All nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). The monoclonal antibodies developed by H.F. Epstein were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biology, University of Iowa. N. Shemesh was supported by Fay and Bert Harbour award. A.B.-Z. was supported by an Israeli Council for Higher Education Alon Fellowship, by a Marie Curie International Reintegration grant, and by grants from the Binational Science Foundation and the Israeli Science Foundation (Grant No. 91/11).

Using behavioral assays to monitor protein folding in a living organism
1. Synchronizing Animals for Behavioral Assays
<ol>
	<li>Follow standard methods for maintenance of C. elegans36, including media and plates preparation, food preparation and animal growth conditions. Carefully control cultivation conditions, including temperature (15-25 &#176;C), population size and food availability. Discard any plate that shows bacterial or fungal contamination.</li>
	<li>Pick 15-30 embryos, transfer them to new plates and grow at the desired temperature (15-25 &#176;C) for the duration of the experiment. Alternatively, set 10-20 gravid adults on a plate, let them lay eggs for 30-60 min and then remove them from the plate. Both direct picking of embryos and setting gravid adults will give some variability in age (&#177;6 hr). To reduce this variability, either pick embryos at a specific stage, for example comma stage, or set 10-20 adults who are just starting to lay eggs (day 2 of adulthood). Avoid synchronization by bleaching whenever possible, given that this treatment is stressful and can affect proteostasis.</li>
	<li>Pick synchronized animals, transfer to an assay plate for scoring and then discard.</li>
</ol>
2. Movement Assay
Pick synchronized animals and transfer to a clean plate at the desire temperature for movement scoring (15-25 &#176;C). For each biological replicate, score &#62;20 animals and repeat assay at least 3x per experimental condition. Use the Wilcoxon Mann-Whitney rank sum test to compare two independent experimental conditions.
<ol>
	<li>To assay for mild movement impairment, set 5-7 animals together at the center of a clean bacterial lawn and allow the animals to move on the plate for 2 min. This time interval was determined based on the time it took 90% of wild type animals to clear a 1 cm circle.</li>
	<li>Count the number of animals that did not clear a 1 cm circle (e.g. that remained within a 1 cm radius from the point at which they were set) within 2 min as uncoordinated.</li>
	<li>To assay for severe movement impairment, set the animals on a clean bacterial lawn.</li>
	<li>Photograph several animals at time zero (t=0) and again 5 min later (t=5). Score animals that did not move one body length after 5 min as paralyzed.</li>
</ol>
3. Thermo-resistance
<ol>
	<li>Pick &#62;20 synchronized animals and transfer to a 24-well plate containing 450 &#181;l Heat Shock (HS) buffer (Table 1). For each biological replicate, score &#62;20 animals and repeat assay at least 4x per experimental condition.</li>
	<li>Transfer 24-well plate into a heated bath. HS temperature and duration strongly depend on growth conditions, in particular, the cultivation temperature.</li>
	<li>Supplement the HS buffer with 9 &#181;l SYTOX orange.</li>
	<li>Score animal survival by monitoring dye uptake, using a fluorescent stereoscope with a TXR filter. Animals that took up the dye are dead. Use the Wilcoxon Mann-Whitney rank sum test to compare two independent experimental conditions.</li>
</ol>
Using immunostaining and tagged proteins to monitor protein folding in specific cells
4. Monitor Localization of Proteins by Immunostaining
<ol>
	<li>At the required stage, transfer at least 30 animals into an Eppendorf tube containing M9 buffer36.</li>
	<li>Wash the animals a few times with M9 buffer by performing a short, low speed centrifugation step (3,000 rpm/900 x g, 2 min) and resuspending.</li>
