We thank the Caenorhabditis Genetics Center, funded by the NIH National Center for Research Resources (NCRR), for some of the nematode strains. 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. This research was supported by a grant from the Israel Science Foundation (grant No. 278/18) and by a grant from the Israel Ministry of Science &#38; Technology, and the Ministry of Foreign Affairs and International Cooperation, General Directorate for Country Promotion, Italian Republic (grant No. 3-14337). We thank members of the Ben-Zvi laboratory for help in preparing this manuscript.

1. Preparation of nematode growth media plates for RNAi
<ol>
	<li>To a 1 L bottle, add 3 g of NaCl, 2.5 g of Bacto-Peptone, 17 g of agar and distilled water up to 1 L and autoclave.</li>
	<li>Cool bottle to 55 &#176;C.</li>
	<li>Add 25 mL of 1 M KH2PO4, pH 6.0, 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 1 mL of cholesterol solution (Table 1) to make nematode growth media (NGM).</li>
	<li>Add 1 mL of ampicillin (100 mg/mL) and 0.5 mL of 1 M IPTG (Table 1) to make NGM-RNAi solution. 
	NOTE: Commonly used HT115(DE3) E. coli bacteria contain an IPTG-inducible T7 DNA polymerase used for expressing the dsRNA-encoding plasmids. These plasmids also encode for ampicillin resistance.</li>
	<li>Mix the warm solution by swirling the bottle.</li>
	<li>In the hood or using sterile procedures, pour the NGM solution into plates or using a peristaltic pump, dispense the solution into plates. Use 6-well, 12-well, 40 mm or 60 mm plates for this screen. Agar should fill about 2/3 of the plate depth.</li>
	<li>Let the plates dry overnight on the bench at room temperature, keeping the plates covered. NGM plates for RNAi can be stored at 4 &#176;C for up to a month.</li>
</ol>
2. Growing RNAi bacteria and seeding the plates
<ol>
	<li>In the hood or using sterile procedures, add 1 mL of ampicillin (100 mg/mL) and 2.5 mL of tetracycline (5 mg/mL) (Table 1) to a pre-autoclaved 1 L of LB solution and mix. 
	NOTE: Commonly used HT115(DE3) E. coli bacteria are tetracycline-resistant.</li>
	<li>In the hood or using sterile procedures, add 600 &#181;L of LB solution to each well in 2 mL-deep 96-well sterile plates. It is best to use a multichannel pipet for dispensing the media.</li>
	<li>Using sterile procedures, inoculate wells with HT115(DE3) E. coli bacteria transformed with a dsRNA-encoding plasmid targeting a gene of interest or an empty plasmid, as a control. Cover the plates and incubate at 37 &#176;C overnight. Libraries that consist of bacterial clones expressing dsRNA, corresponding to ~94% of predicted C. elegans genes were previously constructed22,23 and are commercially available. The chaperone library used here was constructed by Dr. Richard Morimoto laboratory26.</li>
	<li>Using sterile procedures, seed 75, 150 or 250 &#181;L of bacteria onto the 12-well, 40 mm and 6-well or 60 mm NGM RNAi plates, respectively. Clearly mark the name of the target gene on the plate. Bacteria should cover 30-50% of the agar surface and should not touch the edges of the plate.</li>
	<li>Allow plates to dry for at least 2 days on the bench at room temperature, keeping the plates covered. 
	NOTE: Plates can be incubated at 37 &#176;C overnight. Make sure the inner wells are dry before using or storing the plates. For all long-term purposes (i.e., drying or storage) keep the plates in the dark. Dried, seeded plates can be stored at 4 &#176;C for up to a month.</li>
</ol>
3. Non-stressful synchronization of embryos
<ol>
	<li>Use a worm pick to move about 100 eggs from an unsynchronized worm plate to a newly seeded NGM plate.</li>
	<li>Cultivate animals for 5 days at 15 &#176;C, 3.5 days at 20 &#176;C or 2.5 days at 25 &#176;C. Animals should reach the first day of egg laying. 
	NOTE: Worms are commonly cultivated at 20 &#176;C. However, different chaperone mutant strains may require specific cultivation temperatures. For example, many temperature-sensitive strains are cultivated at 15 &#176;C but shifted to 25 &#176;C to expose their phenotype.</li>
	<li>Add 1 mL of M9 buffer (Table 1) slowly and away from the bacterial lawn. Rotate the plate so that the buffer completely covers the plate. Then tilt it to one side and remove the liquid from the plate and wash the animals off the plate. 
