4 . TNF induces death in multiple primary cell types , and is marked by caspase-3 and -8 cleavage in primary keratinocytes and mouse dermal fibroblasts in vitro .\",\n \"( A and B )\",\n \"Primary keratinocytes ( A ) and mouse dermal fibroblasts ( MDFs ) ( B ) were treated with 100 ng/ml human Fc-TNF , 50 µM Nec-1 or 10 µM Q-VD-OPh for 24 hr as indicated , then viability was assessed by propidium iodide ( PI ) uptake and flow cytometry .\",\n \"Cells were generated from three different mice for each group .\",\n \"Control: Shpn+/+ or +/m .\",\n \"( C and D )\",\n \"Western blot analysis of primary keratinocytes ( C ) and MDFs ( D ) treated as indicated ( concentrations as in A and B ) then lysed and lysates separated on SDS/PAGE and western blotted with indicated antibodies .\",\n \"Shpn+/m: n = 1; Shpnm/m: n = 2 ( n = 1 for MDFs ) mice analyzed shown above .\",\n \"* Indicates non-specific band .\",\n \"Data from additional mice is shown in Figure 4—figure supplement 1 .\",\n \"( E ) Neutrophils , monocytes and bone-marrow-derived macrophages were treated with 100 ng/ml human Fc-TNF , 50 µM Nec-1 , 20 µM Q-VD-OPh ( 10 µM for macrophages ) or 500 nM Compound A ( CpdA ) for 20 hr ( 24 hr for macrophages ) as indicated .\",\n \"Viability was assessed by PI uptake and flow cytometry .\",\n \"The Smac mimetic CpdA sensitizes cells to TNF-induced cell death and serves as a control .\",\n \"Cells were generated from three different mice for each group except for macrophages , where six to eight mice were analyzed .\",\n \"( A , B , E )\",\n \"Data are represented as mean + SEM , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 00610 . 7554/eLife . 03464 . 007Figure 4—figure supplement 1 . TNF induces caspase-3 and -8 cleavage in primary keratinocytes and mouse dermal fibroblasts in vitro . Western blot analysis of primary keratinocytes and mouse dermal fibroblasts ( MDFs ) treated as indicated ( reagent concentrations as per Figure 4A , B ) then lysed and lysates separated on SDS/PAGE and western blotted with indicated antibodies .\",\n \"* Indicates non-specific band .\",\n \"Lysates generated from different mice to those shown in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 007 Given the appearance of cleaved caspase-3-positive cells in the Shpnm/m dermis ( Figure 2C ) , we sought to determine whether some of these might be non-fibroblast cells such as immune cells .\",\n \"Since lymphoid cells are not required for cpdm dermatitis ( Potter et al . , 2014 ) and macrophage infiltration appears early , we tested whether myeloid cells were also sensitive to TNF-induced death .\",\n \"Shpnm/m neutrophils , monocytes and bone-marrow-derived macrophages ( BMDMs ) were all sensitive to killing by TNF , whereas the wild-type cells were not ( Figure 4E ) .\",\n \"Neutrophil and monocyte cell death was more efficiently blocked by a combination of Nec-1 and Q-VD , whereas in macrophages Nec-1 was sufficient to block cell death .\",\n \"These data suggest that myeloid cell death may contribute to the inflammatory skin phenotype .\",\n \"We did not detect a defect in p38 , JNK or NF-κB pro-survival signaling in SHARPIN-deficient keratinocytes and MDFs , as has been shown for other cell types ( Gerlach et al . , 2011; Ikeda et al . , 2011; Tokunaga et al . , 2011 ) .\",\n \"Consistent with earlier reports ( Gerlach et al . , 2011; Peltzer et al . , 2014 ) , however , using an antibody that specifically recognises linear ubiquitin we detected substantially reduced linear ubiquitylation in the native TNFR1 signaling complex obtained from Shpnm/m vs wild-type mouse embryonic fibroblasts ( MEFs ) ( Figure 5A ) .\",\n \"We also found that HaCaT keratinocytes ( a human immortalized cell line ) stably expressing catalytically inactive HOIPC885S were sensitive to TNF-induced cell death ( Figure 5B ) , indicating a requirement for LUBAC and its linear-ubiquitin-chain-forming activity in preventing TNF-induced keratinocyte death . 10 . 7554/eLife . 03464 . 008Figure 5 . Sharpin is required for normal linear ubiquitylation of the TNF-R1 signaling complex , and HOIP protects keratinocytes from TNF-induced cell death .\",\n \"( A ) Anti Flag Immuno-Precipitation ( IP ) of the TNF receptor signaling complex in immortalised mouse embryonic fibroblasts ( MEFs ) treated with Flag-TNF for the times indicated .\",\n \"( B ) HaCaT human keratinocytes stably expressing HOIP , the catalytically inactive HOIPC885S , or an empty vector ( control ) were treated with 100 ng/ml TNF for 24 hr .\",\n \"Viability was assessed by propidium iodide ( PI ) uptake and flow cytometry .\",\n \"Data are presented as mean + SEM , n = 3 , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 008 To investigate the importance of the caspase-8-mediated apoptotic pathway in vivo we generated Shpnm/mCasp8+/− mice .\",\n \"We did not attempt to generate Shpnm/mCasp8−/− mice because Casp8 deletion results in embryonic lethality at around E10 . 5 ( Varfolomeev et al . , 1998 ) .\",\n \"In contrast to Shpnm/m mice , 12-week-old Shpnm/mCasp8+/− mice had almost no epidermal hyperplasia and largely normal keratin 6 and 14 expression ( Figure 6A , B ) .\",\n \"By 15 weeks of age , however , significant hyperplasia was observed in one of these mice ( Figure 6B ) .\",\n \"Shpnm/mCasp8+/− mice retained other aspects of the phenotype: splenomegaly with disrupted splenic architecture , liver inflammation , and a lack of intestinal Peyer's patches ( Figure 6C ) .\",\n \"Some Shpnm/mCasp8+/− mice succumbed to a pulmonary infection with Pasteurella and required euthanasia while littermates were unaffected , suggesting that Shpnm/mCasp8+/− mice are partially immunocompromised .\",\n \"Mice with obvious signs of infection were not used for the spleen , blood , or liver analyses but were included in the skin analysis .\",\n \"Occult infection , however , in Shpnm/mCasp8+/− mice used for hematopoietic , liver , and spleen analyses cannot be completely excluded . 10 . 7554/eLife . 03464 . 009Figure 6 . Protection from cpdm dermatitis with Casp8 heterozygosity but not Bid deletion .\",\n \"( A ) Histological and immunofluorescence skin analysis .\",\n \"( B ) Epidermal thickness of mice of indicated age and genotypes determined as in Figure 1B , by an investigator blinded to genotype .\",\n \"Top panel is a repeat of data in 1B for reference purposes only .\",\n \"Dotted lines are drawn at 30 µm and 14 weeks .\",\n \"Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .\",\n \"Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mBid−/− , and Shpn+/+ or +/mCasp8+/− in upper , middle and lower graphs , respectively .\",\n \"*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .\",\n \"( C ) Histological analysis of spleen , liver , and small intestine .\",\n \"Black arrows in liver images point to areas of periportal inflammation .\",\n \"Black arrow in small intestine image points to Peyer's patch .\",\n \"( D ) Average spleen weights of mice of indicated genotypes .\",\n \"Spleen weights were taken from 12–14-week-old mice , or younger mice if they required euthanasia due to their dermatitis .\",\n \"Data are represented as mean + SEM , ***p ≤ 0 . 005 .\",\n \"( A and C )\",\n \"Control mice are Shpn+/+ or +/m , n ≥ 3 mice analyzed each genotype or group .\",\n \"Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .\",\n \"H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 009 In certain cell types such as hepatocytes , BID can be cleaved by caspase-8 to generate truncated BID ( tBID ) that , in turn , mediates caspase-9- and mitochondria-dependent apoptosis ( Czabotar et al . , 2014 ) .\",\n \"BID is a key regulator of UV-induced apoptosis in keratinocytes , indicating that this pathway is of importance in the skin ( Pradhan et al . , 2008 ) .\",\n \"Because cleaved caspase-9 is found in the Shpnm/m epidermis ( Ikeda et al . , 2011 ) and Shpnm/m keratinocyte extracts contained caspase-9-substrate-cleaving activity ( Liang and Sundberg , 2011 ) , we hypothesized that TNF-induced BID-dependent apoptosis may be a driver of the cpdm phenotype and , hence , generated Shpnm/mBid−/− mice .\",\n \"We observed no protection from any aspects of the cpdm phenotype ( Figure 6A–D ) , however , indicating that caspase-8 , but not BID , is an important driver of this disease .\",\n \"Caspase-8 heterozygosity significantly delayed epidermal hyperplasia , but other aspects of the cpdm phenotype ( e . g . splenomegaly and liver inflammation ) remained .\",\n \"We therefore sought to test the role of necroptosis in the inflammatory disease by generating Shpnm/mRipk3−/− mice .\",\n \"Whereas control mice invariably developed severe dermatitis by 12 weeks of age , roughly half of the 12-week-old Shpnm/mRipk3−/− mice had a less severe epidermal phenotype at this point ( Figure 7A , B ) .\",\n \"When aged over 12 weeks , all Shpnm/mRipk3−/− animals went on to develop severe disease and were euthanized due to their skin phenotype before 18 weeks of age .\",\n \"The remainder of Shpnm/mRipk3−/− mice developed skin disease at the same rate as control mice .\",\n \"12 week-old Shpnm/mRipk3−/− mice had no signs of liver inflammation and significantly less splenomegaly , although they still lacked Peyer's patches ( Figure 7C , D ) .\",\n \"We also generated Shpnm/mMlkl−/− mice and found they had a similar epidermal phenotype to Shpnm/m mice ( Figure 7B ) .\",\n \"Like Shpnm/mRipk3−/− mice , however , 12-week-old Shpnm/mMlkl−/− animals had reduced splenomegaly and only one out of 12 mice showed signs of liver inflammation ( Figure 7C , D ) .\",\n \"Collectively these results indicate that RIPK3 and MLKL are important drivers of the liver and splenic cpdm phenotype , and that RIPK3 contributes to the epidermal phenotype , importantly in a non-MLKL-dependent ( hence , most likely , necroptosis-independent ) manner . 10 . 7554/eLife . 03464 . 010Figure 7 . Ripk3 deficiency slightly delays cpdm dermatitis onset , and Ripk3 and Mlkl deficiency partially protects against the cpdm splenic phenotype and markedly attenuates liver inflammation .\",\n \"( A ) Histological and immunofluorescence skin analysis .\",\n \"( B ) Epidermal thickness of mice of indicated age and genotypes determined as in Figure 1B , by an investigator blinded to genotype .\",\n \"Top panel is a repeat of data in 1B for reference purposes only .\",\n \"Dotted lines are drawn at 30 µm and 14 weeks .\",\n \"Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .\",\n \"Control mice are Shpn+/+ or +/m , Shpn+/+ or +/mRipk3−/− , and Shpn+/+ or +/mMlkl−/− in upper , middle and lower graphs , respectively .\",\n \"*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 , # significantly different to Shpnm/m mice ( Fisher's exact test ) , p ≤ 0 . 05 .\",\n \"( C ) Histological analysis of spleen , liver , and small intestine .\",\n \"Black arrows in liver image points to areas of periportal inflammation .\",\n \"Black arrows in small intestine image points to Peyer's patches .\",\n \"( D ) Average spleen weights of mice of indicated genotypes .\",\n \"Spleen weights were taken from 12-week-old mice , or younger mice if they required euthanasia due to their dermatitis .\",\n \"Data are represented as mean + SEM , ***p ≤ 0 . 005 .\",\n \"( A and C )\",\n \"Control mice are Shpn+/+ or +/m , n ≥ 3 mice analyzed each genotype or group .\",\n \"Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .\",\n \"H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 010 Since deletion neither of one allele of Casp8 nor of two alleles of Ripk3 was able to fully rescue all the multi-organ pathology in cpdm mice , we sought to generate Shpnm/mCasp8−/−Ripk3−/− mice , taking advantage of the fact that Ripk3 deletion prevents Casp8−/−-mediated embryonic lethality ( Kaiser et al . , 2011; Oberst et al . , 2011 ) .\",\n \"We generated these mice independently at two separate facilities .\",\n \"At one facility , most of these triple-deficient mice died perinatally , typically in a window between E17 and 1 to 2 days after birth , and no Shpnm/mCasp8-/-Ripk3−/− animals were obtained at weaning ( Figure 8A , B; numbers in A refer to mice analyzed at first facility only ) .\",\n \"Most Shpnm/mCasp8−/−Ripk3−/− embryos obtained by Caesarean section at E19 appeared edematous or were dead .\",\n \"Some were able to breathe; however , these died within the first 2 days after birth .\",\n \"Hematopoietic analysis at E19 did not reveal any consistent differences ( Figure 8C ) .\",\n \"At a separate facility , two viable Shpnm/mCasp8−/−Ripk3−/− mice were obtained from a limited number of matings .\",\n \"By roughly 3 months of age these mice appeared runted and were euthanized .\",\n \"Histologically these mice had no epidermal phenotype , but disrupted splenic architecture was apparent ( Figure 8D ) . 10 . 7554/eLife . 03464 . 011Figure 8 . Shpnm/mCasp8−/−Ripk3−/− mice are prone to perinatal lethality .\",\n \"( A ) Table of segregation of expected and observed genotypes from Shpn+/mCasp8-/-Ripk3−/− or Shpnm/mCasp8+/-Ripk3−/− intercrosses at various developmental stages .\",\n \"E10 . 5 controls: Shpn+/+ or +/mCasp8-/-Ripk3−/−; E19 controls: Shpnm/mCasp8+/+ or +/−Ripk3−/−; weaning controls: Shpn+/+ or +/mCasp8−/−Ripk3−/− or Shpnm/mCasp8+/+ or +/−Ripk3−/− .\",\n \"*Significantly different to expected value , Fisher's exact test p < 0 . 0005 .\",\n \"( B ) Photos of E19 embryos obtained by Caesarian section .\",\n \"The Shpnm/mCasp8−/−Ripk3−/− mouse on the right was recovered dead , all others were alive .\",\n \"Other embryos are control mice: Shpnm/mCasp8+/+ or +/−Ripk3−/− .\",\n \"( C ) ADVIA blood analysis from E19 embryos .\",\n \"RBC: red blood cells; WBC: white blood cells; MCV: mean cell volume .\",\n \"Horizontal lines depict data means .\",\n \"Control mice: Shpnm/mCasp8+/+ or +/−Ripk3−/− .\",\n \"( D ) Histological analysis of tissue from 12-week-old mice of indicated genotypes .\",\n \"Mice were from a separate facility to those in A–C and are not included in the table in A . Two mice were analyzed for each genotype .\",\n \"Scale bars: skin 100 µm , spleen 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 011 While the reason for the lethality of the triple-deficient animals is unknown , we were readily able to obtain viable Shpnm/mCasp8+/−Ripk3−/− mice .\",\n \"These mice were indistinguishable from control mice at 12 weeks of age and had no skin , liver , or spleen pathology ( Figure 9A–E ) .\",\n \"Remarkably , Peyer's patches were present ( Figure 9C , Figure 9—figure supplement 1 ) .\",\n \"One 67-week-old and three 45-week-old Shpnm/mCasp8+/−Ripk3−/− mice had no signs of any skin phenotype , however another mouse developed dermatitis at 42 weeks ( Figure 9A , B , D; note that epidermal thickness was not quantified for the 42-week-old mouse ) . 10 . 7554/eLife . 03464 . 012Figure 9 . Ripk3 deletion and Casp8 heterozygosity markedly delays emergence of cpdm dermatitis and liver inflammation and restores Peyer’s patches .\",\n \"( A ) Representative photos of mice of indicated genotypes and age .\",\n \"Three Shpnm/mCasp8+/-Ripk3−/− mice were analyzed at 45 weeks and one at 67 weeks with no detectable dermatitis , however another mouse developed dermatitis by 42 weeks of age .\",\n \"( B ) Histological and immunofluorescence skin analysis .\",\n \"( C ) Histological analysis of spleen , liver , and small intestine .\",\n \"Black arrows in liver image point to areas of periportal inflammation .\",\n \"Black arrows in small intestine images point to Peyer's patches .\",\n \"( D ) Epidermal thickness of mice of indicated age and genotypes measured as described in Figure 1B .\",\n \"Top panel is a repeat of data in 1B for reference purposes only .\",\n \"Dotted lines are drawn at 30 µm and 14 weeks .\",\n \"Red numbers correspond to proportion of Shpnm/m mice with epidermal thickness >30 µm at < 14 weeks of age ( upper left quadrant ) .\",\n \"Control mice are Shpn+/+ or +/m and Shpn+/+ or +/mCasp8+/−Ripk3−/− in upper and lower graphs , respectively .\",\n \"*** Significantly different to control group ( Fisher's exact test ) , p ≤ 0 . 005 .\",\n \"( E ) Average spleen weights of mice of indicated genotypes .\",\n \"Spleen weights were taken from 12-week-old mice , or younger mice if they required euthanasia due to their dermatitis .\",\n \"Data are represented as mean + SEM , ***p ≤ 0 . 005 .\",\n \"( B and C )\",\n \"Control mice are Shpn+/+ or +/m , n ≥ 3 mice analysed each genotype or group .\",\n \"Scale bars: skin and liver 100 µm , spleen 500 µm , small intestine 1 mm .\",\n \"H&E: hematoxylin and eosin . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 01210 . 7554/eLife . 03464 . 013Figure 9—figure supplement 1 . Restoration of Peyer's patches in 8-week-old Shpnm/mCasp8+/−Ripk3−/− mice . Numbers of Peyer's patches per small intestine counted from 8-week-old mice of indicated genotypes .\",\n \"Horizontal lines depict data means . DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 013 To investigate the effect of the various genetic crosses on Shpnm/m leukocytosis , we analyzed white blood cell levels in peripheral blood using an ADVIA hematological analyzer ( Figure 10 ) .\",\n \"Tnfr1 deletion was more effective at reducing leukocyte numbers than Tnfr2 or Il1r1 deletion , although all of these compound knock-out mice had leukocyte subsets that were elevated compared to controls .\",\n \"Caspase-8 heterozygosity or Bid deletion did not prevent the leukocytosis , whereas Ripk3 and Mlkl deletion markedly reduced it , suggesting the hematopoietic phenotype is driven predominantly by necroptosis .\",\n \"Shpnm/mRipk3−/− and Shpnm/mMlkl−/− mice still had elevated neutrophils , however Shpnm/mCasp8+/−Ripk3−/− mice had no white blood cell elevation . 10 . 7554/eLife . 03464 . 014Figure 10 . Peripheral blood counts from various crosses . Peripheral blood was collected from 11–14-week-old mice ( reconstituted mice: Shpnm/m → wild type were 12 months old and Tnfr1−/− mice were 35 weeks old ) , or younger if the mice were euthanized due to severe dermatitis .\",\n \"Shpn+/+ or +/m and Shpnm/m mice are highlighted in blue and red for reference purposes .\",\n \"Blood was analyzed using an ADVIA 2120 hematological analyzer .\",\n \"RBC: red blood cells .\",\n \"Horizontal lines depict data means .\",\n \"Control mice are Shpn+/+ or +/m , for example Tnfr1−/− control mice are Shpn+/+ or +/mTnfr1−/− .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 03464 . 014\"\n ],\n [\n \"LUBAC , composed of three proteins , HOIL-1 , HOIP and SHARPIN , has recently emerged as a regulator of a diverse set of signaling complexes ( Zak et al . , 2011; Schmukle and Walczak , 2012; Tokunaga , 2013 ) .\",\n \"Lack of HOIP causes mid-gestational embryonic lethality ( embryonic day 10 . 5 ) , and this is prevented by simultaneous loss of Tnfr1 ( Peltzer et al . , 2014 ) .\",\n \"Together , these data demonstrate that LUBAC is required for full-strength IL-1ß and TNF signaling and deficiency in these signaling pathways would be expected to impair an inflammatory response .\",\n \"The consequence , however , of Sharpin deficiency in mice is multi-organ inflammation ( HogenEsch et al . , 1993 ) .