	<li>Place the tubes on ice for a few minutes to cool down.</li>
	<li>Remove excess liquid and incubate with 500 &#181;l ice-cold 4% paraformaldehyde solution (Table 2). The time of incubation needs to be calibrated for each protein examined and should be around 5-30 min at room temperature. Prolonged incubation at 4 &#176;C can also be performed but this may impair sample permeabilization.</li>
	<li>Wash fixed animals 3x with PBS-Tween (pH 7.2) buffer by centrifugation (3,000 rpm, 1-2 min). The animals can be stored at this stage at 4 &#176;C for a few weeks.</li>
	<li>Collect the animals by centrifugation (3,000 rpm, 1-2 min).</li>
	<li>In the hood, remove most of the supernatant and resuspend the pellet in 1 ml&#160;&#946;-mercaptoethanol (&#946;-ME) solution (Table 2).</li>
	<li>Incubate at 37 &#176;C overnight with gentle rocking on a nutator.</li>
	<li>In the hood, wash animals 3x with PBS-Tween (pH 7.2) buffer.</li>
	<li>Resuspend the animals in 50-100 &#181;l&#160;of PBS-Tween (pH 7.2). The animals can be stored at this stage at 4 &#176;C for a few weeks.</li>
	<li>Collect the animals by centrifugation (3,000 rpm, 1-2 min) and remove most of the PBS-Tween buffer.</li>
	<li>Add 100-150 &#181;l&#160;of collagenase solution to each tube and incubate at 37 &#176;C with strong agitation (using a Thermo-mixer or a shaking incubator).</li>
	<li>Examine the animals frequently under a stereomicroscope, starting after 5 min of incubation. When 20% of the animals are broken, stop the reaction by placing the tube on ice. This step is highly sensitive! The duration of treatment depends on fixation conditions and can vary widely between strains. A prolonged incubation could result in complete degradation of the animals. Conditions must be calibrated for each enzyme batch used and degradation should be monitored carefully.</li>
	<li>Wash the animals twice in PBS-Tween (pH 7.2) and then collect the animals by centrifugation (3,000 rpm, 1-2 min) and remove most of the PBS-Tween buffer.</li>
	<li>Add 1 ml&#160;AbA solution (Table 2) and incubate with gentle rocking for 1 hr at room temperature. The animals can be stored at this stage at 4 &#176;C for a few weeks.</li>
	<li>Collect the animals by centrifugation (3,000 rpm, 1-2 min) and resuspend in 200 &#956;l&#160;AbA.</li>
	<li>Add the primary antibody diluted to 1:100-1:1,000 in AbA solution and incubate with gentle rocking. The temperature and duration of incubation can vary between antibodies and should be calibrated for each protein examined. We begin with an overnight incubation at room temperature.</li>
	<li>Wash the animals by centrifugation (3,000 rpm, 1-2 min).</li>
	<li>Add AbA buffer (1 ml) and incubate with gentle mixing for 15-60 min at room temperature. The animals can be stored at this stage at 4 &#176;C for a few weeks.</li>
	<li>Optional: The sample can also be stained with fluorescent dyes. To do so, add the dye (e.g. DAPI (2 mg/ml) or Phalloidin (1:100)) to 200 &#956;l AbA and incubate with gentle rocking for up to 1 hr. This step can be performed during one of the steps for washing the secondary antibody.</li>
	<li>Wash the animals 3x by repeating steps 4.18-4.19.</li>
	<li>Add 100 &#956;l&#160;AbA with the appropriate fluorescently-tagged secondary antibody, diluted 1:100.</li>
	<li>Maintain the samples covered (or in a dark tube) and incubate with rocking for several hours at room temperature.</li>
	<li>Wash the animals 3x by incubating in AbA buffer (1 ml) with gentle mixing for 15-60 min at room temperature followed by centrifugation (3,000 rpm, 1-2 min). The animals can be stored at this stage at 4 &#176;C for a few weeks.</li>
	<li>To mount animals on slides, remove all liquid and resuspend in 10 &#956;l&#160;of the same buffer.</li>
	<li>Cut the end of the pipette tip to avoid sheering of the animals and transfer 1-2 &#956;l&#160;of the sample onto a slide.</li>
	<li>Add mounting medium in a 1:1 ratio.</li>