	NOTE: When using temperature-sensitive animals or chaperone mutants, it is best to maintain the buffers used in the protocol at the animals&#8217; cultivation temperature.</li>
	<li>Repeat step 3.3 three times or until all the animals are washed off the plate.</li>
	<li>Using a standard plastic tip, cut a square of agar from the washed plate where eggs are concentrated and place the piece of agar onto a newly seeded NGM plate. 
	NOTE: ~200 eggs are required to produce enough egg-laying animals; too many animals consume the bacteria too quickly. Low food levels can impact proteostasis27.</li>
	<li>Cultivate the animals for 5 days at 15 &#176;C, 3.5 days at 20 &#176;C or 2.5 days at 25 &#176;C. At this point, the plates should be covered with synchronized eggs. 
	NOTE: Animals can be shifted to a new plate for a short duration for a more stringent synchronization. However, it is important to only use adults at the early stages of egg-laying as animals can retain eggs in their uterus impacting synchronization.</li>
	<li>Add 1 mL of M9 buffer slowly and away from the bacterial lawn.</li>
	<li>Rotate the plate so that the buffer completely covers the plate. Then tilt it to one side and remove the liquid from the plate and wash the animals off the plate.</li>
	<li>Repeat step 3.7 three times or until all animals are washed off the plate.</li>
	<li>Add 1 mL of M9 buffer and use a cell scraper to release the eggs from the plate.</li>
	<li>Collect the M9 buffer containing the eggs from the plates.</li>
	<li>Centrifuge the M9 buffer containing the eggs at 3,000 x g for 2 min.</li>
	<li>Remove the supernatant and add M9 buffer to reach a volume of 1 mL.</li>
	<li>Resuspend the eggs to disrupt any chunks of eggs and bacteria.</li>
	<li>Repeat the washing procedure described in steps 3.11-3.13 five times. The egg pellet should appear white. If it is still yellow/brown, repeat the wash until a white pellet is attained. 
	NOTE: Bacteria that remain on the eggs can contaminate the dsRNA-expressing bacteria.</li>
	<li>Remove most of the supernatant, leaving about 200 &#181;L. Synchronized eggs can be used for RNAi screens.</li>
</ol>
4. Common phenotypic assays
<ol>
	<li>Cultivation of animals during experiments
	<ol>
		<li>Place a drop of ~30 eggs close to the bacterial lawn in each RNAi-seeded plate. For reference, also place ~30 eggs on plates seeded with empty vector-containing (L4440) bacteria.</li>
		<li>Cultivate age-synchronized animals on NGM RNAi-seeded plates. The duration of the experiment will depend on the stage at which the animals are to be monitored and the temperature of cultivation. Adjust the cultivation temperature when using temperature-sensitive mutant animals. Adjust the cultivation duration when using developmentally delayed mutant animals. 
		NOTE: While timing can vary, once animals reach adulthood, egg laying (and the resulting rapid food consumption), as well as age-dependent proteostsis collapse28, could impact the results. It is thus recommended to score animals before the onset of egg laying (day 1 of adulthood). Wild type animals reach this stage after 4.5 days at 15 &#176;C, 3 days at 20 &#176;C or 2 days at 25 &#176;C.</li>
		<li>The number of repeats used will depend on the size of the gene set examined. For the chaperone library (97 genes), repeat experiments at least four times. The size of the population depends on the assay used. In the behavioral assays discussed here, score &#62;15 animals per experimental condition in each repeat. Data and statistical analyses also strongly depend on the type of assay used. Data in the assays discussed here can be presented as means &#177; SEM.</li>
		<li>Compare the RNAi- and empty vector control-treated animals as independent populations. P values can be calculated using one-way or two-way ANOVA, depending on the changes examined, namely aggravating or alleviating alone or both. When examining a single RNAi treatment vs. control, P values can be calculated using one-way or two-way Mann-Whitney rank sum test. Other than statistical significance, consider a threshold for hits based on the degree of impact on the phenotype.</li>
	</ol>
	</li>
	<li>Developmental arrest/delay
	<ol>
		<li>Cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-containing control bacteria reach adulthood but before egg laying starts.</li>
		<li>Monitor animals using a stereomicroscope and count the number of larvae and adults to score the percent of developmentally delayed animals. For reference, compare to mutant animals grown on empty vector-containing control bacteria. hsp-1 or hsp-90 RNAi treatment results in developmental arrest of wild type animals and can be used as a positive control.</li>
		<li>To score the percent of developmentally delayed animals over time, repeat step 4.2.2. 