\",\n \"Likewise , loss-of-expression and loss-of-function mutations in HOIL-1 result in a fatal , human , inherited disorder characterized by chronic inflammation; consistent with loss of the major defensive inflammatory signaling pathways , affected patients suffer from invasive bacterial infections ( Boisson et al . , 2012 ) .\",\n \"In vitro experiments suggest that HOIL-1 is an essential component of LUBAC , which is present in many cell types .\",\n \"With this in mind it is surprising that HOIL-1-deficient mice were reported to be normal ( Tokunaga et al . , 2009 ) .\",\n \"If , however , we set this observation to one side , it seems that in humans and mice loss of LUBAC can precipitate an inflammatory disease .\",\n \"This could be because the loss of the homeostatic component of TNFR1 , IL-1R1 , and TLR signaling reduces the ability of an organism to resist infection , which , even in the weakened signaling environment of LUBAC deficiency , drives inflammatory signaling .\",\n \"Another possibility is that full-strength signaling from TNF , IL-1ß , or TLR ligands is required to upregulate essential negative feedback regulators such as IκBα and A20 .\",\n \"Signaling , therefore , although reduced , is constitutively active .\",\n \"This explanation seems less likely because IκBα and A20 transcripts were still upregulated in response to TNF or Pam3CSK4 in HOIL-1/HOIP-deficient and cpdm cells ( Haas et al . , 2009; Tokunaga et al . , 2009; Gerlach et al . , 2011; Zak et al . , 2011 ) .\",\n \"The fact that the phenotype from the cpdm mice could not be transferred by the hematopoietic compartment , and that the skin phenotype is maintained following skin transplantation onto nude mice , indicates that these animals suffer from an intrinsic skin defect ( HogenEsch et al . , 1993; Gijbels et al . , 1995 ) .\",\n \"This is not unprecedented; loss of Notch signaling in the skin causes a dermatitis disease in mice that is sufficient to drive systemic inflammation with similar features to the cpdm phenotype ( Dumortier et al . , 2010 ) .\",\n \"The earlier hematopoietic reconstitution experiments , however , may not have allowed sufficient time for the inflammatory phenotype to develop .\",\n \"The work described here excludes this caveat because reconstituted mice did not develop signs of the cpdm phenotype even a year after reconstitution; thus , the inflammatory skin phenotype in the Shpnm/m mutant mice is the result of an intrinsic skin defect .\",\n \"It is , however , noteworthy that loss of Mlkl did not affect the Shpnm/m skin phenotype but significantly reduced or prevented splenomegaly and liver inflammation .\",\n \"Conversely , reduction in caspase-8 markedly ameliorated the skin phenotype but did not prevent splenomegaly or liver inflammation .\",\n \"This shows that the skin and systemic phenotype are separable , although the exact mechanism is unclear .\",\n \"The fact that cpdm keratinocytes are sensitized to TNF-induced apoptosis and necroptosis led us to hypothesize that TNF/TNFR1-dependent cell death was causative for the skin phenotype ( Gerlach et al . , 2011 ) .\",\n \"Because of the systemic inflammation we suspected that necroptotic cell death and the release of DAMPs was the driver .\",\n \"Consistent with this hypothesis , we show here that Tnfr1 deficiency ( as with Tnf deficiency ) suppresses the cpdm phenotype , whereas Tnfr2 deficiency had no effect on the phenotype .\",\n \"Loss of IL-1 signaling has been shown to suppress the cpdm phenotype ( Liang et al . , 2010 ) , but because the environment of the mice likely plays a large part in the onset and severity of the inflammatory disease it was not possible to determine whether loss of IL-1 signaling is as potent as Tnf deficiency at preventing the phenotype .\",\n \"We therefore generated Shpnm/mIl1r1−/− mice , which succumbed to the inflammatory disease much later than Shpnm/m mice , but were far more inflammation prone than any of the Shpnm/mTnf−/+ , Shpnm/mTnf−/− , or Shpnm/mTnfr1−/− mice .\",\n \"These data are again consistent with our original hypothesis , suggesting that TNF/TNFR1 signaling is the main driver of inflammation and that IL-1 contributes to exacerbating the disease .\",\n \"Casp8 deficiency results in early embryonic lethality in mice ( Varfolomeev et al . , 1998 ) , and loss of Casp8 or Fadd in the skin leads to keratinocyte hyperplasia and inflammatory skin disease ( Kovalenko et al . , 2009; Bonnet et al . , 2011 ) .\",\n \"Therefore , we considered it unlikely that loss of caspase-8 would diminish the chronic proliferative dermatitis of the Shpnm/m mutant mice .\",\n \"To our surprise , however , loss of a single allele of Casp8 was strikingly effective at reducing the skin phenotype and appearance of cleaved caspase-3 in the skin .\",\n \"It has been proposed that the intrinsic mitochondrial apoptosis pathway mediated by caspase-9 and caspase-3 plays a role in the keratinocyte hyperplasia observed in Shpnm/m mutant mice based on the presence of disrupted mitochondria in cpdm skin and in vitro experiments ( Liang and Sundberg , 2011 ) .\",\n \"Given the prominent role that caspase-8 plays in the dermatitis , it would be expected that if the intrinsic apoptosis pathway is engaged it should be downstream of caspase-8 and require cleavage of the BH3 protein , BID ( Czabotar et al . , 2014 ) .\",\n \"Cells that require BID cleavage by caspase-8 in order to undergo apoptosis are known as type II cells ( Barnhart et al . , 2003 ) , but it is unclear whether keratinocytes are type I or type II ( Pradhan et al . , 2008; Geserick et al . , 2014 ) .\",\n \"Genetic deletion of Bid did not suppress the dermatitis or any other aspect of the cpdm phenotype , therefore we conclude that the intrinsic mitochondrial pathway is unlikely to play a significant role in the disease .\",\n \"Because necroptotic cell death can be inflammatory , by provoking the release of DAMPs , we expected that deficiency in RIPK3 or MLKL , essential effectors of the necroptotic cell death pathway , would reduce the severity of the inflammation in Shpnm/m mice .\",\n \"Whilst there was a modest delay in the appearance of the dermatitis in Shpnm/mRipk3−/− mice , all animals went on to develop severe skin disease .\",\n \"In contrast to RIPK3 deficiency , deletion of Mlkl did not even delay appearance of the dermatitis .\",\n \"Together , this suggests that RIPK3 may exacerbate the skin phenotype independently of necroptosis , possibly via a direct role in cytokine production .\",\n \"Combined with the markedly ameliorated dermatitis seen in Shpnm/mCasp8+/− mice , this indicates that apoptosis is the main driver of Shpnm/m dermatitis .\",\n \"Although apoptosis is generally regarded as being immunologically inert , when in excess it can result in severe inflammation .\",\n \"For example , conditional deletion of cFLIP in adult keratinocytes caused severe inflammation in the epidermis ( Panayotova-Dimitrova et al . , 2013 ) .\",\n \"Like the cpdm phenotype , this inflammation was TNF dependent .\",\n \"An excess of apoptotic cells may cause disease by overwhelming phagocytosis and clearance of apoptotic bodies , leading to secondary necrosis and DAMP release .\",\n \"It has also been proposed that in certain contexts , such as viral infections , apoptosis may be inflammatory ( Cullen et al . , 2013 ) .\",\n \"In other Shpnm/m organs such as the liver , spleen , and hematopoietic system , Ripk3 and Mlkl deletion ameliorated or protected against disease , indicating that Sharpin deficiency triggers apoptosis in some tissues but necroptosis in others .\",\n \"Because it has been shown that RIPK3 or MLKL can mediate cytokine production induced by the absence of caspase-8 ( Kang et al . , 2013 ) , and potentially other , as yet undescribed , pathways , we cannot completely exclude the possibility that Ripk3 or Mlkl deletion affords protection by blunting cytokine production .\",\n \"Absence of SHARPIN , however , leads to increased , not decreased , caspase-8 activity , so it is not obvious whether these observations apply in this case .\",\n \"Furthermore , whereas it is clear that RIPK3 plays a role in promoting cytokine production in response to a number of stimuli ( Vince et al . , 2012 ) , we have so far only observed a defect in necroptosis and not in inflammatory cytokine production in Mlkl−/− cells ( Murphy et al . , 2013; Allam et al . , 2014; Rickard et al . , 2014 ) .\",\n \"Whilst Shpnm/mCasp8+/− , Shpnm/mRipk3−/− , and Shpnm/mMlkl−/− mice all developed significant disease in either the liver , spleen , hematopoietic compartment , or skin , Shpnm/mCasp8+/−Ripk3−/− mice were almost completely protected from disease for approximately 10–11 months , and in one case 15 months .\",\n \"One of these mice developed the skin phenotype at 10 months of age , indicating that the single allele of Casp8 is enough to eventually cause dermatitis .\",\n \"This is supported by the development of epidermal phenotype in 14–15-week-old Shpnm/mCasp8+/− mice .\",\n \"SHARPIN has been shown to regulate LTß/LTßR signaling , and LTßR is required for the development of Peyer's patches ( De Togni et al . , 1994 ) , therefore we had assumed that the absence of Peyer's patches was due to defective LTßR signaling .\",\n \"A recent study , however , demonstrated that rudimentary Peyer's patches formed in Shpnm/m embryos , but that these regressed post-natally ( Seymour et al . , 2013 ) .\",\n \"Our data show that whilst neither Casp8 heterozygosity nor Ripk3 deletion in isolation restored intestinal Peyer's patches , the combination was able to do so .\",\n \"This suggests that the post-natal regression of these secondary lymphoid organs in Shpnm/m mice is due to deregulated cell death .\",\n \"In a wider sense , our results are particularly important when interpreting the recent finding that crossing cpdm mice with Ripk1K45A/K45A mice ( which lack RIPK1 kinase activity ) completely suppresses the cpdm phenotype ( Berger et al . , 2014 ) .\",\n \"RIPK1 has a promiscuous role in immune signaling , regulating pro-survival , necroptotic , and apoptotic pathways .\",\n \"However , while the RIPK1 kinase domain is well known for its ability to activate the necroptotic pathway , it is not believed to be required to cause apoptosis ( Vandenabeele et al . , 2010 ) .\",\n \"Yet we show here that the Shpnm/m skin phenotype is associated with the appearance of processed caspase-3 in the skin , and both the epidermal hyperplasia and appearance of processed caspase-3 is markedly reduced by loss of a single allele of Casp8 .\",\n \"Consistent with the hypothesis that Shpnm/m keratinocyte hyperplasia is due to apoptosis , it is not prevented by loss of the key necroptotic effector MLKL .\",\n \"Furthermore , purified Shpnm/m keratinocytes and dermal fibroblasts rapidly activate caspase-8 and caspase-3 in response to TNF .\",\n \"Taken together with the work of Berger et al . our work inescapably suggests that in the context of the Shpnm/m epidermis the dominant role of the RIPK1 kinase domain is to activate apoptosis .\",\n \"Unexpectedly , but supporting this conclusion , we showed that Nec-1 ( the RIPK1 kinase inhibitor ) blocked TNF-induced caspase-8 and caspase-3 activation in Shpnm/m dermal fibroblasts .\",\n \"This conclusion is particularly confronting because we and others have shown that loss of RIPK1 in the skin results in a RIPK3/MLKL-dependent hyperplasia that is presumably dependent on necroptosis ( Dannappel et al . , 2014; Rickard et al . , 2014 ) .\",\n \"Thus RIPK1 is able to both activate and inhibit either apoptosis or necroptosis in a highly context-dependent manner .\",\n \"Ripk3 deletion prevents the embryonic lethality seen with either Fadd or Casp8 deletion and the mice survive to adulthood ( Kaiser et al . , 2011; Oberst et al . , 2011; Dillon et al . , 2012 ) .\",\n \"Given this , the perinatal lethality we observed in Shpnm/mCasp8−/−Ripk3−/− mice was completely unexpected .\",\n \"At E19 some of these mice were recovered alive by Caesarean section and established regular breathing , some failed to establish normal breathing , some appeared edematous and were not recovered alive , whilst others were in the process of being resorbed from earlier embryonic lethality .\",\n \"To complicate the picture , two viable Shpnm/mCasp8−/−Ripk3−/− mice were obtained at a separate facility .\",\n \"At 3 months of age these mice had no epidermal phenotype but were runted .\",\n \"One potential explanation for the different penetrance of the phenotype may be a different genetic background because the mice described in Figure 8A were exon 3-deleted caspase-8 mice ( Beisner et al . , 2005 ) , whereas the mice in Figure 8D were exon 3- and 4-deleted caspase-8 ( Salmena et al . , 2003 ) .\",\n \"Exon 3- and 4-deleted caspase-8 mice in yet a third facility ( not shown ) , however , did not survive past weaning , indicating that environmental differences undoubtedly also contribute to the variable penetrance .\",\n \"Future efforts aimed at understanding the lethality of Shpnm/mCasp8−/−Ripk3−/− mice should yield important insights into not only the biology of SHARPIN and linear ubiquitin , but also that of caspase-8 and RIPK3 .\",\n \"In summary , we provide strong evidence that Sharpin deficiency sensitizes keratinocytes to TNF/TNFR1-induced , caspase-8-mediated apoptosis , and that this defect appears to drive the cpdm dermatitis .\",\n \"Ripk3 deletion provided only a modest and variable delay in the presentation of dermatitis .\",\n \"Unlike Casp8 heterozygosity , Ripk3 and Mlkl deletion ameliorated many aspects of the systemic cpdm phenotype .\",\n \"This indicates a tissue-specific role for Sharpin in regulating cell death pathways .\",\n \"Only combined Casp8 heterozygosity and Ripk3 deficiency was able to almost completely prevent all aspects of the cpdm systemic phenotype that we evaluated , including the early loss of Peyer's patches .\",\n \"Whilst inflammation is a known sequelae to necroptotic DAMP release , these findings provide further evidence that excessive apoptosis can also cause inflammatory disease .\",\n \"Furthermore , these results indicate that the suppression of cpdm dermatitis seen by crossing to mice lacking RIPK1 kinase activity ( Berger et al . , 2014 ) may , surprisingly , be due to RIPK1’s kinase activity being upstream of caspase-8 in mediating TNF-induced apoptosis .\"\n ],\n [\n \"Mice were maintained at the Walter and Eliza Hall Institute of Medical Research ( WEHI ) and University College London ( UCL ) .\",\n \"C57BL/Ka Sharpincpdm/cpdm mice were obtained from Jax ( Bar Harbor , ME ) , and then either backcrossed one to two times onto C57BL/6 or crossed with C57BL/6 Ripk3−/− , Mlkl−/− , Casp8+/− , Casp8+/-Ripk3−/− , Bid−/− , Il1r1−/− , Tnfr1−/− , or Tnfr2−/− mice .\",\n \"For timed matings , mice were analyzed by Caesarean section .\",\n \"For E19 timed matings , pregnant females were injected at E17 and E18 with progesterone .\",\n \"Primary keratinocytes and MDFs were isolated and cultured as described previously ( Gerlach et al . , 2011; Etemadi et al . , 2013 ) .\",\n \"Cell lysates were prepared using DISC buffer ( 1% NP-40 , 10% glycerol , 150 mM NaCl , 20 mM Tris pH 7 . 5 , 2 mM EDTA , cOmplete protease inhibitor cocktail ( Roche; Penzberg , Germany ) , 2 mM sodium orthovanadate , 10 mM sodium fluoride , β-glycerophosphate , N2O2PO7 ) .\",\n \"Cell lysates were loaded in NuPAGE Bis-Tris gels ( Life Technologies/Thermo Fisher Scientific; Waltham , MA ) and transferred on to Immobilon-P PVDF membranes ( Millipore; Billerica , MA ) or Hybond-C Extra ( GE Healthcare; Little Chalfont , UK ) .\",\n \"Membranes were blocked and antibodies diluted in 5% skim milk powder or Bovine Serum Albumin ( BSA ) in 0 . 1% PBS or TBS-Tween20 .\",\n \"Antibodies used for western blot: cleaved caspase-3 ( 9661 ) and −8 ( 8592 ) , phospho-JNK1/2 ( 4668P ) , phospho-p38 ( 4511 ) , p38 ( 9212 ) , caspase-8 ( 4927 ) , JNK1/2 ( 9252 ) , IκBα ( CN: 9242 ) , and phospho-p65 ( 3033 ) from Cell Signaling Technology ( Danvers , MA ) , β-actin ( A-1978; Sigma Aldrich; St . Louis , MO ) , RIPK1 ( 610458; BD Biosciences; Franklin Lakes , NJ ) , cFLIP ( AG-20B-0005; Adipogen; Liestal , Switzerland ) , FADD ( generated in-house; gift from Lorraine O’Reilly ) and MLKL ( generated in house; Murphy et al . , 2013 ) .\",\n \"Signals were detected by chemoluminescence ( Millipore ) after incubation with secondary antibodies conjugated to horseradish peroxidase .\",\n \"For isolation of neutrophils and monocytes , red blood cells were lysed and bone marrow cells were stained with flurochrome-conjugated anti-mouse Ig antibodies ( CD11b [Mac-1] and Ly6G [1A8] ) and sorted using a FACS ARIA instrument ( BD Biosciences ) .\",\n \"Neutrophils ( CD11b+ Ly6G+ ) and monocytes ( CD11b+Ly6G− ) were cultured in 5% FCS RPMI at 1 × 105 and 0 . 5 × 105 cells per well , respectively , in a 96-well u-bottom tissue culture plate .\",\n \"BMDMs were isolated and cultured as described previously ( Wong et al . , 2014 ) .\",\n \"Keratinocytes , MDFs , neutrophils , monocytes , and BMDMs were stimulated with TNF ( 100 ng/ml ) , Nec-1 ( 50 μM ) , QVD-Oph ( 10 μM , 20 μM for neutrophils and monocytes ) and CpdA ( 911 , 500 nM ) .\",\n \"After 24 hr ( 20 hr for neutrophils and monocytes ) all cells except keratinocytes were stained with propidium iodide ( PI ) and cell death analyzed on a FACScalibur instrument ( BD Biosciences ) .\",\n \"For the keratinocyte MTS viability assay phenazine methosulfate ( PMS; 0 . 92 mg/ml in PBS; Sigma-Aldrich ) and 3- ( 4 , 5-dimethylthiazol-2-yl ) -5- ( 3-carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) -2H-tetrazolium ( MTS; 2 mg/ml in PBS; Promega; Fitchburg , WI ) were combined in a 1:20 ratio .\",\n \"The mixture was added to cell culture media in a 1:5 ratio and incubated for 1–4 hr at 37°C in a humidified 5% CO2 incubator .\",\n \"Media was transferred to a flat-bottom 96-well plate and absorbance was measured at 490 nm .\",\n \"Viability was calculated relative to the untreated sample .\",\n \"For HaCaT experiments , cells stably expressing HOIP , HOIPC885S ( catalytically inactive ) , or an empty vector were seeded and incubated the next day with 100 ng/ml histidine-tagged TNF for 24 hr .\",\n \"Supernatant and adherent cells were harvested and resuspended in PBS containing 5 mg/ml PI .\",\n \"PI-positive cells were measured by flow cytometry ( BD Accuri; BD Biosciences ) .\",\n \"Paraffin-embedded tissue was fixed in 10% neutral buffered formalin then processed and stained with hematoxylin and eosin ( H&E ) according to standard practices .\",\n \"Immunohistochemistry and immunofluorescence analysis was performed as described previously ( Rickard et al . , 2014 ) .\",\n \"Ly5 . 1 mice were irradiated with 2 × 550 rads spaced 3 hr apart .\",\n \"Following red blood cell lysis , ∼ 5 × 106 BM cells from Shpnm/m ( Ly5 . 2 ) mice were intravenously injected .\",\n \"Mice were maintained on 2 mg/ml neomycin in drinking water for 3 weeks post irradiation .\",\n \"Reconstitution efficiency was assessed 6 weeks and 12 months post reconstitution by staining for Ly5 . 1 and Ly5 . 