	<li>Cover the slide gently with a coverslip and seal with nail polish. If the slide is not monitored immediately, the slides can be stored at -20 &#176;C for a prolonged period of time.</li>
</ol>
5. Monitor Localization of Tagged Proteins
<ol>
	<li>At the required stage, collect at least 15 animals into a 10 ml&#160;drop of M9 buffer on a slide.</li>
	<li>Using a &#34;worm pick&#34;, move the animals into a 10 ml&#160;drop of paraformaldehyde solution (4%) (Table 2) and wait until all animals stop moving (approximately&#160;5 min).</li>
	<li>Using a &#34;worm pick&#34;, move the animals into a 2 ml&#160;drop of M9 buffer and mounting medium (1:1) (Table 2).</li>
	<li>Gently cover the animals with a coverslip and seal with nail polish.</li>
</ol>
Using ex vivo assays to monitor protein folding and stability
6. Partial Digestion
<ol>
	<li>Wash animals from 15 plates (60 mm) using 1-2 ml&#160;M9 buffer in a microfuge tube. Please note that animals should not be crowded and sufficient food should be available throughout the duration of the experiment.</li>
	<li>Wash the animals a few times with M9 buffer by centrifuging (3,000 rpm, 2 min) and resuspending animals.</li>
	<li>After the final spin, resuspend animals in 100 &#956;l M9 buffer.</li>
	<li>Flash-freeze in liquid N2. Samples can be stored at -80 &#176;C.</li>
	<li>Using a chilled pestle and motorized drill (a hand held tissue culture drill can also used), grind the sample. Keep sample on ice and avoid over-heating.</li>
	<li>Examine the animals frequently under a stereomicroscope to determine if grinding is complete. If intact animals remain, repeat the procedure.</li>
	<li>Estimate the volume by pipetting the worm extract.</li>
	<li>Add 1:1 ratio of (2x) worm lysis solution (Table 3). Do not use protease inhibitors of any kind.</li>
	<li>Incubate on ice for 15 min.</li>
	<li>Remove debris by centrifugation (1,000 x g for 2 min) in a precooled centrifuge (4 &#176;C).</li>
	<li>Transfer the supernatant to a clean microfuge tube. Samples can be used directly or stored at -80 &#176;C.</li>
	<li>Prepare chymotrypsin solution (Table 3) and maintain on ice until use.</li>
	<li>Determine total protein concentration using the Bradford assay.</li>
	<li>Dilute the sample if necessary such that the total protein concentration is 3-3.5 mg/ml.</li>
	<li>Add chymotrypsin solution to the total protein lysate (1:1,200).</li>
	<li>To determine time of incubation, incubate the sample with chymotrypsin for 60 min. Remove 20 &#956;l aliquots from the sample at different time points (e.g. 0, 5, 20, 40, and 60 min), mix immediately with SDS sample buffer and boil for 5 min at 96 &#176;C. *Alternatively, incubate the sample with different concentrations of chymotrypsin and stop the reaction after a specific time by adding SDS sample buffer and boiling for 5 min at 96 &#176;C.</li>
	<li>Run samples on a SDS-PAGE gel.</li>
	<li>Perform western blot analysis using appropriate antibodies to determine the relative stability of the protein.</li>
	<li>Determine the optimal conditions (chymotrypsin concentration and digestion time) that show significant change between samples and use these conditions for future experiments.</li>
	<li>Determine the intensity of different digest products (lower molecular weight fragments) using densitometry software, such as the NIH image gel module.</li>
</ol>

The authors declare that they have no competing financial interests.

Probes of the cellular proteostasis capacity must be highly sensitive to changes in the folding environment and be easily monitored so as to provide real-time assessment of protein folding quality control capabilities. Using metastable proteins as probes of proteostasis capacity, we monitored changes in phenotype, subcellular protein localization,&#160;and protein conformation. The protocols presented here focus on muscle protein-associated phenotypes, including motility, muscle filament organization and myosin stability...