		NOTE: If there are egg-laying adults on the plate, transfer the developmentally delayed animals to a new NGM-RNAi plate labeled for the same target gene to avoid confusion with progeny.</li>
	</ol>
	</li>
	<li>Sterility or egg laying defects
	<ol>
		<li>Cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-control bacteria begin to lay eggs.</li>
		<li>Monitor animals using a stereomicroscope and score the percent of animals with no visible eggs in their uterus. For reference, compare to mutant animals grown on empty vector-control bacteria.</li>
		<li>Alternately, monitor animals using a stereomicroscope and score the percent of animals with a uterus full of eggs, defined as EGg Laying defective (Egl-d) phenotype29.</li>
	</ol>
	</li>
	<li>Embryonic lethality
	<ol>
		<li>Cultivate age-synchronized animals as in step 4.1 until the animals begin to lay eggs.</li>
		<li>Transfer ~100 eggs to an empty plate. Spread the eggs in rows to simplify counting.</li>
		<li>Score the percent of unhatched eggs on the plate after 24-48 hours. For reference, compare to eggs of animals treated with empty vector-control bacteria.</li>
	</ol>
	</li>
	<li>Paralysis assay
	<ol>
		<li>For day 1 adults, cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-control bacteria reach adulthood but before egg laying starts.</li>
		<li>Draw a line on the back of a regular NGM agar plate using a fine marker.</li>
		<li>Place 5-10 animals on the marked line.</li>
		<li>Set a timer and wait for 10 min.</li>
		<li>Score the percent of animals remaining on the line as paralyzed worms. For reference, compare to mutant animals grown on empty vector-control bacteria. Wild type animals treated with unc-45 RNAi show severe paralysis phenotype and can be used as a positive control. 
		NOTE: This assay highlights animals showing medium to severe paralysis. Such animals usually lie straight on the plate, rather than presenting the common curved shape. Moreover, a patch cleared of bacteria is visible around the heads of paralyzed worms.</li>
	</ol>
	</li>
	<li>Thrashing assay
	<ol>
		<li>Cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-control bacteria reach adulthood but before egg laying starts.</li>
		<li>Pipet 100 &#181;L of M9 buffer at the animals&#8217; cultivation temperature into a 96-well plate.</li>
		<li>Place ~15 worms, one per well, into the M9 buffer-containing wells.</li>
		<li>Let the animals adjust for 5 min.</li>
		<li>Examine each animal under the stereomicroscope, start a timer counting down 15 s, and count the number of body bends each animal performs in that timespan. The values counted can be normalized to body bends per min. For reference, compare to mutant animals grown on empty vector-control bacteria. 
		NOTE: This motility assay is very sensitive and can detect very mild differences between treatments. However, motility in liquid and motility on agar can differ.</li>
	</ol>
	</li>
</ol>
5. Validation of protein knockdown
<ol>
	<li>Place 250-300 synchronized eggs onto a 60 mm NGM-RNAi plate seeded with the relevant dsRNA-expressing or empty vector-containing (L4440) bacteria. 
	NOTE: RNAi knockdown could result in aberrant accumulation of embryos, in a lack of embryos or in developmental arrest that could impact gene expression. This should be considered when determining the age of the animals to be examined.</li>
	<li>For day 1 adults, cultivate animals for 4.5 days at 15 &#176;C, 3 days at 20 &#176;C or 2 days at 25 &#176;C.</li>
	<li>Pick and transfer a total of 200 young adult animals into the cap of a 1.5 mL tube fill with 200 &#181;L of PBS-T. 
	NOTE: When using temperature-sensitive animals or chaperone mutants, it is best to maintain the buffers used in the protocol at the animals&#8217; cultivation temperature.</li>
	<li>Close the cap carefully and centrifuge at 1,000 x g for 1 min.</li>
	<li>Add 800 &#181;L of PBS-T (Table 1) and centrifuge at 1,000 x g for 1 min.</li>
	<li>Carefully remove the top 900 &#181;L.</li>
	<li>Repeat steps 5.4-5.6 three times.</li>
	<li>Remove 900 &#181;L, leaving 100 &#181;L of solution containing the 200 worms.</li>
	<li>Add 25 &#181;L of 5x sample buffer (Table 1).</li>
	<li>Heat the samples for 10 min at 92&#176;C while shaking at 1000 rpm. Samples can then be frozen and kept at -20 &#176;C.</li>
	<li>Load 20 &#181;L of each sample and run on an SDS-PAGE gel.</li>
	<li>Perform western blot analysis using appropriate antibodies to determine the relative stability of the protein.</li>
	<li>Determine the intensity of the bands using densitometric software, such as the freely available ImageJ gel module. Normalize all values to those measured in the control sample(s). 