2 in peripheral blood obtained from retro-orbital bleeding .\",\n \"Cytokines were analyzed using a BioPlex Pro mouse cytokine 23-plex kit ( Bio-Rad; Hercules , CA ) , or for analysis of TNF levels a mouse TNF ELISA kit ( eBioscience; San Diego , CA ) was used .\",\n \"Skin lysates were prepared by homogenizing skin in ice cold protein lysis buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 2 mM EDTA , 1% Triton-X100 , 10% glycerol ) using a Tissue Lyser II ( QIAGEN; Hilden , Germany ) for 12 cycles of 30 s at 30 Hz .\",\n \"A BCA kit ( Thermo Fisher Scientific ) was used to normalise protein levels .\",\n \"Values below the reference range were assigned the value of the lowest standard .\",\n \"Peripheral blood was collected into EDTA tubes ( Sarstedt; Nümbrecht , Germany ) and analyzed using an ADVIA 2120 hematological analyzer .\",\n \"For TNF-RSC isolation , immortalised MEFs were stimulated with 3xFlag-2xStrep-TNF at 0 . 5 μg/ml for the indicated times , or left untreated .\",\n \"Cells were subsequently solubilized in lysis buffer ( 30 mM Tris–HCl ( pH 7 . 4 ) , 150 mM NaCl , 2 mM EDTA , 2 mM KCl , 10% Glycerol , 1% Triton X100 , EDTA-free proteinase inhibitor cocktail ( Roche ) and 1x phosphatase inhibitor cocktail 2 ( Sigma Aldrich ) ) at 4°C for 30 min .\",\n \"The lysates were cleared by centrifugation , and 3xFlag-2xStrep-TNF was added to the untreated samples .\",\n \"Next , lysates were subjected to anti-Flag immunoprecipitation using M2 beads ( Sigma Aldrich ) for 16 hr .\",\n \"The beads were washed three times with lysis buffer , proteins were eluted in 50 μl of LDS buffer ( Life Technologies/Thermo Fisher Scientific ) containing 50 mM DTT .\",\n \"Samples were analyzed by western blotting .\",\n \"Antibodies used were: HOIP ( custom-made , Thermo Fisher Scientific ) , SHARPIN ( 14626-1-AP; ProteinTech; Chicago , IL ) , TNFR1 ( ab19139; Abcam; Cambridge , UK ) , and linear ubiquitin ( Genentech; South San Francisco , CA ) .\",\n \"Pearson chi-square and Fisher's exact test were used to assess frequencies of observed vs expected genotypes during development , at birth , and at weaning .\",\n \"Fisher's exact test was used for epidermal thickness statistical calculations .\",\n \"Student's t test was used to calculate statistical significance shown for all other data .\"\n ]\n]","isConversational":false},"abstract":{"kind":"list like","value":["SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro .","The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation , characterized by dermatitis , liver inflammation , splenomegaly , and loss of Peyer's patches .","TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1 , but not Tnfr2 , deficiency suppresses the phenotype ( and it does so more efficiently than Il1r1 loss ) .","TNFR1-induced apoptosis can proceed through caspase-8 and BID , but reduction in or loss of these players generally did not suppress inflammation , although Casp8 heterozygosity significantly delayed dermatitis .","Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype , and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it , even restoring Peyer's patches .","Unexpectedly , Sharpin , Ripk3 and Casp8 triple deficiency caused perinatal lethality .","These results provide unexpected insights into the developmental importance of SHARPIN ."],"string":"[\n \"SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro .\",\n \"The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation , characterized by dermatitis , liver inflammation , splenomegaly , and loss of Peyer's patches .\",\n \"TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1 , but not Tnfr2 , deficiency suppresses the phenotype ( and it does so more efficiently than Il1r1 loss ) .\",\n \"TNFR1-induced apoptosis can proceed through caspase-8 and BID , but reduction in or loss of these players generally did not suppress inflammation , although Casp8 heterozygosity significantly delayed dermatitis .\",\n \"Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype , and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it , even restoring Peyer's patches .\",\n \"Unexpectedly , Sharpin , Ripk3 and Casp8 triple deficiency caused perinatal lethality .\",\n \"These results provide unexpected insights into the developmental importance of SHARPIN .\"\n]","isConversational":false},"summary":{"kind":"list like","value":["In response to an injury or infection , areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body .","At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apoptosis and necroptosis .","Apoptosis is a highly controlled process during which the contents of the cell are safely destroyed in order to prevent damage to surrounding cells .","Necroptosis , on the other hand , is not controlled: the cell bursts and releases its contents into the surroundings .","Inflammation is activated by a protein called TNFR1 , which is controlled by a complex that includes a protein called SHARPIN .","Mice that lack the SHARPIN protein develop inflammation on the skin and internal organs , even in the absence of injury or infection .","However , it is not clear how SHARPIN controls TNFR1 to prevent inflammation .","Rickard et al . and , independently Kumari et al . have now studied this process in detail .","Rickard et al . cross bred mice that lack SHARPIN with mice lacking other proteins involved in inflammation and cell death .","The experiments show that apoptosis is the main form of cell death in skin inflammation , but necroptosis has a bigger role in the inflammation of internal organs .","Mice that lack both the apoptotic and necroptotic cell-death pathways can develop relatively normally , but they die shortly after birth if they also lack SHARPIN .","Experiments on these mice could help us to understand how SHARPIN works ."],"string":"[\n \"In response to an injury or infection , areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body .\",\n \"At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apoptosis and necroptosis .\",\n \"Apoptosis is a highly controlled process during which the contents of the cell are safely destroyed in order to prevent damage to surrounding cells .\",\n \"Necroptosis , on the other hand , is not controlled: the cell bursts and releases its contents into the surroundings .\",\n \"Inflammation is activated by a protein called TNFR1 , which is controlled by a complex that includes a protein called SHARPIN .\",\n \"Mice that lack the SHARPIN protein develop inflammation on the skin and internal organs , even in the absence of injury or infection .\",\n \"However , it is not clear how SHARPIN controls TNFR1 to prevent inflammation .\",\n \"Rickard et al . and , independently Kumari et al . have now studied this process in detail .\",\n \"Rickard et al . cross bred mice that lack SHARPIN with mice lacking other proteins involved in inflammation and cell death .\",\n \"The experiments show that apoptosis is the main form of cell death in skin inflammation , but necroptosis has a bigger role in the inflammation of internal organs .\",\n \"Mice that lack both the apoptotic and necroptotic cell-death pathways can develop relatively normally , but they die shortly after birth if they also lack SHARPIN .\",\n \"Experiments on these mice could help us to understand how SHARPIN works .\"\n]","isConversational":false},"year":{"kind":"string","value":"2014"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":2,"numItemsPerPage":100,"numTotalItems":4346,"offset":200,"length":100}},"jwt":"eyJhbGciOiJFZERTQSIsImtpZCI6IjVHZDBvd0g5MTM2eDZjc1FvbE1zcktNWUZoRVFoUm5PVVVybEpjOUhaUEEifQ.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc4NjE3OTgxNiwianRpIjoiNTA0YWRjZTktMjNjNC00ZmU1LTkyZmEtODg5OTQ4NDlmZDMzIiwic3ViIjoiL2RhdGFzZXRzL3NhbmR5MzcvZWxpZmUiLCJleHAiOjE3ODYxODM0MTYsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.b9T2RyF9k24bE655VW5JepBIQOrkvI-jtkAK9i9Xd-hMtL7NQSXzZfSltOMLoqKxd7EXlOnETowb_0VMBxeKCQ","displayUrls":true,"splitSizeSummaries":[{"config":"default","split":"train","numRows":4346,"numBytesParquet":131728263},{"config":"default","split":"test","numRows":241,"numBytesParquet":7238778},{"config":"default","split":"validation","numRows":241,"numBytesParquet":7230553}]},"discussionsStats":{"closed":0,"open":1,"total":1},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
headings list | keywords list | title stringlengths 30 189 | id stringlengths 14 14 | sections list | abstract list | summary list | year stringclasses 11
values |
|---|---|---|---|---|---|---|---|
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits | elife-08153-v2 | [
[
"Protein degradation by the ubiquitin-proteasome system ( UPS ) fulfills essential roles in eukaryotic cells in maintaining proteome homeostasis ( proteostasis ) , signaling , and cell cycle progression .",
"The 26S proteasome is a large macromolecular machine composed of the 20S catalytic core and the 19... | [
"Hallmarks of cancer , including rapid growth and aneuploidy , can result in non-oncogene addiction to the proteostasis network that can be exploited clinically .",
"The defining example is the exquisite sensitivity of multiple myeloma ( MM ) to 20S proteasome inhibitors , such as carfilzomib .",
"However , MM ... | [
"Cells have several mechanisms for removing proteins that have been damaged or are no longer needed .",
"One of these mechanisms is carried out by a large protein complex called the proteasome .",
"Drugs that block the proteasome are toxic to all cells , and a type of blood cancer called multiple myeloma is par... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Dual functions of TAF7L in adipocyte differentiation | elife-00170-v1 | [
[
"Adipose tissue plays a central role in energy homeostasis by acting as a major lipid storage site as well as an important endocrine tissue .",
"Excess food intake vs energy expenditure invariably leads to obesity , an increasingly prevalent condition in modern societies .",
"Obesity is , in turn , ti... | [
"The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood .",
"Here we report that TAF7L , a paralogue of TFIID subunit TAF7 , is enriched in adipocytes and white fat tissue ( WAT ) in mouse .",
"Depletion of TAF7L reduced adipocyte-... | [
"The development of a single fertilized egg into a highly complex animal is determined by its genome , with a process called differential gene regulation exerting exquisite control over gene expression to ensure that various specialized cells are generated and that many types of tissue are produced .",
"However ,... | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"chromosomes and gene expression"
] | Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation | elife-02104-v2 | [
[
"Pluripotent mouse embryonic stem cells ( mESCs ) have the ability to self-renew or to differentiate into all cell types .",
"Specific transcription factors like Oct4 , Sox2 , and Nanog form a core transcriptional network , which is required for the maintenance of mESC pluripotency ( Orkin et al . , 2008 ... | [
"The histone acetyltransferase ( HAT ) Mof is essential for mouse embryonic stem cell ( mESC ) pluripotency and early development .",
"Mof is the enzymatic subunit of two different HAT complexes , MSL and NSL .",
"The individual contribution of MSL and NSL to transcription regulation in mESCs is not well unders... | [
"Embryonic stem cells are special cells that have the ability to become many different types of cells , such as skin , muscle , or neuronal cells .",
"This process is called differentiation .",
"They can also undergo a process called self-renewal to produce more embryonic stem cells .",
"These processes are c... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"physics of living systems"
] | Metabolic constraints drive self-organization of specialized cell groups | elife-46735-v3 | [
[
"During the course of development , groups of isogenic cells often form spatially organized , interdependent communities .",
"The emergence of such phenotypically heterogeneous , spatially constrained sub-populations of cells is considered a requisite first step towards multicellularity .",
"Here , cl... | [
"How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains unresolved .",
"We find that within a mature , clonal yeast colony developing in low glucose , cells arrange into metabolically disparate cell groups .",
"Using this system , we model and experimentally identify ... | [
"Under certain conditions , single-celled microbes such as yeast and bacteria form communities of many cells .",
"In some cases , the cells in these communities specialize to perform specific roles .",
"By specializing , these cells may help the whole community to survive in difficult environments .",
"These ... | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"tools and resources"
] | An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish | elife-64744-v1 | [
[
"Lipid droplets are cellular organelles that act as storage sites for neutral lipids and are key regulators of cellular metabolism ( Wilfling et al . , 2013 ) .",
"Lipid droplets are present in most cell types and are characterized by a monophospholipid membrane surrounding a hydrophobic lipid core ( Tauc... | [
"Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells .",
"Although increasingly implicated in disease , current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes , limiting investigation of their rapid ... | [
"Organisms need fat molecules as a source of energy and as building blocks , but these ‘lipids’ can also damage cells if they are present in large amounts .",
"Cells guard against such toxicity by safely sequestering lipids in specialized droplets that participate in a range of biological processes .",
"For ins... | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis | elife-17877-v3 | [
[
"The highly conserved exosome complex , an RNA-degrading and processing machine , is expressed in all eukaryotic cells ( Januszyk and Lima , 2014; Kilchert et al . , 2016 ) .",
"The first subunit was discovered from an analysis of mechanisms controlling yeast rRNA synthesis .",
"Mutations of Exosc2 ( ... | [
"Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs , it almost certainly controls diverse biological processes .",
"How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood .",
"Previous... | [
"Red blood cells supply an animal’s tissues with the oxygen they need to survive .",
"These cells circulate for a certain amount of time before they die .",
"To replenish the red blood cells that are lost , first a protein called stem cell factor ( SCF ) instructs stem cells and precursor cells to proliferate ,... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cancer biology"
] | STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells | elife-53003-v3 | [
[
"Protein phosphorylation plays a regulatory role in nearly all biological processes and dysregulation of protein phosphorylation contributes to many diseases .",
"Both kinases and phosphatases have been implicated in the pathogenesis of specific cancers , and several small molecule kinase inhibitors are s... | [
"Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers , and partial loss of PP2A function contributes to cell transformation .",
"Displacement of regulatory B subunits by the SV40 Small T antigen ( ST ) or mutation/deletion of PP2A subunits alters the abundance and ty... | [
"Cells maintain a fine balance of signals that promote or counter cell growth and division .",
"Two sets of enzymes – called kinases and phosphatases – contribute to this balance .",
"In general , kinases “switch on” other proteins by tagging them with a phosphate molecule .",
"This process is called phosphor... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Thrombospondin-4 controls matrix assembly during development and repair of myotendinous junctions | elife-02372-v3 | [
[
"Cellular structure and function depend on dynamic interactions with extracellular matrix ( ECM ) proteins , defects in which cause many diseases such as muscular dystrophies and osteoarthritis ( Emery , 2002; Mayer , 2003; Kanagawa and Toda , 2006; Carmignac and Durbeej , 2012; Maldonado and Nam , 2013 ) .",... | [
"Tendons are extracellular matrix ( ECM ) -rich structures that mediate muscle attachments with the skeleton , but surprisingly little is known about molecular mechanisms of attachment .",
"Individual myofibers and tenocytes in Drosophila interact through integrin ( Itg ) ligands such as Thrombospondin ( Tsp ) , ... | [
"Tendons , the tough connective tissues that link muscles to bones , are essential for lifting , running and other movements in animals .",
"A matrix of proteins , called the extracellular matrix , connects the cells in a tendon , giving it the strength it needs to prevent muscles from detaching from bones during... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands | elife-00205-v1 | [
[
"The capacity to segregate and functionalize regulatory elements within large and complex vertebrate genomes relies on the activity of chromatin modifying and epigenetic systems ( Kouzarides , 2007 ) .",
"Nevertheless , it remains very poorly understood how the enzymes responsible for initiating these fun... | [
"CpG islands ( CGIs ) are associated with most mammalian gene promoters .",
"A subset of CGIs act as polycomb response elements ( PREs ) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development .",
"How CGIs function mechanistically as nucleation sites f... | [
"Gene expression in eukaryotic cells can be controlled in a number of different ways , including various epigenetic mechanisms that do not involve making changes to DNA sequences that define the genes themselves .",
"A well-known epigenetic mechanism for silencing genes in vertebrates is DNA methylation—the addit... | 2012 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"neuroscience"
] | Noise promotes independent control of gamma oscillations and grid firing within recurrent attractor networks | elife-06444-v2 | [
[
"Cognitive processes are mediated by computations in neural circuits and are often associated with gamma frequency oscillations in circuit activity .",
"Gamma activity and cognitive performance often co-vary within tasks and between individuals , while cognitive deficits in psychiatric disorders such as a... | [
"Neural computations underlying cognitive functions require calibration of the strength of excitatory and inhibitory synaptic connections and are associated with modulation of gamma frequency oscillations in network activity .",
"However , principles relating gamma oscillations , synaptic strength and circuit com... | [
"When electrodes are placed on the scalp , or lowered into the brain itself , rhythmic waves of electrical activity are seen that reflect the coordinated firing of large numbers of neurons .",
"The pattern of the waves varies between different brain regions , and according to what the animal or person is doing ."... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | A shared numerical representation for action and perception | elife-16161-v1 | [
[
"Animals , including humans , estimate spontaneously and reasonably accurately the approximate quantity of arrays of objects , without recourse to other forms of representation , such as density ( Cicchini et al . , 2016 ) .",
