text stringlengths 1 654k |
|---|
2. If different fluorophores are excited by the same laser, use the line scanning feature. |
3. A Galvano scanner enables more sensitivity, but has a higher risk for photobleaching. A Resonant scanner enables faster acquisition, thus lowering the risk for photobleaching, but is less sensitive. |
4. Averaging between 2 and 16 images, it enhances signal to noise ratio, but makes acquisition slower and, of course, involves more exposure of the sample to the laser. |
5. Duration of acquisition- depends on the biological question (e.g JUNQ and IPOD formation takes ~2 hr). We have imaged yeast with the resonant scanner for up to 30 hr in 3D time-lapse. |
6. Time lapse intervals- Smaller intervals will create a more coherent movie but might cause photo bleaching and therefore loss of signal. |
Representative Results |
Figure 1 |
Figure 1. Model: Sub-cellular compartmentalization of misfolded proteins. Quality control machinery directs misfolded proteins into distinct compartments with distinct functions: Soluble proteins, targeted for degradation, undergo poly-ubiquitination and are sent to the Juxta-NuclearQuality control compartment (JUNQ). ... |
Figure 2 |
Figure 2. Modeling protein misfolding with GFP-Ubc9ts. Under normal conditions, GFP-Ubc9ts (green) is natively folded, and is localized diffusely in the nucleus and the cytosol. The nucleus is labeled by NLS-TFP (red). Expression of Ubc9ts was shut off by addition of 2% glucose before imaging in all experiments. Click ... |
1. Upon temperature shift to 37 °C, GFP-Ubc9ts (green) is misfolded and forms cytosolic Stress Foci. The nucleus is labeled by NLS-TFP (red). |
2. Upon recovery from heat shock at 23 °C, the thermally denaturated GFP-Ubc9ts is degraded, as indicated by decreased fluorescence level. |
3. Upon temperature shift to 37 °C and proteasome inhibition with 80 Mm MG132, GFP-Ubc9ts is misfolded and processed into JUNQ and IPOD inclusions. The nucleus is labeled by NLS-TFP (red). |
4. Upon temperature shift to 37 °C and ubiquitination inhibition, GFP-Ubc9ts is misfolded and processed into the IPOD inclusion. The nucleus is labeled by NLS-TFP (red). The Ubiquitin Protease 4 (Ubp4) is overexpressed to block Ubc9ts ubiquitination. |
Figure 3 |
Figure 3. Time lapse of JUNQ and IPOD formation. Upon temperature shift to 37 °C and proteasome inhibition with 80 Mm MG132, GFP-Ubc9ts Stress Foci are processed into JUNQ and IPOD inclusions. The nucleus is labeled by NLS-TFP (red). 3D images were acquired at 4 min intervals. (also see Movie 1). Click here to view lar... |
Discussion |
Our intuitions about biochemical processes derive from bench top experiments in which a well-mixed solution of reactants and products is allowed to reach equilibrium in a beaker. In such a setting, the concentration of a given chemical species may be expressed as a single number, which is the ratio of a molar quantity ... |
As the technology we use to look at cells under magnification improves by leaps and bounds, it becomes ever clearer that the conditions in which most biochemical reactions take place in vivo bear only the slightest resemblance to those of the classic bench top scenario. Not only is the interior of the cell a densely pa... |
Nowhere are intuitions stemming from classical in vitro biochemical experiments more prone to mislead as in questions pertaining to the in vivo folding, misfolding, and aggregation of proteins. Whereas studies of protein chemistry in bulk reactions can treat the issue of folding for a given protein as a simple yes or n... |
In the newly emerging paradigm for understanding biochemistry, concentration becomes a variable property of each specific nano-environment in the cell, and the molecular events that underlie biological processes must be assayed not only in time, but also in space. The 4D imaging approach presented here enables sensitiv... |
4D imaging is also essential for accurately determining protein localization or colocalization between two different proteins, and for detecting phenomena which maybe be transient but important. For example, especially in a small spherical organism such as yeast, it may appear to be the case that a structure or aggrega... |
In the example experiment we present here, we demonstrate the use of a model misfolded protein, Ubc9ts, to follow aggregation quality control over time and space in the cytosol. At the permissive temperature, Ubc9ts is folded and diffused in the nucleus and cytosol. Upon heat-induced misfolding, it initially forms rapi... |
Disclosures |
No conflicts of interest declared. |
Materials |
Name Company Catalog Number Comments |
MG132 Mercury mbs474790 |
con A Sigma C2010 |
Glass bottom plates ibidi ibd81158 |
4D Fluorescence Imaging of Protein Aggregation |
Confocal 3D movies were acquired using a Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), using a 60x water objective NA 1.27, 0.3 micron slices, 0.5% laser power (from 65 mW 488 laser and 50 mW 561 laser). z-stacks were acquired every 5 min for 90 min. Each z-series was acquired with 0.5 micron step s... |
DOWNLOAD MATERIALS LIST |
References |