	NOTE: The aim of RNAi knockdown in our screens is to lower protein levels of a specific chaperone/co-chaperone. Thus, the best way to assess the efficiency of the RNAi knockdown is by western blot analysis. This requires specific antibodies. Alternately, qPCR can be used to quantify mRNA levels.</li>
</ol>

The authors have nothing to disclose.

An integrated picture of the proteostasis network reflecting how it is organized and functions in different metazoan cells and tissues remains lacking. To address this shortcoming, specific information on the interactions of various components of this network, such as molecular chaperones, in specific tissues during the course of development and aging is required. Here, we showed how the use of tissue-specific perturbations enabled us to examine the chaperone network in a given tissue. To explore tissue-specific chaperone genetic interactions, three different approaches were considered. In the first approach, UNC-45, a chaperone that is highly expressed in muscle cells, was used to screen for chaperone interactions via feeding-RNAi25. While the use of a specialized chaperone allows for discerning tissue-specificity, it can only report on that highly focused sub-network to which it contributes. Note that most C. elegans neurons are resistant to feeding-based RNAi delivery50 and thus using this approach to identify genetic interactions in neuronal cells requires crossing the mutant chaperone examined with an RNAi-enhanced strain51. In a second approach, a tissue-specific promoter was used to drive over-expression of a chaperone in muscle and thus specifically affect the muscle folding environment40. However, over-expressing a single chaperone could be generally beneficial to the folding environment and thus mask disruption caused by the two chaperones together. The third approach relied on tissue-specific RNAi knockdown to examine chaperone interactions in a given a tissue41. One advantage of this approach is that it allows for targeting neuronal cells that are resistant to RNAi delivery via feeding42. Still, this approach requires the use of a mild mutant (although not specialized), as well as that this mutant be crossed into a rde-1 null mutant carrying a tissue-specific rde-1 rescue gene. Importantly, these approaches can be combined so as to potentially modulate chaperone function in a single tissue or even a single cell.
The quality control machinery can impact the function of many gene products by perturbing proteostasis. This is a major challenge when using genetic interactions to explore the quality control network18. For example, chronic expression of aggregation-prone proteins or proteostasis collapse in aging resulted in phenotypic aggravation of many unrelated metastable proteins in C. elegans and yeast45,52,53. Moreover, clathrin-mediated endocytosis in mammalian cells was inhibited upon functional sequestration of Hsc70 to protein aggregates. Yet, it was shown that endocytosis could be rescued by Hsc70 over-expression, while aggregation could not54,55. Likewise, a genetic screen in Drosophila designed to uncover regulators of the heat shock response identified a missense mutation in flight muscle actin that constitutively activated the heat shock response56. Taken together, perturbation of the proteostasis network can expose metastable proteins or induce stress that can impact result specificity. Nonetheless, analyzing genetic interactions can yield highly specific and functional insight. For example, epistatic analyses of yeast genes required for folding in the endoplasmic reticulum identified specific genetic interactions between molecular chaperones that were subsequently validated19. Likewise, an aggravating screen for chaperones that enhance the toxicity of two aggregation-prone models (as measured by motility) identified a specific subset of 18 chaperones, orthologs of which impacted Huntingtin aggregation in human cells26. Here, we showed that various perturbations of the proteostasis network can uncover specific and functional chaperone interactions.
The main advantage of using RNAi feeding-based genetic interaction screens is the relative simplicity of the method. Even employing a general behavioral output, such as motility, can reveal novel genetic interactions (Figure 3). However, variability and partial effects of expression knockdown can limit the robustness and specificity of the results57. Moreover, genetic interactions are not indicative of physical interactions and the relationship between two genes could thus be indirect. Exploring the nature of the interactions and discarding non-specific interactions can be time consuming57,58,59. This is a concern that needs to be addressed in the screen setup and validation. For example, using null alleles in genetic screening allows for determining whether these genes function in the same or distinct pathways in a given biological process. However, using partial loss of gene function, hypomorphic alleles, such as temperature-sensitive alleles, or RNAi-dependent down-regulation of expression, results in residual activities that can yield aggravating or alleviating phenotypes, regardless of whether the genes act in the same or in parallel pathways59. Thus, the nature of any interaction requires further analysis. However, using hypomorphic alleles and RNAi could identify a broad range of interactors, including genes in the same protein complex or pathway or in a redundant pathway59. While this larger scope of possible interactions can yield more hits in a genetic screen, it can also lead to non-specific interactions, such as, for example, the non-specific exposure of temperature-sensitive alleles in proteostasis collapse45,53.