"Even newborn infants of less than 3 days show selective habituation to number ... | [
"Humans and other species have perceptual mechanisms dedicated to estimating approximate quantity: a sense of number .",
"Here we show a clear interaction between self-produced actions and the perceived numerosity of subsequent visual stimuli .",
"A short period of rapid finger-tapping ( without sensory feedbac... | [
"Humans and many other animals have the ability to make spontaneous and rapid estimates of the approximate number of items that they can see .",
"This sense of number , or “numbersense” , is particularly important in humans , as evidence suggests that it lays the groundwork for acquiring mathematical skills .",
... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"microbiology and infectious disease"
] | Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake | elife-65282-v3 | [
[
"Malaria parasites evade host immunity by replicating within vertebrate erythrocytes .",
"In humans , the virulent Plasmodium falciparum pathogen uses multiple ligands for erythrocyte invasion ( Cowman et al . , 2012 ) and then remodels its host cell to achieve tissue adherence and nutrient acquisition ( ... | [
"Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake .",
"As the member proteins are unique to Plasmodium spp .",
", how they interact and traffic through subcellular sites to serve these essential functions is unknown .",
"We show that RhopH is synthesized as... | [
"Malaria is an infectious disease caused by the family of Plasmodium parasites , which pass between mosquitoes and animals to complete their life cycle .",
"With one bite , mosquitoes can deposit up to one hundred malaria parasites into the human skin , from where they enter the bloodstream .",
"After increasin... | 2021 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"evolutionary biology"
] | A Mesozoic clown beetle myrmecophile (Coleoptera: Histeridae) | elife-44985-v1 | [
[
"A pervasive feature of colony-forming insect societies is the profusion of intruder arthropods that have evolved to exploit their rich resources ( Kistner , 1979; Kistner , 1982; Hölldobler and Wilson , 1990; Parker , 2016 ) .",
"The diversity of such organisms is impressive , with ~10 , 000 species hypo... | [
"Complex interspecies relationships are widespread among metazoans , but the evolutionary history of these lifestyles is poorly understood .",
"We describe a fossil beetle in 99-million-year-old Burmese amber that we infer to have been a social impostor of the earliest-known ant colonies .",
"Promyrmister kistn... | [
"Many animals live lives that are closely intertwined with those of other species .",
"While a clown fish sheltering within the tentacles of a sea anemone may be a textbook example , ‘symbiotic’ interactions that occur inside ant nests are among some of the most dramatic .",
"Known as myrmecophiles – after the ... | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | A spiral attractor network drives rhythmic locomotion | elife-27342-v1 | [
[
"The increasing availability of large scale recordings of brain networks at single neuron resolution provides an unprecedented opportunity to discover underlying principles of motor control .",
"However , such long-sought data sets are revealing a new challenge - the joint activity of large neural populat... | [
"The joint activity of neural populations is high dimensional and complex .",
"One strategy for reaching a tractable understanding of circuit function is to seek the simplest dynamical system that can account for the population activity .",
"By imaging Aplysia’s pedal ganglion during fictive locomotion , here w... | [
"In all animals , neurons in the brain work together to generate movement .",
"From a slug’s ability to crawl , to your ability to move your hand , movement is dependent on hundreds or thousands of neurons being active at the same time .",
"Rhythmic movements such as crawling or swimming show this clearly: grou... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol | elife-25466-v2 | [
[
"Animal cells carefully control both their overall content of cholesterol as well as its distribution among organelles .",
"Although present in the membrane of every organelle , 60–90% of a cell’s cholesterol is concentrated in its plasma membrane ( PM ) ( De Duve , 1971; Lange et al . , 1989 ) .",
"I... | [
"Cells employ regulated transport mechanisms to ensure that their plasma membranes ( PMs ) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins ( LDL ) and synthesis .",
"To date , all inhibitors of cholesterol transport block steps in lysosomes , limiting our understanding of p... | [
"Cells are surrounded by a plasma membrane made mostly from oily molecules known as lipids .",
"One of these lipids , called cholesterol , is essential for keeping this membrane stable .",
"Cholesterol is partly produced within the cells at a specialized structure called the endoplasmic reticulum , and partly i... | 2017 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"cell biology",
"short report",
"biochemistry and chemical biology"
] | Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates | elife-36620-v2 | [
[
"N-BPs are the most commonly prescribed drugs used to treat osteoporosis ( Drake et al . , 2008 ) .",
"They have two negatively charged phosphonate groups that bind to hydroxyapatite crystals with high affinity and enable efficient accumulation of N-BPs on the bone surface ( Drake et al . , 2008 ) .",
... | [
"Nitrogen-containing-bisphosphonates ( N-BPs ) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases .",
"Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts , the mechanism by which N-BPs enter the cytosol from ... | [
"As some people age , their bones may become weak , brittle , and break easily .",
"This condition is called osteoporosis .",
"To treat osteoporosis , doctors often prescribe drugs called nitrogen-containing bisphosphonates ( NBPs ) .",
"These drugs destroy cells called osteoclasts , which break down bone .",... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Early patterning and specification of cardiac progenitors in gastrulating mesoderm | elife-03848-v2 | [
[
"Mammalian heart development involves the allocation of cardiac progenitors in a discrete spatial and temporal order ( Evans et al . , 2010; Bruneau , 2013 ) .",
"Understanding the identity and regulation of these progenitors is critical to understanding the origins of congenital heart defects and may lea... | [
"Mammalian heart development requires precise allocation of cardiac progenitors .",
"The existence of a multipotent progenitor for all anatomic and cellular components of the heart has been predicted but its identity and contribution to the two cardiac progenitor ‘fields’ has remained undefined .",
"Here we sho... | [
"Most internal organs in the body are made up of several different kinds of cells .",
"Understanding where these cells come from and how these different cells develop from a single cell in an embryo could help to guide regenerative therapies , where tissues grown in the laboratory are used to repair damage that t... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health",
"tools and resources",
"microbiology and infectious disease"
] | Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure | elife-40183-v2 | [
[
"The potential of super-resolution microscopy ( SRM ) to unravel details of the structure and replication of viruses was recognised early on in the development of the methodology ( Betzig et al . , 2006; Müller and Heilemann , 2013 ) .",
"Since then , SRM has been used to provide unprecedented insights in... | [
"Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses .",
"Here , we report on a new methodology which combines Structured Illumination... | [
"Viruses are like the Trojan horses of the biological world; they sneak their genetic code into a living cell and then hijack it , forcing that cell to produce their own viral proteins .",
"Yet , if scientists replace the harmful genes in a virus with other genes , the virus can be transformed into a powerful too... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | α8β1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism | elife-13063-v2 | [
[
"Coordinated gastrointestinal motility orchestrates nutrient absorption by mixing ingested food with digestive enzymes ( Armand et al . , 1994; Kong and Singh , 2008 ) and by propelling the food bolus from the stomach to the small intestine , the primary site of nutrient absorption .",
"Dysfunctional gast... | [
"Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders .",
"In this work , we demonstrate a critical role for the RGD-binding integrin α8β1 in promoting nutrient absorption through regulation of gastrointestinal motility .... | [
"Animals absorb nutrients from the food they eat in a complicated process that involves multiple steps .",
"In the mouth , teeth break down the food into smaller chunks .",
"Then the food passes through the stomach and small intestine , where enzymes break it down into individual molecules that are small enough... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Arid1b haploinsufficient mice reveal neuropsychiatric phenotypes and reversible causes of growth impairment | elife-25730-v3 | [
[
"It is becoming clear that SWI/SNF chromatin-remodeling complexes have a major impact on human diseases , from cancer to neuropsychiatric disorders to body size regulation ( Kadoch and Crabtree , 2015; Ronan et al . , 2013 ) .",
"SWI/SNF chromatin-remodeling complexes use the energy of ATP to remodel nucl... | [
"Sequencing studies have implicated haploinsufficiency of ARID1B , a SWI/SNF chromatin-remodeling subunit , in short stature ( Yu et al . , 2015 ) , autism spectrum disorder ( O'Roak et al . , 2012 ) , intellectual disability ( Deciphering Developmental Disorders Study , 2015 ) , and corpus callosum agenesis ( Halg... | [
"DNA does not just float freely inside our cells .",
"Instead , it is wound around proteins called histones and packaged tidily into a form called chromatin .",
"This packaging allows genes to be switched on or off by making it easier or harder to access different stretches of the genetic code .",
"A group of... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology"
] | Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist | elife-13828-v2 | [
[
"Glucagon , a 29-amino acid-long hormone synthetized in pancreatic α-cells through cleavage of its precursor , proglucagon , by prohormone convertase 2 ( PC2 ) , counterbalances the effects of insulin on blood glucose homeostasis by stimulating hepatic glycogenolysis and gluconeogenesis ( Gromada et al . , 20... | [
"Glucagon secretion dysregulation in diabetes fosters hyperglycemia .",
"Recent studies report that mice lacking glucagon receptor ( Gcgr-/- ) do not develop diabetes following streptozotocin ( STZ ) -mediated ablation of insulin-producing β-cells .",
"Here , we show that diabetes prevention in STZ-treated Gcgr... | [
"After meals , digested food causes sugar to accumulate in the blood .",
"This triggers the release of the hormone insulin from beta cells in the pancreas , which allows liver cells , muscle cells and fat cells to use and store the sugar for energy .",
"Other cells in the pancreas , called alpha cells , release... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"genetics and genomics"
] | MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly | elife-57068-v1 | [
[
"The glucose-sensing Mondo pathway is a key regulator of energy metabolism ( Abdul-Wahed et al . , 2017; Richards et al . , 2017 ) .",
"It consists of three basic helix-loop-helix/leucine zipper ( bHLH/Zip ) family transcription factors ( TFs ) : the two Mondo family factors ‘Mlx interacting protein’ ( Ml... | [
"The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals .",
"Here , we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development .",
"We showed that knockdown of mondoa impaired the early morphogenetic movement o... | [
"In most animals , a protein called MondoA closely monitors the amount of glucose in the body , as this type of sugar is the fuel required for many life processes .",
"Glucose levels also act as a proxy for the availability of other important nutrients .",
"Once MondoA has detected glucose molecules , it turns ... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine"
] | Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration | elife-19484-v2 | [
[
"The regenerative capacity of adult skeletal muscle is endowed in a population of Pax7-expressing resident stem cells called satellite cells ( SCs ) ( Brack et al . , 2012 ) .",
"Genetic studies utilizing lineage labeling , as well as cell ablation , have established that Pax7-expressing SCs are essential... | [
"Skeletal muscle regenerative potential declines with age , in part due to deficiencies in resident stem cells ( satellite cells , SCs ) and derived myogenic progenitors ( MPs ) ; however , the factors responsible for this decline remain obscure .",
"TGFβ superfamily signaling is an inhibitor of myogenic differen... | [
"Even in adulthood , injured muscles can repair themselves largely because they contain groups of stem cells known as satellite cells .",
"These cells divide to produce progenitor cells that later develop , or differentiate , into new muscle fibers .",
"However as muscles get older , this repair process becomes... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Synaptic memory requires CaMKII | elife-60360-v2 | [
[
"The mechanism by which the brain stores information has fascinated neuroscientists for well over a century .",
"Two general ideas have emerged .",
"The first proposes that information is held by ongoing activity in neuron ensembles .",
"This , indeed , underlies short-term working memory ( D’Espo... | [
"Long-term potentiation ( LTP ) is arguably the most compelling cellular model for learning and memory .",
"While the mechanisms underlying the induction of LTP ( ‘learning’ ) are well understood , the maintenance of LTP ( ‘memory’ ) has remained contentious over the last 20 years .",
"Here , we find that Ca2+-... | [
"How the brain stores information is a question that has fascinated neuroscientists for well over a century .",
"Two general ideas have emerged .",
"The first is that groups of neurons hold information by staying active .",
"The second is that they hold information by strengthening their connections to one an... | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser | elife-53514-v2 | [
[
"Phytochrome photosensor proteins are crucial for the optimal development of all vegetation on Earth ( Butler et al . , 1959; Gan et al . , 2014; Quail et al . , 1995 ) .",
"Prototypical phytochromes can exist in two photochemical states with differential cellular signaling activity , called red light-abs... | [
"Phytochrome proteins control the growth , reproduction , and photosynthesis of plants , fungi , and bacteria .",
"Light is detected by a bilin cofactor , but it remains elusive how this leads to activation of the protein through structural changes .",
"We present serial femtosecond X-ray crystallographic data ... | [
"Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction .",
"To detect the level of light , plant cells use proteins called phytochromes , which are also found in some bacteria and fungi .",
"Phytochrome proteins change shape when t... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | A tension-adhesion feedback loop in plant epidermis | elife-34460-v4 | [
[
"As our understanding of the role of forces in development deepens , assessing accurate stress patterns in tissues has become increasingly important ( Roca-Cusachs et al . , 2017 ) .",
"Stress patterns can be revealed through three approaches: 1- Computational models , for example with spring networks or ... | [
"Mechanical forces have emerged as coordinating signals for most cell functions .",
"Yet , because forces are invisible , mapping tensile stress patterns in tissues remains a major challenge in all kingdoms .",
"Here we take advantage of the adhesion defects in the Arabidopsis mutant quasimodo1 ( qua1 ) to dedu... | [
"The parts of a plant that protrude from the ground are constantly shaken by the wind , applying forces to the plant that it must be able to resist .",
"Indeed , mechanical forces are crucial for the development , growth and life of all organisms and can trigger certain behaviours or the production of particular ... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Oxyntomodulin regulates resetting of the liver circadian clock by food | elife-06253-v2 | [
[
"Extended night or rotating shift work is associated with an elevated risk for developing cancer , cardiovascular disease , immune deficiency , mood disorders , and metabolic alterations ( Rosenberg and Doghramji , 2011; Herichova , 2013 ) .",
"One major factor believed to contribute to this adverse healt... | [
"Circadian clocks coordinate 24-hr rhythms of behavior and physiology .",
"In mammals , a master clock residing in the suprachiasmatic nucleus ( SCN ) is reset by the light–dark cycle , while timed food intake is a potent synchronizer of peripheral clocks such as the liver .",
"Alterations in food intake rhythm... | [
"Humans and other animals have adapted their behavior and their biology to the daily cycle of light and dark .",
"Groups of genes are reliably switched on or off at different times of the day , and act as internal , or ‘circadian’ , clocks that help these organisms to stay on a 24-hour cycle .",
"External signa... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Cytoplasmic sharing through apical membrane remodeling | elife-58107-v3 | [
[
"Throughout the tree of life , there are upper limits to the size of individual cells .",
"This size limitation is imposed by genome content , which impacts biosynthetic capacity and cell growth ( Conlon and Raff , 1999; Mueller , 2015 ) .",
"In diverse tissues and organisms , the existence of ‘giant ... | [
"Multiple nuclei sharing a common cytoplasm are found in diverse tissues , organisms , and diseases .",
"Yet , multinucleation remains a poorly understood biological property .",
"Cytoplasm sharing invariably involves plasma membrane breaches .",
"In contrast , we discovered cytoplasm sharing without membrane... | [
"Most cells are self-contained – they have a cell membrane that delimits and therefore defines the cell , separating it from other cells and from its environment .",
"But sometimes several cells interconnect and form collectives so they can pool their internal resources .",
"Some of the best-known examples of t... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Columnar processing of border ownership in primate visual cortex | elife-72573-v2 | [
[
"One of the deepest and most enduring mysteries of visual perception is how the brain constructs an internal model of the ever-changing world that falls before our eyes .",
"An essential step in this dynamic process of constructive perception is the assignment of border ownership .",