1. Gershenson, A., Gierasch, L. M. Protein folding in the cell: challenges and progress. Current opinion in structural biology. 21, 32-41 (2011). |
2. Aguzzi, A., Calella, A. M. Prions: protein aggregation and infectious diseases. Physiological reviews. 89, 1105-1152 (2009). |
3. Morimoto, R. I. Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes & development. 22, 1427-14 (2008). |
4. Cohen, E., Dillin, A. The insulin paradox: aging, proteotoxicity and neurodegeneration. Nature reviews. Neuroscience. (2008). |
5. Hershko, A., Ciechanover, A. The ubiquitin system. Annual review of biochemistry. 67, (1998). |
6. Tyedmers, J., Mogk, A., Bukau, B. Cellular strategies for controlling protein aggregation. Nature reviews. Molecular cell biology. 11, 777-788 (2010). |
7. Treusch, S., Cyr, D. M., Lindquist, S. Amyloid deposits: Protection against toxic protein species? Cell cycle (Georgetown, Tex.). 8, 1668-1674 (2009). |
8. Spokoini, R., et al. Confinement to Organelle-Associated Inclusion Structures Mediates Asymmetric Inheritance of Aggregated Protein in Budding Yeast. Cell Rep. (2012). |
9. Weisberg, S. J., et al. Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity. Proc Natl Acad Sci U S A. 109, 15811-15816 (2012). |
10. Kaganovich, D., Kopito, R., Frydman, J. Misfolded proteins partition between two distinct quality control compartments. Nature. 454, 1088 (2008). |
11. Betting, J., Seufert, W. A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway. The Journal of biological chemistry. 271, 25790 (1996). |
12. Tongaonkar, P., Beck, K., Shinde, U. P., Madura, K. Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal. Analytical biochemistry. 272, 263 (1999). |
Comments |
0 Comments |
Post a Question / Comment / Request |
You must be signed in to post a comment. Please or create an account. |
Usage Statistics |
2018 |
Pumpkins (2018) |
0 |
Pumpkins (2018) |
Watch Pumpkins Movie Online on Bmovies, Fmovies, BmoviesFree, A teen Horror Movie set in the 80’s in the North of England. |
The 11th Patient (2018) |
0 |
The 11th Patient (2018) |
Watch The 11th Patient Movie Online on Bmovies, Fmovies, BmoviesFree, Steven, A young teenage boy, is found in a comatose state deep in the woods ... |
Slime (2018) |
0 |
Slime (2018) |
Watch Slime Movie Online on Bmovies, Fmovies, BmoviesFree, In Atlanta Yo Money name is gaining respek in the streets when it comes to his music, ... |
Robbery (2018) |
0 |
Robbery (2018) |
Watch Robbery Movie Online on Bmovies, Fmovies, BmoviesFree, When his criminal father is diagnosed with dementia, a young thief plans a series of ... |
Fronteras (2018) |
0 |
Fronteras (2018) |
Watch Fronteras Movie Online on Bmovies, Fmovies, BmoviesFree, A Hispanic Border Patrol Agent has his morals challenged when a life-threatening ... |
Gundermann (2018) |
0 |
Gundermann (2018) |
Watch Gundermann Movie Online on Bmovies, Fmovies, BmoviesFree, The real life story of East German singer and writer Gerhard Gundermann and his ... |
Wake (2018) |
0 |
Wake (2018) |
Watch Wake Movie Online on Bmovies, Fmovies, BmoviesFree, A widowed mortician, struggling with agoraphobia, is given a birthday gift from her ... |
30 Nights (2018) |
0 |
30 Nights (2018) |
Watch 30 Nights Movie Online on Bmovies, Fmovies, BmoviesFree, In a last ditch effort to save their marriage, Nick and Willa visit a renowned, ... |
Draug (2018) |
0 |
Draug (2018) |
Watch Draug Movie Online on Bmovies, Fmovies, BmoviesFree, In the 11th century a missionary goes missing somewhere in the huge forests bordering ... |
South Africa whip Trinidad & Tobago 12-0New Delhi: Coetzee Pietie marked her return from retirement by slamming four goals as South Africa pounded Trinidad and Tobago 12-0 in a pool A match of the women's hockey competition in the 19th Commonwealth Games here Monday. |
Pietie, the 32-year-old forward and penalty corner specialist, came out of a self-imposed five-year absence from competitive hockey and showed she was none the worse for it by slotting home the goals as the South Africans ran riot at the Major Dhyan Chand National Stadium. |
"Everything went to plan today (Monday) and it is a privilege to score a hat-trick in an international match," said Pietie, who showcased her versatility with two penalty corner conversions and as many field goals. |
"I took a five-year break because I was exhausted, but I was playing in local matches. Then, my coach convinced me to return and this is my third month," she added. |
Pietie began the flood of goals with two conversions in the first 10 minutes and thereafter, the Trinidadians were helpless against the South African onslaughts that were magnified by the tottering defence. |
"We need to remain focused on our next match. We made too many mistakes in the defence, but we can bounce back," said a brave Trinidad and Tobago skipper Patricia Wright-Alexis. |
The other goal-scorers for South Africa, who led 3-0 at the break, were: Dirkie Chamberlain (3), Jennifer Wilson (2), Kathleen Taylor, Lesle Anne George and Farah Fredericks. |
Source: IANS |
Continue Reading... |
Vintage Rose Gold Engagement Rings Art Deco |
Vintage Rose Gold Engagement Rings Art Deco |
Photo Detail |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.