The interplay of the various steps of protein biosynthesis, such as mRNA transcription, processing, and translation, as well as protein folding, translocation, and assembly/disassembly, determines the load of metastable proteins that depend on the cellular protein quality control machineries for their function1. The absence or malfunction of protein quality control machineries can, therefore, result in the functional decline of diverse types of cellular machineries and in the onset of protein misfolding diseases2-7. When the capacity of protein folding and clearance machineries is balanced with the load of metastable proteins, protein homeostasis (proteostasis) is achieved, a state that ultimately prevents the accumulation of misfolded proteins and aggregation within cells6. Accumulation of misfolded proteins is thought to be the signal that activates stress-inducible transcription factors, such as heat shock factor (HSF-1), and results in the activation of cyto-protective stress responses6,8.
Our understanding of the functions of proteostasis networks in metazoans has mostly been derived from in vitro reconstitution studies and from observations made with tissue culture cells and unicellular organisms9. For example, research on molecular chaperones that prevent and resolve protein damage has focused on biochemical and cell biological studies of the mechanisms of chaperone-mediated protein folding, disaggregation and translocation10-13. In comparison, only limited information is available on the integrated function of various proteostasis components in the different cells and tissues of metazoans under &#34;normal&#34; growth conditions and in response to stress11. The discovery that in C. elegans, cellular protein quality control can also be regulated cell nonautonomously, as reflected in experiments showing that mutations in the two neurons that perceive temperature can block the activation of the heat shock response and reduce thermotolerance, demonstrated the need to study proteostasis regulation in multicellular organism14-17. What is missing, however, is a cohesive picture of how proteostasis networks, such as the various molecular chaperone families, function in the tissues of an intact metazoan and how dynamic are these networks during development and aging. To meet this goal, reliable sensors for monitoring proteome maintenance in living animals are needed to determine the proteostatic capacity of different cells in a multicellular organism during the course of development and aging.
For a given protein to function as a sensor of cellular proteostasis, it must respond to changes in the cellular folding environment while only minimally interfering with the folding of unrelated proteins in the cell. To explore the maintenance and recovery of cellular proteostasis in a living organism, two complementary approaches that depend on folding sensors can be taken. The first relies on designed folding-sensors, based on experimentally identified metastable proteins that are known to depend on proteostasis machinery, such as firefly luciferase18-20 or GFP tagged with a degron21-24. In the second approach, endogenous metastable proteins, such as temperature-sensitive(ts) or age-dependent aggregating proteins that respond to incremental changes in the cellular environment, are traced25-27. Designed folding-sensors serve no essential biological function yet offer the advantage of being detectable by powerful reporting assays, such as GFP-tagged proteins, and can be employed with many different cellular and animal models18. However, because introducing a single foreign protein can affect the folding environment27, such polypeptides can overload the cellular proteostasis machinery. Alternatively, designed folding-sensors that are not native to the cell on which they report may not be affected by changes in the proteostasis capacity of the cell. For example, one GFP-tagged proteasome reporter substrate required ~90% of the proteasome to be inhibited before a phenotype could be detected23. In contrast, endogenous metastable proteins that rely on the proteostasis machineries of the cell offer the advantage of being within the cellular sensitivity range. However, the loss-of-function associated with the misfolding of such proteins can also impact cellular function and organismal viability. Here, we will focus on the use of endogenous C. elegans folding-sensors.