"Consider panel 1... | [
"To understand a visual scene , the brain segregates figures from background by assigning borders to foreground objects .",
"Neurons in primate visual cortex encode which object owns a border ( border ownership ) , but the underlying circuitry is not understood .",
"Here , we used multielectrode probes to recor... | [
"To understand a visual scene , the brain needs to identify objects and distinguish them from background .",
"A border marks the transition from object to background , but to differentiate which side of the border belongs to the object and which to background , the brain must integrate information across space ."... | 2021 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"short report",
"genetics and genomics"
] | Global biogeographic sampling of bacterial secondary metabolism | elife-05048-v2 | [
[
"Soil-dwelling bacteria produce many of the most important members of our pharmacy , including the majority of our antibiotics as well as many of the cytotoxic compounds used in the treatment of cancers ( Cragg and Newman , 2013 ) .",
"The traditional approach for characterizing the biosynthetic potential... | [
"Recent bacterial ( meta ) genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment .",
"Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ket... | [
"Many of the most useful medicinal drugs—including antibiotics and cancer drugs—are derived from bacteria living in the soil that produce these chemicals as part of their natural life cycle .",
"Many of these chemicals have been found by culturing bacteria in the laboratory , but this approach is limited because ... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ | elife-11134-v2 | [
[
"In Escherichia coli , DNA replication is highly efficient with speeds of up 600–1000 nucleotides per second ( Mok and Marians , 1987; Mcinerney et al . , 2007 ) , >100 , 000 basepairs ( bp ) synthesized per binding event ( Yao et al . , 2009 ) , and an error rate of ∼1 per million ( Bloom et al . , 1997 ) ."... | [
"The replicative DNA polymerase PolIIIα from Escherichia coli is a uniquely fast and processive enzyme .",
"For its activity it relies on the DNA sliding clamp β , the proofreading exonuclease ε and the C-terminal domain of the clamp loader subunit τ .",
"Due to the dynamic nature of the four-protein complex it... | [
"DNA replication is complicated because the two strands that form its “double helix” structure run in opposite directions and need to be replicated at the same time .",
"One of the new strands , the leading strand , is built continuously .",
"While the other strand , called the lagging strand , is made in stret... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Human VMPFC encodes early signatures of confidence in perceptual decisions | elife-38293-v3 | [
[
"Our everyday lives involve situations where we must make judgments based on noisy or incomplete sensory information – for example deciding whether crossing the street on a foggy morning , in poor visibility , is safe .",
"Being able to rely on an internal estimate of whether our perceptual judgments are ... | [
"Choice confidence , an individual’s internal estimate of judgment accuracy , plays a critical role in adaptive behaviour , yet its neural representations during decision formation remain underexplored .",
"Here , we recorded simultaneous EEG-fMRI while participants performed a direction discrimination task and r... | [
"While waiting to cross the road on a foggy morning , you see a shape in the distance that appears to be an approaching car .",
"How do you decide if it is safe to cross ?",
"We often have to make important decisions about the world based on imperfect information .",
"What guides our subsequent actions in the... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | The microbiota protects from viral-induced neurologic damage through microglia-intrinsic TLR signaling | elife-47117-v1 | [
[
"Viral infection of the CNS can cause permanent neurologic damage and psychiatric disorders ( Bergmann et al . , 2006; Koyuncu et al . , 2013; van den Pol , 2009 ) .",
"Indeed , multiple viruses including Zika and West Nile virus have been associated with Guillain-Barre syndrome and memory deficits , resp... | [
"Symbiotic microbes impact the function and development of the central nervous system ( CNS ) ; however , little is known about the contribution of the microbiota during viral-induced neurologic damage .",
"We identify that commensals aid in host defense following infection with a neurotropic virus through enhanc... | [
"Trillions of bacteria , fungi and viruses live inside us , forming what is known as our microbiota .",
"Far from causing problems , these microbes benefit our health in many ways .",
"Most of our microbiota lives in our gut , yet there is increasing evidence that it can influence how our central nervous system... | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | The ‘de novo’ DNA methyltransferase Dnmt3b compensates the Dnmt1-deficient intestinal epithelium | elife-12975-v2 | [
[
"DNA methylation patterns are established by DNA methyltransferase enzymes ( Dnmts ) , for which two categories have been defined based on in vitro assays .",
"The ‘de novo’ methyltransferases , Dnmt3a and Dnm3b , establish novel patterns of DNA methylation , and prefer to bind unmethylated DNA in vitro (... | [
"Dnmt1 is critical for immediate postnatal intestinal development , but is not required for the survival of the adult intestinal epithelium , the only rapidly dividing somatic tissue for which this has been shown .",
"Acute Dnmt1 deletion elicits dramatic hypomethylation and genomic instability .",
"Recovery of... | [
"Genes in a cell can be switched on or off at different times depending on the cell’s requirements .",
"Small chemical groups can be attached to the gene’s DNA , which dictates whether it is activated or inactivated .",
"For example , methyl groups can be attached to DNA by enzymes known as DNA methytransferase... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"medicine",
"genetics and genomics"
] | PTPRG is an ischemia risk locus essential for HCO3–-dependent regulation of endothelial function and tissue perfusion | elife-57553-v2 | [
[
"Inadequate tissue perfusion relative to metabolic demand is a fundamental pathophysiological cause of acute ( e . g . myocardial infarction and stroke ) and chronic ( e . g . heart failure and neurodegenerative disorders ) cardiovascular disease .",
"Newer treatment options improve the prognosis for some... | [
"Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear .",
"We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort .",
"We show that endothelial cells express the putative... | [
"Restricted blood flow in the heart or brain can deprive these vital organs of oxygen , thereby causing a heart attack or stroke .",
"However , the body has compensatory mechanisms to mitigate damage: if the blood flow is reduced in one blood vessel , acidic waste accumulates locally .",
"This causes nearby blo... | 2020 |
[
"Introduction",
"Material and methods"
] | [
"developmental biology",
"short report",
"computational and systems biology"
] | Temporal modulation of collective cell behavior controls vascular network topology | elife-13212-v2 | [
[
"The unique vascular topology of different organs exists for organ-specific functions .",
"Different vascular network densities determine the specific amount of oxygen and nutrients to be delivered to each host tissue .",
"Development of new vascular networks depends upon two types of specialized endo... | [
"Vascular network density determines the amount of oxygen and nutrients delivered to host tissues , but how the vast diversity of densities is generated is unknown .",
"Reiterations of endothelial-tip-cell selection , sprout extension and anastomosis are the basis for vascular network generation , a process gover... | [
"Many animals have a network of blood vessels that supplies oxygen and nutrients to every part of the body .",
"Each organ contains a unique pattern of blood vessels; some have lots of densely packed vessels , while others have fewer vessels that are more widely spaced .",
"New blood vessels typically form by s... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Material and methods"
] | [
"neuroscience"
] | A Cryptochrome 2 mutation yields advanced sleep phase in humans | elife-16695-v3 | [
[
"Sleep is vital for all animals .",
"Sleep-wake timing is regulated by the internal biological clock driving physiological rhythms with a period of approximately 24 hr ( Takahashi , 1995 ) .",
"The circadian clock is composed of interlocked transcriptional and translational negative feedback loops ( L... | [
"Familial Advanced Sleep Phase ( FASP ) is a heritable human sleep phenotype characterized by very early sleep and wake times .",
"We identified a missense mutation in the human Cryptochrome 2 ( CRY2 ) gene that co-segregates with FASP in one family .",
"The mutation leads to replacement of an alanine residue a... | [
"Sleep is an essential process in animals .",
"In humans , the disturbance of normal sleep-wake cycles through shift-work or long-term sleep disorders increases the risk of developing conditions including mental illness , cancer and metabolic syndromes .",
"Understanding how sleep-wake behavior is controlled wi... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"neuroscience"
] | Somatostatin binds to the human amyloid β peptide and favors the formation of distinct oligomers | elife-28401-v2 | [
[
"The amyloid beta ( Aβ ) peptide represents a 37 to 49 amino acids endoproteolytic fragment of the amyloid precursor protein ( APP ) , a single-span transmembrane protein , which in humans is coded on the long arm of Chromosome 21 within cytogenetic band q21 . 3 ( Kang et al . , 1987 ) .",
"The cellular b... | [
"The amyloid β peptide ( Aβ ) is a key player in the etiology of Alzheimer disease ( AD ) , yet a systematic investigation of its molecular interactions has not been reported .",
"Here we identified by quantitative mass spectrometry proteins in human brain extract that bind to oligomeric Aβ1-42 ( oAβ1-42 ) and/or... | [
"Treating Alzheimer’s disease and related dementias is one of the major challenges currently facing healthcare providers worldwide .",
"A hallmark of the disease is the formation of large deposits of a specific molecule , known as amyloid beta ( Aβ ) , in the brain .",
"However , more and more research suggests... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology"
] | Unravelling druggable signalling networks that control F508del-CFTR proteostasis | elife-10365-v3 | [
[
"Cystic fibrosis ( CF ) is the most common lethal genetic disease in Caucasians .",
"It is caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene that encodes a chloride channel localised to the apical membrane of several epithelial cells .",
"Mutations that cause CFTR loss of... | [
"Cystic fibrosis ( CF ) is caused by mutations in CF transmembrane conductance regulator ( CFTR ) .",
"The most frequent mutation ( F508del-CFTR ) results in altered proteostasis , that is , in the misfolding and intracellular degradation of the protein .",
"The F508del-CFTR proteostasis machinery and its homeo... | [
"Cystic fibrosis is a genetic disease that commonly affects people of European descent .",
"The condition is caused by mutations in the gene encoding a protein called “cystic fibrosis transmembrane conductance regulator” ( or CFTR for short ) .",
"CFTR forms a channel in the membrane of cells in the lungs that ... | 2015 |
[
"Introduction",
"Results",
"Discussion"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Community effects in regulation of translation | elife-10965-v1 | [
[
"Translation happens when a ribosome assembles productively on an mRNA .",
"A number of molecular interactions influence the proper convergence of these parts , often involving RNA elements or factors at the opposite ends of the mRNA ( Jackson et al . , 2010 ) .",
"For example , Poly ( A ) binding pro... | [
"Certain forms of translational regulation , and translation itself , rely on long-range interactions between proteins bound to the different ends of mRNAs .",
"A widespread assumption is that such interactions occur only in cis , between the two ends of a single transcript .",
"However , certain translational ... | [
"Genes encode the instructions needed to make proteins and other molecules .",
"To make a protein , the DNA within a gene is copied to produce molecules of messenger ribonucleic acid ( mRNA ) that are then used as templates to build proteins via a process called translation .",
"This process – which involves pr... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"short report",
"structural biology and molecular biophysics",
"computational and systems biology"
] | Precise assembly of complex beta sheet topologies from de novo designed building blocks | elife-11012-v2 | [
[
"Modular domains constitute the primary structural and functional units of natural proteins .",
"Multi-domain proteins likely evolved through simple linear concatenation of successive domains onto the polypeptide chain or through the insertion of one or more continuous sequences into the middle of another... | [
"Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements .",
"A similar challenge presumably limited the emergence of large and complex protein topologies in evolution .",
"Here , we demonstrate that protein topologies with six and seven-... | [
"A protein is made up of a sequence of amino acids and must fold into a specific three-dimensional structure if it is to work correctly .",
"The structure is formed by segments of the protein adopting specific shapes , the two most common shapes being alpha helices and beta strands .",
"Beta strands commonly in... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"microbiology and infectious disease"
] | Polo-like kinase Cdc5 regulates Spc72 recruitment to spindle pole body in the methylotrophic yeast Ogataea polymorpha | elife-24340-v2 | [
[
"Segregation of sister chromatids into two daughter cells is pivotal to the proliferation of eukaryotic cells .",
"Chromosome segregation is followed by cytokinesis , which results in physical separation of two daughter cells .",
"In many organisms , the position of the mitotic spindle dictates the si... | [
"Cytoplasmic microtubules ( cMT ) control mitotic spindle positioning in many organisms , and are therefore pivotal for successful cell division .",
"Despite its importance , the temporal control of cMT formation remains poorly understood .",
"Here we show that unlike the best-studied yeast Saccharomyces cerevi... | [
"Before a cell divides , it needs to duplicate its genetic material to provide the new daughter cell with a full set of genetic information .",
"To do so , the cell forms a complex of proteins called the spindle apparatus , which is made up of string-like microtubules that divide the chromosomes evenly .",
"In ... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | PLK4 promotes centriole duplication by phosphorylating STIL to link the procentriole cartwheel to the microtubule wall | elife-46054-v2 | [
[
"Centrioles are microtubule-based structures that recruit a surrounding pericentriolar material ( PCM ) to form the centrosome ( Nigg and Holland , 2018; Gönczy , 2012 ) .",
"Centrosomes nucleate the formation of the microtubule cytoskeleton in interphase cells and form the poles of the mitotic spindle du... | [
"Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia .",
"Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 ( PLK4 ) .",
"Although significant progress has been made in understanding centriole compos... | [
"A cell’s DNA is the chemical instruction manual for everything it does .",
"Each cell in our bodies contains over two meters of DNA , which is divided into 46 packages of information called chromosomes .",
"When the body needs to make more cells , for example during growth or repair , existing cells divide in ... | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"biochemistry and chemical biology"
] | Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39 | elife-08378-v2 | [
[
"The conserved core of the archaeal and eukaryotic transcription machineries encompasses a highly complex multisubunit RNAP as well as evolutionary conserved transcription factors that govern its activities through the transcription cycle .",
"The minimal requirements for promoter-directed and start site-... | [
"Transcription initiation of archaeal RNA polymerase ( RNAP ) and eukaryotic RNAPII is assisted by conserved basal transcription factors .",
"The eukaryotic transcription factor TFIIE consists of α and β subunits .",
"Here we have identified and characterised the function of the TFIIEβ homologue in archaea that... | [
"Life on Earth is often categorized into three domains: the eukaryotes ( which include plants , animals and fungi ) , the bacteria and a group of unusual , single-celled microorganisms called the archaea .",
"But several recent discoveries suggest that the origin of the eukaryotes lies within the archaeal domain ... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"tools and resources",
"neuroscience"
] | Quantitative isoform-profiling of highly diversified recognition molecules | elife-07794-v2 | [
[
"The remarkable anatomical and functional complexity of nervous systems relies on molecular programs for cell intrinsic properties and selective cellular interactions .",
"Major advances in transcriptomics have enabled the identification of gene regulatory programs and mRNA targets that underlie specifica... | [
"Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners .",
"Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for... | [
"To create a protein , a gene is first copied to form an RNA molecule that contains regions known as introns and exons .",
"Splicing removes the introns and joins the exons together to form a molecule of ‘messenger RNA’ , which is translated into a protein .",
"Over the course of evolution , many groups—or fami... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Ingression-type cell migration drives vegetal endoderm internalisation in the Xenopus gastrula | elife-27190-v2 | [
[
"The basic body plan of metazoans is established by gastrulation , and at its core is the movement of endoderm and mesoderm from the surface to the interior of the embryo .",
"Among invertebrates , the pre-gastrulation embryo typically consists of a single-layered epithelium , and a common mechanism of ge... | [
"During amphibian gastrulation , presumptive endoderm is internalised as part of vegetal rotation , a large-scale movement that encompasses the whole vegetal half of the embryo .",
"It has been considered a gastrulation process unique to amphibians , but we show that at the cell level , endoderm internalisation e... | [
"In most animals , the early embryo consists of a single layer of cells that forms a hollow sphere .",
"This simple structure first gains complexity by organising into multiple layers that are fated to become specialised tissues in the adult , such as muscle or skin .",
"To form the primitive gut , a group of c... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"physics of living systems",
"neuroscience"
] | Variability in locomotor dynamics reveals the critical role of feedback in task control | elife-51219-v2 | [
[
"Animals routinely exhibit dramatic variations in morphophysiology between individuals but can nevertheless achieve similar performance in sensorimotor tasks ( Sponberg et al . , 2015; Bullimore and Burn , 2006 ) .",
"Further , individual animals can experience rapid changes in their own morphophysiologic... | [
"Animals vary considerably in size , shape , and physiological features across individuals , but yet achieve remarkably similar behavioral performances .",
"We examined how animals compensate for morphophysiological variation by measuring the system dynamics of individual knifefish ( Eigenmannia virescens ) in a ... | [
"People come in different shapes and sizes , but most will perform similarly well if asked to complete a task requiring fine manual dexterity – such as holding a pen or picking up a single grape .",
"How can different individuals , with different sized hands and muscles , produce such similar movements ?",
"One... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Structural development and dorsoventral maturation of the medial entorhinal cortex | elife-13343-v3 | [
[
"The representation of space in the rodent brain has been investigated in detail .",
"The functional development of spatial response properties has also been investigated in the cortico-hippocampal system ( Ainge and Langston , 2012; Wills et al . , 2014 ) , with studies suggesting the early emergence of ... | [
"We investigated the structural development of superficial-layers of medial entorhinal cortex and parasubiculum in rats .",
"The grid-layout and cholinergic-innervation of calbindin-positive pyramidal-cells in layer-2 emerged around birth while reelin-positive stellate-cells were scattered throughout development ... | [
"Many animals , from rats to humans , need to navigate their environments to find food or shelter .",
"This ability relies on a kind of memory known as spatial memory , which provides a map of the outside world within the animal’s brain .",
"Specifically , cells in a part of the brain called the medial entorhin... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"biochemistry and chemical biology"
] | Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes | elife-24542-v2 | [
[
"In eukaryotic translation initiation , initiator tRNA ( Met-tRNAi ) recognises AUG start codons in mRNA as part of a larger complex bound to the 40 S ribosome .",
"Eukaryotic translation initiation factor 2 ( eIF2 ) is the key factor that delivers Met-tRNAi to ribosomes ( Hinnebusch , 2014; Dever et al .... | [
"Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes ( TC ) .",
"This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response .",
"Key to controlling the activity of eIF2 are transla... | [
"All cells sense and react to changes in the world around them .",
"One way that cells react to threats to their health is by switching off genes required for their normal activity and diverting this energy to switching on genes that deal with the specific stress .",
"A protein called eIF2 controls this general... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Potentiation of cerebellar Purkinje cells facilitates whisker reflex adaptation through increased simple spike activity | elife-38852-v2 | [
[
"Active touch is important for exploring our environment , allowing us to assess the shape , substance and movements of objects and organisms around us ( Prescott et al . , 2011 ) .",
"Throughout the animal kingdom , various systems have evolved for this purpose; these include for example the antennae of ... | [
"Cerebellar plasticity underlies motor learning .",
"However , how the cerebellum operates to enable learned changes in motor output is largely unknown .",
"We developed a sensory-driven adaptation protocol for reflexive whisker protraction and recorded Purkinje cell activity from crus 1 and 2 of awake mice .",... | [
"Rodents use their whiskers to explore the world around them .",
"When the whiskers touch an object , it triggers involuntary movements of the whiskers called whisker reflexes .",
"Experiencing the same sensory stimulus multiple times enables rodents to fine-tune these reflexes , e . g . , by making their movem... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Distinct insulin granule subpopulations implicated in the secretory pathology of diabetes types 1 and 2 | elife-62506-v2 | [
[
"Blood glucose levels are maintained in a narrow range by secretion of the hormone insulin from pancreatic β-cells .",
"Insufficient insulin secretion leads to elevated levels of blood glucose resulting in diabetes ( Rorsman and Ashcroft , 2018 ) .",
"Type 1 diabetes ( T1D ) results primarily from imm... | [
"Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes .",
"Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes , accompanying changes to insulin granules have not been established .",
"To address thi... | [
"Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin .",
"People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the β-cells in their pancreas that make this hormone .",
"People with type 2 diabetes d... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Synthetic enzyme-substrate tethering obviates the Tolloid-ECM interaction during Drosophila BMP gradient formation | elife-05508-v1 | [
[
"Bone Morphogenetic Proteins ( BMPs ) represent a conserved family of secreted signalling proteins that regulate diverse processes during development and homeostasis ( Wu and Hill , 2009 ) .",
"Multiple tiers of regulation within the BMP signalling pathway exist , including extracellular modulators that f... | [
"Members of the Tolloid family of metalloproteinases liberate BMPs from inhibitory complexes to regulate BMP gradient formation during embryonic dorsal-ventral axis patterning .",
"Here , we determine mechanistically how Tolloid activity is regulated by its non-catalytic CUB domains in the Drosophila embryo .",
... | [
"The body of an animal is a highly organised structure of tissues and organs that contain cells with specialised roles .",
"To achieve this level of organisation , it is important that the cells in the embryo know their location and receive the correct instructions on how to develop , when to divide or move .",
... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"immunology and inflammation"
] | Context-enriched interactome powered by proteomics helps the identification of novel regulators of macrophage activation | elife-37059-v2 | [
[
"Pro-inflammatory macrophage activation plays a prominent role in a large number of disorders including cardiovascular disease ( CVD ) ( Aikawa and Libby , 2004; Glass and Olefsky , 2012; Glass and Witztum , 2001; Gregor and Hotamisligil , 2011; Liang et al . , 2007; Randolph , 2014; Ridker and Lüscher , 2014... | [
"The role of pro-inflammatory macrophage activation in cardiovascular disease ( CVD ) is a complex one amenable to network approaches .",
"While an indispensible tool for elucidating the molecular underpinnings of complex diseases including CVD , the interactome is limited in its utility as it is not specific to ... | [
"When human cells or tissues are injured , the body triggers a response known as inflammation to repair the damage and protect itself from further harm .",
"However , if the same issue keeps recurring , the tissues become inflamed for longer periods of time , which may ultimately lead to health problems .",
"Th... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Material and methods"
] | [
"immunology and inflammation"
] | Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells | elife-01659-v1 | [
[
"Immune cells migrate throughout the body where they search tissues for pathological events induced by invading pathogens or emerging tumors , for example .",
"Upon encounter with these events , circulating immune cells stop and respond in collaboration with yet other immune cells , often in organized lym... | [
"Natural killer ( NK ) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines .",
"Most studies of mouse NK cells , however , have focused on conventional NK ( cNK ) cells in the spleen .",
"Recently , we described t... | [
"Our immune system has white blood cells that migrate throughout the body in search of invading microbes or diseased and damaged cells .",
"When these events are encountered , the white blood cells move into the affected tissue and launch an immune response to eliminate the threat .",
"Natural killer cells are ... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Material and methods"
] | [
"cell biology",
"computational and systems biology"
] | EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling | elife-12223-v2 | [
[
"Signaling by the epidermal growth factor receptor ( EGFR ) converts diverse external stimuli into specific cellular responses .",
"EGFR signaling is implicated in embryonic development , tissue homeostasis and wound healing ( Yu et al . , 2010; Sibilia et al . , 2007 ) , while EGFR overexpression and hyp... | [
"Autocatalytic activation of epidermal growth factor receptor ( EGFR ) coupled to dephosphorylating activity of protein tyrosine phosphatases ( PTPs ) ensures robust yet diverse responses to extracellular stimuli .",
"The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signa... | [
"In living tissue , the ability of individual cells to grow is influenced by signal molecules in the environment around each cell .",
"For example , after an injury , a molecule called epidermal growth factor can stimulate cells to grow to repair the wound .",
"Epidermal growth factor binds to and activates a r... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"structural biology and molecular biophysics"
] | Massive endocytosis triggered by surface membrane palmitoylation under mitochondrial control in BHK fibroblasts | elife-01293-v1 | [
[
"Endocytic processes that do not use classical endocytic proteins remain poorly understood because they are mechanistically inhomogeneous and difficult to study in isolation ( Mayor and Pagano , 2007; Doherty and McMahon , 2009 ) .",
"Nevertheless , they are involved in a wide range of cellular processes ... | [
"Large Ca transients cause massive endocytosis ( MEND ) in BHK fibroblasts by nonclassical mechanisms .",
"We present evidence that MEND depends on mitochondrial permeability transition pore ( PTP ) openings , followed by coenzyme A ( CoA ) release , acyl CoA synthesis , and membrane protein palmitoylation .",
... | [
"Cells use a process called endocytosis to absorb proteins and other molecules .",
"There are many forms of endocytosis , but they usually involve the molecule of interest becoming tucked into a bud that forms in the cell membrane .",
"This bud is then pinched off to leave the molecule inside a vesicle that is ... | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"immunology and inflammation"
] | AXL receptor tyrosine kinase is required for T cell priming and antiviral immunity | elife-12414-v1 | [
[
"AXL is a member of the TAM ( TYRO3 , AXL , and MERTK ) subfamily of RTK that potently inhibits the production of type I IFNs ( Bhattacharyya et al . , 2013; Rothlin et al . , 2015; Rothlin et al . , 2007; Zagorska et al . , 2014 ) .",
"In DCs , AXL is an IFN-stimulated gene ( ISG ) and hijacks molecular ... | [
"The receptor tyrosine kinase ( RTK ) AXL is induced in response to type I interferons ( IFNs ) and limits their production through a negative feedback loop .",
"Enhanced production of type I IFNs in Axl-/- dendritic cells ( DCs ) in vitro have led to speculation that inhibition of AXL would promote antiviral res... | [
"The immune system must be ever vigilant to ward off infections .",
"Immune cells called T-cells can identify and eliminate microbes , but if they are too aggressive , they can damage the body .",
"To prevent this , the body has systems that control immune responses .",
"For example , another type of immune c... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health",
"microbiology and infectious disease"
] | Two complement receptor one alleles have opposing associations with cerebral malaria and interact with α+thalassaemia | elife-31579-v2 | [
[
"Complement Receptor One ( CR1 ) plays a key role in the control of complement activation and the immune clearance of C3b/C4b-coated immune complexes ( Krych-Goldberg and Atkinson , 2001 ) .",
"CR1 is expressed on a range of cells including red blood cells ( RBCs ) , leucocytes and glomerular podocytes ( ... | [
"Malaria has been a major driving force in the evolution of the human genome .",
"In sub-Saharan African populations , two neighbouring polymorphisms in the Complement Receptor One ( CR1 ) gene , named Sl2 and McCb , occur at high frequencies , consistent with selection by malaria .",
"Previous studies have bee... | [
"Malaria kills more than half a million children in Africa every year .",
"The disease is caused by the Plasmodium falciparum parasite , and mosquitos infected with the parasites spread them to humans when they bite .",
"Once inside a human , the parasites infect the red blood cells .",
"In severe cases , the... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine437 | elife-30760-v2 | [
[
"Cdk5 shares strong homology with other members of the family of cyclin-dependent kinases ( Cdks ) , but it is distinct in its modes of regulation and function ( Dhavan and Tsai , 2001; Pozo and Bibb , 2016 ) .",
"Unlike other Cdks , Cdk5 is best known for its function in post-mitotic cells rather than ce... | [
"Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health .",
"The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood .",
"Here , we show that in Drosophila melanogaster Cdk5 regulates basal autophagy , a key mechanism suppressing neurodeg... | [
"Cells have a problem that we recognize from our own homes: if nobody cleans up , garbage accumulates .",
"Unwanted material in cells can include proteins that clump together and can no longer carry out their normal tasks .",
"If left to build up , these protein aggregates can damage the cell and even kill it .... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"genetics and genomics"
] | Expansion of the circadian transcriptome in Brassica rapa and genome-wide diversification of paralog expression patterns | elife-58993-v2 | [
[
"The transition from basic research in Arabidopsis to new model systems for monocot and dicot crops has focused attention on the implications of polyploidy for current models of genetic processes developed in Arabidopsis .",
"The expansion of gene content through whole genome duplication ( WGD ) , tandem ... | [
"An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops .",
"Here we describe the circadian transcriptome in the polyploid crop Brassica rapa .",
"Strikingly , almost three-quarters of the expressed genes exhibited circadian rhythmicity .",
"Genetic redundan... | [
"Like animals , plants have internal biological clocks that allow them to adapt to daily and yearly changes , such as day-night cycles or seasons turning .",
"Unlike animals , however , plants cannot move when their environment becomes different , so they need to be able to weather these changes by adjusting whic... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"biochemistry and chemical biology"
] | Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation | elife-07777-v2 | [
[
"Intrinsically disordered proteins/regions ( IDPs/IDRs ) constitute a significant fraction of the metazoan proteome ( Liu et al . , 2006; Uversky , 2013 ) .",
"By virtue of their structural malleability and propensity to interact with multiple-binding partners , these peptide stretches of ∼30 or more amin... | [
"Intrinsically disordered proteins/regions ( IDPs/IDRs ) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins .",
"They are abundant in eukaryotic proteomes and are often associated with human diseases , but their biological functions have been elus... | [
"DNA contains instructions to make all the proteins and other molecules that drive essential processes in cells .",
"To issue such specific sets of instructions , a section of DNA—called a gene—is first copied to make molecules of messenger ribonucleic acid ( or mRNA for short ) in a process called transcription ... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"immunology and inflammation"
] | ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity | elife-33057-v2 | [
[
"Immune responses require precise , dynamic gene regulation that must activate rapidly as threats rise and resolve efficiently as they clear .",
"Post-transcriptional control of mRNA abundance and expression by RNA binding proteins ( RBPs ) is a key layer of this response that can enact rapid , signal-res... | [
"Dynamic post-transcriptional control of RNA expression by RNA-binding proteins ( RBPs ) is critical during immune response .",
"ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer , but functions in adaptive immunity are less clear .",
"We used HITS-CLIP to define ZFP36 targets i... | [
"The immune system must quickly respond to anything that may cause disease – from cancerous cells to viruses .",
"For instance , a type of white blood cell called a T cell patrols the body , looking for potential threats .",
"If a T cell identifies such a threat , it “activates” and undergoes various changes so... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"microbiology and infectious disease"
] | Extended low-resolution structure of a Leptospira antigen offers high bactericidal antibody accessibility amenable to vaccine design | elife-30051-v2 | [
[
"The molecular details of how surface antigens of a pathogen are exposed to the host’s defenses are highly relevant to rational vaccine development ( Rappuoli et al . , 2016; Dormitzer et al . , 2012; Dormitzer et al . , 2008 ) .",
"The structural context of immunologically accessible epitopes allows for ... | [
"Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection .",
"These obligatory structures exposed to the host immune system provide important targets for rational vaccine design .",
"Here , we use a systematically designed series of multi-domain constructs in combinati... | [
"Vaccines encourage the immune system to develop a protection against disease-causing bacteria and viruses .",
"Some types of immune cells release antibodies , which recognize particular proteins on the surface of the invading microbe .",
"A vaccine that contains these surface proteins allows immune cells to de... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3 | elife-21337-v2 | [
[
"RNA granules are concentrated assemblies of RNA and RNA-binding proteins that form without a limiting membrane in the cytoplasm or nucleoplasm of cells ( Courchaine , 2016 ) .",
"RNA granules are ubiquitous cellular structures and several classes of cytoplasmic RNA granules have been described , includin... | [
"RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins .",
"The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood .",