C. elegans is a well-established metazoan model for the study of both development and aging that utilizes many conserved biological pathways and can be used to follow protein folding in the cell, using a combination of cell biology, biochemical and genetic approaches. We employed metastable proteins as probes of proteostatic capacity by monitoring changes in their phenotype, localization and stability. A variety of protein functions can, moreover, be studied by simple behavioral analysis. Likewise, substantial mislocalization of proteins occurs when cellular protein quality control networks fail to adjust to cellular demands. Proteins can be easily visualized in cells of living animals using fluorescently-tagged proteins or via immunostaining. Finally, using ex vivo methods, it is possible to monitor protein expression and stability. This allows for fast and simple screening of behavioral and physiological changes, coupled with in depth analysis of protein localization and stability, allowing for the monitoring of proteostasis modifiers. By combining these different methods, a broad view of the protein-folding environment of a cell can be obtained. Indeed, this strategy has been successfully used to monitor proteostasis perturbation in C. elegans, yeast, tissue culture and bacteria15,25-35.

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			<td height="238" rowspan="7" style="height:238px;width:267px;">Antibody staning and fiaxtion</td>
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			<td height="34" style="height:34px;">DABCO</td>
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			<td style="width:264px;">8034560100</td>
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			<td height="34" style="height:34px;">Triton X-100</td>
			<td style="width:215px;">Alfa Aesar</td>
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			<td height="34" style="height:34px;">&#946; -ME</td>
			<td style="width:215px;">Sigma</td>
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			<td height="34" style="height:34px;">BSA</td>
			<td style="width:215px;">Di Cam</td>
			<td style="width:264px;">000-40-100</td>
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			<td height="34" style="height:34px;">&#160;Partial digest&#160;</td>
			<td>Chymotrypsin</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:264px;">C4129-250MG</td>
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			<td height="68" rowspan="2" style="height:68px;">Thermo-resistance</td>
			<td>SYTOX Orange</td>
			<td>Invitrogen</td>
			<td>S11368</td>
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			<td height="34" style="height:34px;">Cholesterol</td>
			<td style="width:215px;">Amaresco</td>
			<td style="width:264px;">0433-250G</td>
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			<td height="20" style="height:20px;">EQUIPMENT</td>
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			<td height="43" style="height:43px;width:267px;">Material Name</td>
			<td style="width:272px;">Company</td>
			<td style="width:215px;">Catalog Number</td>
			<td style="width:264px;">Comments (optional)</td>
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		<tr height="59">
			<td height="59" style="height:59px;width:267px;">M165 FC fluorescent stereoscope</td>
			<td style="width:272px;">Leica</td>
			<td style="width:215px;"></td>
			<td style="width:264px;">TXR filter</td>
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			<td height="118" rowspan="2" style="height:118px;width:267px;">EXi Blue Fluorescence Microscopy Camera</td>
			<td rowspan="2">QImaging</td>
			<td rowspan="2" style="width:215px;">EXI-BLU-R-F-M-14-C&#160;</td>
			<td rowspan="2" style="width:264px;"></td>
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			<td height="59" style="height:59px;width:267px;">ConfoCor 3/510 META confocal microscope</td>
			<td style="width:272px;">Zeiss</td>
			<td style="width:215px;"></td>
			<td style="width:264px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Pellet pestles</td>
			<td>Sigma</td>
			<td>Z359947-100EA</td>
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			<td height="20" style="height:20px;">Pellet Pestle Cordless Motor</td>
			<td>Kontes</td>
			<td>K749540-0000</td>
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			<td height="20" style="height:20px;">MicroCL 17 Microcentrifuge Series</td>
			<td>Thermo</td>
			<td>75002455</td>
			<td>Refrigerated, , 230 V 50/60 Hz, includes 24x 1.5/2.0 ml rotor with ClickSeal Biocontainment Lid</td>
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using caenorhabditis elegans as a model system to study protein homeostasis in a multicellular organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

A compromised folding environment can lead to protein misfolding and aggregation. Protein misfolding is associated with altered conformation and results in a loss of protein function. We use complementary approaches to monitor protein folding and function in intact animals, specific cells and protein extracts. An intelligent choice of folding-sensor, based on strong behavioral and cellular phenotypes that are associated with a known destabilizing mutation, can then be used to monitor changes in the protein-folding enviro...

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Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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6.0K Aufrufe.  Ben-Gurion University of the Negev. To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.