"During polarization of the C . elegans zygote , germline RNA granules , called P granules ... | [
"Animal cells contain many smaller compartments known as organelles that perform particular roles .",
"For example , a compartment called the nucleus stores most of the cell’s genetic information .",
"The nucleus and many other organelles form inside layers of membrane that physically separate them from the res... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Inverted formin 2 regulates intracellular trafficking, placentation, and pregnancy outcome | elife-31150-v1 | [
[
"Implantation and placentation involve complex synchronization between the developing embryo and decidualization of the uterus .",
"Extravillous trophoblasts ( EVTs ) differentiate from column cytotrophoblasts ( CTBs ) , invade through the endometrium to the myometrium , and remodel decidual spiral arteri... | [
"Healthy pregnancy depends on proper placentation—including proliferation , differentiation , and invasion of trophoblast cells—which , if impaired , causes placental ischemia resulting in intrauterine growth restriction and preeclampsia .",
"Mechanisms regulating trophoblast invasion , however , are unknown .",
... | [
"The placenta is an organ that develops with the baby during pregnancy and links the baby with his or her mother .",
"This connection allows mom and baby to communicate throughout the pregnancy to share nutrition and growth signals , and to coordinate their immune systems .",
"Abnormal placental growth can have... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake | elife-31981-v2 | [
[
"The Target of Rapamycin Complex 1 ( TORC1 ) plays a pivotal role in controlling cell growth in probably all eukaryotic organisms .",
"It operates by integrating upstream signals such as growth factors ( GFs ) and nutrients to modulate , by phosphorylation , multiple downstream effectors , mostly proteins... | [
"The yeast Target of Rapamycin Complex 1 ( TORC1 ) plays a central role in controlling growth .",
"How amino acids and other nutrients stimulate its activity via the Rag/Gtr GTPases remains poorly understood .",
"We here report that the signal triggering Rag/Gtr-dependent TORC1 activation upon amino-acid uptake... | [
"Cells adapt their growth rate depending on the amount of nutrients available .",
"The protein complex called TORC1 plays a central role in this .",
"When nutrients are abundant , TORC1 is very active and stimulates the production of proteins and other molecules needed for the cell to grow .",
"However , when... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods",
"Acknowledgments"
] | [
"neuroscience"
] | Sources of noise during accumulation of evidence in unrestrained and voluntarily head-restrained rats | elife-11308-v2 | [
[
"Subjects performing perceptual decision-making tasks display a large amount of trial-to-trial behavioral variability .",
"Determining the sources of this variability could provide insight into the neural mechanisms of decision-making and produce more accurate predictions of behavior .",
"It has been ... | [
"Decision-making behavior is often characterized by substantial variability , but its source remains unclear .",
"We developed a visual accumulation of evidence task designed to quantify sources of noise and to be performed during voluntary head restraint , enabling cellular resolution imaging in future studies .... | [
"Perceptual decision-making , i . e . making choices based on observed evidence , is rarely perfect .",
"Humans and other animals tend to respond correctly on some trials and incorrectly on others .",
"For over a century , this variability has been used to study the basis of decision-making .",
"Most behavior... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum | elife-02784-v2 | [
[
"Collagens are the most abundant secretory proteins , comprising 25–30% of the human body dry weight ( Pataridis et al . , 2008 ) .",
"They are required for cell attachment , tissue organization and remodeling , and for the differentiation of chondrocytes to produce mineralized bones ( Gelse et al . , 200... | [
"TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum ( ER ) .",
"In this study , we report a connection between the cytoplasmic domain of TANGO1 and SLY1 , a protein that is required for membrane fusion .",
"Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the ... | [
"Collagens are long proteins that join individual cells together to build tissues and organs .",
"They also provide strength and elasticity to bones , tendons , and blood vessels .",
"Like many other proteins , collagens are produced inside cells: they are folded in a compartment called the endoplasmic reticulu... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"neuroscience"
] | Organic electrochemical transistor arrays for real-time mapping of evoked neurotransmitter release in vivo | elife-50345-v2 | [
[
"In the nervous system , neurotransmitters ( NTs ) are released upon arrival of action potentials , and play essential roles in signal transmission for regulating physiological activities ( Kavalali , 2015 ) .",
"Though electrical recording and stimulation of neural activities have offered extremely power... | [
"Though neurotransmitters are essential elements in neuronal signal transduction , techniques for in vivo analysis are still limited .",
"Here , we describe an organic electrochemical transistor array ( OECT-array ) technique for monitoring catecholamine neurotransmitters ( CA-NTs ) in rat brains .",
"The OECT-... | [
"Cells in the nervous system pass messages using a combination of electrical and chemical signals .",
"When an electrical impulse reaches the end of one cell , it triggers the release of chemicals called neurotransmitters , which pass the message along .",
"Neurotransmitters can be either activating or inhibito... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"cancer biology"
] | Synthesizing artificial devices that redirect cellular information at will | elife-31936-v2 | [
[
"A basic ability of living cells is to sense extracellular signals by translating them into changes in regulation of cell signaling genes .",
"They use the natural signaling network to execute complex physiological functions , such as cell survival , behavior and identity .",
"As an interdisciplinary ... | [
"Natural signaling circuits could be rewired to reprogram cells with pre-determined procedures .",
"However , it is difficult to link cellular signals at will .",
"Here , we describe signal-connectors—a series of RNA devices—that connect one signal to another signal at the translational level .",
"We use them... | [
"Cells respond to signals from their surrounding environment .",
"External signals activate a sequence of events inside the cell that can change how it behaves .",
"These events are often called signaling pathways and they typically change the cell’s behavior by changing the activity of its genes .",
"A major... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Social selectivity and social motivation in voles | elife-72684-v2 | [
[
"The brain regions and neurochemicals involved in social behaviors show remarkable conservation across species ( O’Connell and Hofmann , 2011 ) .",
"At the same time , social behavior is not a unified construct , with different species exhibiting distinct social structures and behavioral repertoires .",
... | [
"Selective relationships are fundamental to humans and many other animals , but relationships between mates , family members , or peers may be mediated differently .",
"We examined connections between social reward and social selectivity , aggression , and oxytocin receptor signaling pathways in rodents that natu... | [
"What factors drive the formation of social relationships can vary greatly in animals .",
"While some individuals may be motivated to find social partners , others may just tolerate being around others .",
"A desire to avoid strangers may also lead an individual to seek out acquaintances or friends .",
"Somet... | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Alterations of specific cortical GABAergic circuits underlie abnormal network activity in a mouse model of Down syndrome | elife-58731-v2 | [
[
"Down syndrome ( DS ) is a condition caused by full or partial trisomy of human chromosome 21 , characterized by various physical and neurological features including mild to severe intellectual disability ( Antonarakis et al . , 2020 ) .",
"Individuals with DS present important deficits in cognitive tasks... | [
"Down syndrome ( DS ) results in various degrees of cognitive deficits .",
"In DS mouse models , recovery of behavioral and neurophysiological deficits using GABAAR antagonists led to hypothesize an excessive activity of inhibitory circuits in this condition .",
"Nonetheless , whether over-inhibition is present... | [
"Down syndrome is a genetic disorder caused by the presence of a third copy of chromosome 21 .",
"Affected individuals show delayed growth , characteristic facial features , altered brain development; with mild to severe intellectual disability .",
"The exact mechanisms underlying the intellectual disability in... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"neuroscience"
] | An NMDAR positive and negative allosteric modulator series share a binding site and are interconverted by methyl groups | elife-34711-v1 | [
[
"N-methyl-d-aspartate receptors ( NMDARs ) are a subtype of ionotropic glutamate receptors that are broadly expressed in the brain and are important for normal development , neuronal plasticity and memory formation ( Traynelis et al . , 2010; Paoletti et al . , 2013 ) .",
"NMDARs contribute a slow , Ca2+ ... | [
"N-methyl-d-aspartate receptors ( NMDARs ) are an important receptor in the brain and have been implicated in multiple neurological disorders .",
"Many non-selective NMDAR-targeting drugs are poorly tolerated , leading to efforts to target NMDAR subtypes to improve the therapeutic index .",
"We describe here a ... | [
"The neurons in the brain form networks that can change in response to experience , causing new connections to form between certain neurons and breaking the connections between others .",
"This remodeling process underlies learning and memory .",
"However , in certain neurological disorders , such as schizophre... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | The Lamin B receptor is essential for cholesterol synthesis and perturbed by disease-causing mutations | elife-16011-v2 | [
[
"Lamin B receptor ( LBR ) is an evolutionary conserved , multifunctional protein ( Olins et al . , 2010 ) .",
"The N-terminal moiety of LBR , which resides in the nucleoplasm , contains a chromatin-binding TUDOR domain and associates with the nuclear lamina ( Makatsori et al . , 2004; Pyrpasopoulou et al ... | [
"Lamin B receptor ( LBR ) is a polytopic membrane protein residing in the inner nuclear membrane in association with the nuclear lamina .",
"We demonstrate that human LBR is essential for cholesterol synthesis .",
"LBR mutant derivatives implicated in Greenberg skeletal dysplasia or Pelger-Huët anomaly fail to ... | [
"In humans , mutations in the gene that encodes a protein called Lamin B receptor can lead to diseases ranging from harmless anomalies of blood cells to fatal developmental defects .",
"The severity of the disease depends on the nature of the specific mutation , and whether one or both copies of the gene are affe... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"neuroscience"
] | Tissue acidosis induces neuronal necroptosis via ASIC1a channel independent of its ionic conduction | elife-05682-v1 | [
[
"Acidosis occurs commonly in a variety of neurological disorders and is a main contributing factor to neural injury ( Xiong et al . , 2004; Wemmie et al . , 2013; Friese et al . , 2007; Vergo et al . , 2011 ) .",
"For example , ischemic stroke causes pronounced brain acidosis ( ∼pH 6 . 0 ) , the treatment... | [
"Acidotoxicity is common among neurological disorders , such as ischemic stroke .",
"Traditionally , Ca2+ influx via homomeric acid-sensing ion channel 1a ( ASIC1a ) was considered to be the leading cause of ischemic acidotoxicity .",
"Here we show that extracellular protons trigger a novel form of neuronal nec... | [
"What happens in the minutes and hours after a stroke can determine how much brain damage occurs .",
"In some types of stroke , a blood clot cuts off the blood supply to part of the brain , depriving the brain cells of oxygen and other nutrients , including glucose .",
"One of the consequences is that the blood... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"developmental biology",
"tools and resources"
] | 3D visualization of macromolecule synthesis | elife-60354-v2 | [
[
"The measurement of nascent DNA , RNA , and protein synthesis in animals provides critical information on the state of cells ( dividing , growing ) in relation to their surrounding cells .",
"Traditionally , radiolabeled or brominated nucleosides/amino acids are introduced to live animals , where they are... | [
"Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues .",
"Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine , uridine , methionine , and glucosamine to label nascent synthesis o... | [
"Cells often respond to changes in their environment by producing new molecules and building new cell components , such as proteins , which perform most tasks in the cell , or DNA and RNA , which carry genetic information .",
"Complex tissues – such as limbs , which are made up of muscles , tendons , bones and ca... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | A dynamic mode of mitotic bookmarking by transcription factors | elife-22280-v5 | [
[
"A key component of cell identity is the epigenetic maintenance of cell-type specific transcription programs .",
"However , this maintenance is challenged during each cell cycle .",
"In mitosis , the global transcriptional machinery is inactivated via a cell cycle-dependent phosphorylation cascade ( P... | [
"During mitosis , transcription is shut off , chromatin condenses , and most transcription factors ( TFs ) are reported to be excluded from chromosomes .",
"How do daughter cells re-establish the original transcription program ?",
"Recent discoveries that a select set of TFs remain bound on mitotic chromosomes ... | [
"A kidney cell functions differently from a skin cell despite the fact that all the cells in one organism share the same DNA .",
"This is because not all of the genes encoded within the DNA are active in the cells .",
"Instead , cells can turn on just those genes that are specific to how that cell type works ."... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"immunology and inflammation"
] | TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury | elife-50458-v2 | [
[
"Lung disease is a major contributor to morbidity and mortality worldwide .",
"In the US alone , over 15% of the population suffers from lung disease , at an annual cost of 120 , 000 deaths , and >$50 billion ( Redd , 2002; Kochanek et al . , 2011; Ford et al . , 2015 ) .",
"Environmental lung injury ... | [
"Lung disease causes significant morbidity and mortality , and is exacerbated by environmental injury , for example through lipopolysaccharide ( LPS ) or ozone ( O3 ) .",
"Toll-like receptors ( TLRs ) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns .",
"TL... | [
"Immune cells in the lung help guard against infections .",
"On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria .",
"When the immune cells detect these invaders , the TLR receptors spring into action and trigge... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"neuroscience"
] | Human-specific ARHGAP11B induces hallmarks of neocortical expansion in developing ferret neocortex | elife-41241-v2 | [
[
"The expansion of the neocortex during primate evolution is thought to constitute one important basis for the unparalleled cognitive abilities of humans .",
"The size of the neocortex is mainly regulated by the proliferative capacity of neural progenitor cells during cortical development and the length of... | [
"The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans .",
"ARHGAP11B is a human-specific gene that , based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex , has been proposed... | [
"The human brain owes its characteristic wrinkled appearance to its outer layer , the cerebral cortex .",
"All mammals have a cerebral cortex , but its size varies greatly between species .",
"As the brain evolved , the neocortex , the evolutionarily youngest part of the cerebral cortex , expanded dramatically ... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"cancer biology"
] | The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals | elife-63326-v2 | [
[
"Pro-growth signals in the form of growth factors , hormones , and nutrients impinge on cellular metabolic programs in a coordinated fashion involving both acute , post-translational regulation and transcriptional control of nutrient transporters and metabolic enzymes .",
"The mechanistic target of rapamy... | [
"The mechanistic target of rapamycin complex 1 ( mTORC1 ) stimulates a coordinated anabolic program in response to growth-promoting signals .",
"Paradoxically , recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated... | [
"When building healthy tissue , the human body must carefully control the growth of new cells to prevent them from becoming cancerous .",
"A core component of this regulation is the protein mTORC1 , which responds to various growth-stimulating factors and nutrients , and activates the chemical reactions cells nee... | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cancer biology"
] | Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals | elife-15690-v2 | [
[
"Hedgehog ( Hh ) signaling plays critical roles in embryonic development and tumor growth ( Chen et al . , 2007; Hooper and Scott , 2005; Ingham and McMahon , 2001; Nieuwenhuis and Hui , 2005; Yao and Chuang , 2015 ) .",
"Its misregulation leads to many types of cancers ( Gialmanidis et al . , 2009; Hui a... | [
"Hedgehog signaling plays very important roles in development and cancers .",
"Vertebrates have three transcriptional factors , Gli1 , Gli2 and Gli3 .",
"Among them , Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus ( Ci , in Drosophila ) structurally and functionally as... | [
"Cells in mammals need to be able to communicate with each other to enable them to work together in tissues and organs .",
"A signaling pathway called Hedgehog signaling plays a crucial role in carrying information between cells in developing embryos , but if it is active at other times it can also promote the de... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cancer biology"
] | Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma | elife-57189-v2 | [
[
"Oesophageal cancer is the eighth most common cancer worldwide , and its 5-year survival rate of 15% makes it the sixth most-common cause of cancer-related death ( Ferlay et al . , 2015; Pennathur et al . , 2013 ) .",
"A subtype of oesophageal cancer , oesophageal adenocarcinoma ( OAC ) , is the predomina... | [
"Oesophageal adenocarcinoma ( OAC ) is one of the most common causes of cancer deaths .",
"Barrett’s oesophagus ( BO ) is the only known precancerous precursor to OAC , but our understanding about the molecular events leading to OAC development is limited .",
"Here , we have integrated gene expression and chrom... | [
"Acid fluids present in the gut can sometimes ‘go up’ and damage the oesophagus , the pipe that connects the mouth and the stomach .",
"As a result , a small number of individuals can develop Barrett’s oesophagus , a condition where cells in the lining of the lower oesophagus show abnormal shapes .",
"In certai... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"microbiology and infectious disease"
] | Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate | elife-23217-v2 | [
[
"Malaria is caused by infection of the blood with Apicomplexan parasites of the genus Plasmodium .",
"Critical for the proliferation and survival of Plasmodium in the blood is their ability to quickly penetrate host erythrocytes and acquire nutrients required for rapid growth .",
"To facilitate this ,... | [
"Plasmodium falciparum parasites , the causative agents of malaria , modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste .",
"Here we investigate the contribution of the rhoptry protein RhopH2 , in the formation of new permeability... | [
"Malaria is a life-threatening disease that affects millions of people around the world .",
"The parasites that cause malaria have a complex life cycle that involves infecting both mosquitoes and mammals , including humans .",
"In humans , the parasites spend part of their life cycle inside red blood cells , wh... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis | elife-57282-v1 | [
[
"Following deposition of dehydrated pollen grains on the receptive surface of the female organ , the stigma , pollen rehydrates , germinates and produces a pollen tube that carries the male gametes toward the ovules where the double fertilization takes place .",
"This long itinerary through the different ... | [
"Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules .",
"Pollen tubes first grow within the cell wall of the papilla cells , applying pressure to the cell .",
"Mechanical forces are known to play a major role in plant cell shap... | [
"Flowering plants produce small particles known as pollen that – with the help of the wind , bees and other animals – carry male sex cells ( sperm ) to female sex cells ( eggs ) contained within flowers .",
"When a grain of pollen lands on the female organ of a flower , called the pistil , it gives rise to a tube... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Decoding odor quality and intensity in the Drosophila brain | elife-04147-v1 | [
[
"To navigate the environment in a way that optimizes their survival and reproduction , animals have evolved sensory systems .",
"These have three essential tasks: First , the external world has to be translated into an internal representation in the form of an accurate neural map .",
"Second , the neu... | [
"To internally reflect the sensory environment , animals create neural maps encoding the external stimulus space .",
"From that primary neural code relevant information has to be extracted for accurate navigation .",
"We analyzed how different odor features such as hedonic valence and intensity are functionally... | [
"Organisms need to sense and adapt to their environment in order to survive .",
"Senses such as vision and smell allow an organism to absorb information about the external environment and translate it into a meaningful internal image .",
"This internal image helps the organism to remember incidents and act acco... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase | elife-17578-v1 | [
[
"Alzheimer’s disease ( AD ) is the most common form of dementia and currently the sixth leading cause of death in the United States , with no disease-modifying therapeutics available .",
"A central and pathological hallmark of AD is the deposition of amyloid-beta ( Aβ ) plaques in the brain ( Hardy and Se... | [
"γ-secretase is responsible for the proteolysis of amyloid precursor protein ( APP ) into short , aggregation-prone amyloid-beta ( Aβ ) peptides , which are centrally implicated in the pathogenesis of Alzheimer’s disease ( AD ) .",
"Despite considerable interest in developing γ-secretase targeting therapeutics fo... | [
"Individuals with Alzheimer’s disease generally have deposits known as “amyloid plaques” in the brain .",
"These plaques are made up of a mixture of molecules called amyloid beta peptides that clump together and are thought to be a key cause of the disease .",
"The amyloid beta peptides vary in size; the larger... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | RGS7/Gβ5/R7BP complex regulates synaptic plasticity and memory by modulating hippocampal GABABR-GIRK signaling | elife-02053-v1 | [
[
"Signaling through G protein-coupled receptors for the inhibitory neurotransmitter GABA ( GABABR ) has been recognized to play key roles in mood , nociception , memory , reward , and movement ( Bowery , 2006; Padgett and Slesinger , 2010 ) .",
"In the hippocampus , activation of postsynaptic GABABR on pyr... | [
"In the hippocampus , the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition .",
"Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ ( GIRK ) channels .",
"Here , we sh... | [
"Neurons communicate with one another at junctions called synapses .",
"The arrival of an electrical signal known as an action potential at the first cell causes molecules known as neurotransmitters to be released into the synapse .",
"These molecules diffuse across the gap between the neurons and bind to recep... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"computational and systems biology"
] | Self-establishing communities enable cooperative metabolite exchange in a eukaryote | elife-09943-v2 | [
[
"All living cells possess a system for biochemical reactions , the metabolic network , which supplies cells with their necessary molecular constituents .",
"The reactions participating in this network are highly conserved , so much so that all life is made up of a markedly similar set of metabolites ( Bra... | [
"Metabolite exchange among co-growing cells is frequent by nature , however , is not necessarily occurring at growth-relevant quantities indicative of non-cell-autonomous metabolic function .",
"Complementary auxotrophs of Saccharomyces cerevisiae amino acid and nucleotide metabolism regularly fail to compensate ... | [
"Life is sustained by an array of chemical reactions that is collectively referred to as metabolism .",
"Some of these reactions break down complex substances to release energy and vital compounds , while others make new molecules from smaller building blocks .",
"Bacterial communities are regularly composed of... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"tools and resources",
"neuroscience"
] | An open-source device for measuring food intake and operant behavior in rodent home-cages | elife-66173-v2 | [
[
"Feeding is critical for survival , and dysregulation of food intake underlies medical conditions such as obesity and anorexia nervosa .",
"Quantifying food intake is necessary for understanding these disorders in animal models .",
"However , it is challenging to accurately measure food intake in rode... | [
"Feeding is critical for survival , and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa .",
"It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders .",
"Operant behavioral testing can be u... | [
"Obesity and anorexia nervosa are two health conditions related to food intake .",
"Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is , why animals seek and consume food .",
"Measuring an animal’s food intake is usually done by weighin... | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus | elife-03229-v2 | [
[
"An understanding of ecological interactions and evolutionary history can be beneficial to the understanding of the cellular and developmental processes of an organism .",
"For example , the genes responsible for fruit and bacterial odor preferences in Drosophila melanogaster and Caenorhabditis elegans ar... | [
"Nematodes and insects are the two most speciose animal phyla and nematode–insect associations encompass widespread biological interactions .",
"To dissect the chemical signals and the genes mediating this association , we investigated the effect of an oriental beetle sex pheromone on the development and behavior... | [
"The nematode worm Pristionchus pacificus can live as a parasite inside the oriental beetle , where it waits for the beetle to die so it can feed off the bacteria that live on the beetle's decomposing carcass .",
"This ecologically important interaction is called necromeny .",
"P . pacificus is attracted to a n... | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis | elife-32471-v3 | [
[
"Although many bacteria are rod shaped , the cellular mechanisms that construct and replicate this geometry have remained largely unknown .",
"Bacterial shape is determined by the cell wall sacculus , a giant , encapsulating macromolecule that serves to resist internal osmotic pressure .",
"One of the... | [
"MreB is essential for rod shape in many bacteria .",
"Membrane-associated MreB filaments move around the rod circumference , helping to insert cell wall in the radial direction to reinforce rod shape .",
"To understand how oriented MreB motion arises , we altered the shape of Bacillus subtilis .",
"MreB moti... | [
"Many bacteria are surrounded by both a cell membrane and a cell wall – a rigid outer covering made of sugars and short protein chains .",
"The cell wall often determines which of a variety of shapes – such as rods or spheres – the bacteria grow into .",
"One protein required to form the rod shape is called Mre... | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"short report",
"medicine"
] | Performance of a deep learning based neural network in the selection of human blastocysts for implantation | elife-55301-v2 | [
[
"Assisted reproductive technologies ( ART ) such as in vitro fertilization ( IVF ) , while a solution to many infertile couples have been inefficient with an average success rate of approximately 30% reported in 2015 in the US ( CDC , 2015 ) .",
"IVF is also an expensive solution costing patients well ove... | [
"Deep learning in in vitro fertilization is currently being evaluated in the development of assistive tools for the determination of transfer order and implantation potential using time-lapse data collected through expensive imaging hardware .",
"Assistive tools and algorithms that can work with static images , h... | [
"Around one in seven couples have trouble conceiving , which means there is a high demand for solutions such as in vitro fertilization , also known as IVF .",
"This process involves fertilizing and developing embryos in the laboratory and then selecting a few to implant into the womb of the patient .",
"IVF , h... | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"biochemistry and chemical biology"
] | SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis | elife-06659-v3 | [
[
"Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division .",
"Multi-subunit Structural Maintenance of Chromosomes ( SMC ) protein complexes—such as cohesin and condensin—are major determinants of ch... | [
"Smc–ScpAB forms elongated , annular structures that promote chromosome segregation , presumably by compacting and resolving sister DNA molecules .",
"The mechanistic basis for its action , however , is only poorly understood .",
"Here , we have established a physical assay to determine whether the binding of c... | [
"The genome of any living organism holds all the genetic information that the organism needs to live and grow .",
"This information is written in the sequence of the organism's DNA , and is often divided into sub-structures called chromosomes .",
"Different species have different sized genomes , but even bacter... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Protein aggregates are associated with replicative aging without compromising protein quality control | elife-06197-v1 | [
[
"Aging results in an increasing decline of the organism's fitness over time ( Lopez-Otin et al . , 2013 ) .",
"Remarkably , this process segregates asymmetrically during budding yeast division: the mother cell forms an aging lineage , whereas the daughters generated by these mothers rejuvenate to form ete... | [
"Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells .",
"In budding yeast , various aging factors segregate to the aging ( mother ) -lineage , with poorly understood consequences .",
"In this study , we show that yeast mother cells form a pr... | [
"Aging is a complex process .",
"Studies involving a single-celled organism called budding yeast are commonly used to investigate the factors that contribute to aging .",
"When these yeast cells divide , a small daughter cell buds out from a large mother cell .",
"A mother cell has a limited lifespan and prod... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience",
"genetics and genomics"
] | Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay | elife-12245-v2 | [
[
"Macroautophagy , referred to hereafter as autophagy , is a cellular process by which proteins and organelles are degraded and recycled through sequestration within autophagosomes and delivery to lysosomes ( Levine and Klionsky , 2004 ) .",
"The autophagy pathway is highly conserved and required for organ... | [
"Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health .",
"Defects in the autophagy pathway have been observed in neurodegenerative disorders; however , no genetically-inherited pathogenic mutations in any of the core autophagy-related ( ATG ) gen... | [
"Ataxia is a rare disease that affects balance and co-ordination , leading to difficulties in walking and other movements .",
"The disease mostly affects adults , but some children are born with it and they often have additional cognitive and developmental problems .",
"Mutations in at least 60 genes are known ... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | The auditory representation of speech sounds in human motor cortex | elife-12577-v2 | [
[
"Our motor and sensory cortices are traditionally thought to be functionally separate systems .",
"However , an accumulating number of studies has revealed their roles in action and perception to be highly integrated ( Pulvermüller and Fadiga , 2010 ) .",
"For example , a number of studies have demons... | [
"In humans , listening to speech evokes neural responses in the motor cortex .",
"This has been controversially interpreted as evidence that speech sounds are processed as articulatory gestures .",
"However , it is unclear what information is actually encoded by such neural activity .",
"We used high-density ... | [
"When we speak , we force air out of our lungs so that it passes over the vocal cords and causes them to vibrate .",
"Movements of the jaw , lips and tongue can then shape the resulting sound wave into speech sounds .",
"The brain’s outer layer , which is called the cortex , controls this process .",
"More pr... | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"microbiology and infectious disease"
] | Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals | elife-07861-v3 | [
[
"Fungal secondary metabolites ( SMs ) are often encoded by gene clusters and have been the subject of intensive analysis in recent years .",
"This has resulted from the large number of completed fungal genomes as well as the intrinsic interest of SMs as environmental/pathogenic agents and their potential ... | [
"Secondary metabolites have a great potential as pharmaceuticals , but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer .",
"Here , signals , mediators , and biological effects of terrein production were stud... | [
"Organisms produce a wide variety of small molecules called metabolites through the break down of food and other chemical reactions .",
"Some of these molecules—known as primary metabolites—are required for growth , reproduction and other vital processes .",
"Other molecules called secondary metabolites are not... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Corticostriatal dynamics encode the refinement of specific behavioral variability during skill learning | elife-09423-v2 | [
[
"Animals have the ability to learn novel motor skills , allowing them to perform complex patterns of movement to improve the outcomes of their actions .",
"Acquiring novel skills usually requires exploration of the behavioral space , which is critical for learning ( Skinner , 1981; Sutton and Barto , 1998... | [
"Learning to perform a complex motor task requires the optimization of specific behavioral features to cope with task constraints .",
"We show that when mice learn a novel motor paradigm they differentially refine specific behavioral features .",
"Animals trained to perform progressively faster sequences of lev... | [
"Learning a new motor skill typically involves a degree of trial and error .",
"Movements that achieve the desired outcome—from catching a ball to playing scales—are repeated and refined until they can be produced on demand .",
"This process is made more difficult as the activity of individual neurons and muscl... | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans | elife-21166-v1 | [
[
"In order to effectively adapt to an environment , organisms need to be able to analyze both their internal state and external surroundings to make behavioral decisions that will maximize their chances of survival .",
"It has been hypothesized that decision making in animals is controlled primarily at the... | [
"Animal behavior is directed by the integration of sensory information from internal states and the environment .",
"Neuroendocrine regulation of diverse behaviors of Caenorhabditis elegans is under the control of the DAF-7/TGF-β ligand that is secreted from sensory neurons .",
"Here , we show that C . elegans ... | [
"For almost all species of animal , males and females will often behave differently in similar situations .",
"Little is known about how these sex-specific differences are generated or , for example , how different the nervous system of a male is to that of a female .",
"Moreover , it is also poorly understood ... | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice | elife-03464-v1 | [
[
"Chronic proliferative dermatitis mutation ( cpdm ) mice are deficient in SHARPIN ( Sharpincpdm/cpdm: henceforth referred to as Shpnm/m; protein: SHARPIN ) and develop dermatitis , multi-organ pathology and an immunological phenotype including disrupted lymphoid architecture , splenomegaly , liver inflammatio... | [
"SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro .",
"The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation , characterized by dermatitis , liver inflammation , splenomegaly , and loss... | [
"In response to an injury or infection , areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body .",
"At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apopto... | 2014 |
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