Hit validation and non-specific interactions can be examined in several ways. The number of hits should be low. For example, down-regulation of chaperone expression in an unc-45 mutant background resulted in a small percentage of hits (4%), with most chaperone gene down-regulation not showing any effect on motility. A similar rate was observed when DNJ-24M was over-expressed (6%). As noted above, a screen for chaperone interactions with aggregation-prone proteins identified 18 chaperones of 219 screened (8%).
Several mutant alleles, over-expression lines or disease models should be used. The use of different mutant alleles that have different impacts on chaperone function, as well as different strains with different genetic backgrounds, can support the specificity of any screen hits. Alternatively, using mutation or over-expression of a different chaperone that does not lead to similar perturbation can serve as a negative control. For example, both unc-45(e286) and unc-45(m94) showed aggravating behavior when sti-1, ahsa-1 or daf-41 were down-regulated. Moreover, a similar interaction was observed when animals carrying the ahsa-1(ok3501) deletion mutant were treated with hsp-90 RNAi25.
The identity of the chaperone hits and their known interactions should be examined. For example, chaperones identified in a unc-45 aggravating screen are a highly specific set of chaperones required for the HSP-90 ATPase cycle, including those encoding a client recruiter (STI-1), a remodeling co-chaperone (AHSA-1), and a client maturation co-chaperone (DAF-41). In fact, this set of co-chaperones forms a complete HSP-90 folding cycle60. Likewise, a DNJ-24M screen identified HSP-1, the main chaperone partner of Hsp40s40.
Changes in interactions over the lifespan of the animal should also be examined. For example, chaperones identified in unc-45 aggravating screen strongly impacted motility and myosin organization in adulthood but had a milder effect during development. This could be due to changes in the proteostasis network in adulthood28 or to changes in myosin folding requirements between myo-fiber folding and maintenance.
Use complementary biochemical approaches to directly examine interactions between the proteins, as well as their localization in the cell. For example, the HSP-90 co-chaperones, STI-1, AHSA-1 and DAF-41, are localized to the sarcomere where they interact with myosin25.
C. elegans is a well-established metazoan model for monitoring quality control. It is often used to monitor cellular and organismal proteostasis using a variable toolkit of cell biology, biochemical and genetic approaches. Here, we employed genetic screening approaches and available tools57,58,59, such as a mutant bank, available RNAi libraries22,23 and tissue-specific RNAi strains41,42, to monitor chaperone interactions in a living animal during development and aging. The use of simple behavioral assays, such as motility, simplify the screen of many possible gene pairs to explore novel genetic interactions. This can then serve as a platform to further explore chaperone localization and physical interactions using biochemical tools to mechanistically study their potential interactions in vivo and in vitro. The protocol described here has been successfully used to identify novel chaperone interactions in C. elegans body wall muscle25,40.

Cells cope with protein damage by employing quality control machineries that repair, sequester or remove any damaged proteins1,2. Folding and assembly of protein complexes are supported by molecular chaperones, a diverse group of highly conserved proteins that can repair or sequester damaged proteins3,4,5,6,7. The removal of damaged proteins is mediated by the ubiquitin-proteasome system (UPS)8 or by the autophagy machinery9 in collaboration with chaperones10,11,12. Protein homeostasis (proteostasis) is, therefore, maintained by quality control networks composed of folding and degradation machineries3,13. However, understanding the interactions between the various components of the proteostasis network in vivo is a major challenge. While protein-protein interaction screens contribute important information on physical interactions and chaperone complexes14,15, understanding the organization and compensatory mechanisms within tissue-specific chaperone networks in vivo is lacking.
Genetic interactions are often used as a powerful tool to examine relationship between pairs of genes that are involved in common or compensatory biological pathways16,17,18. Such relationships can be measured by combining pairs of mutations and quantifying the impact of a mutation in one gene on the phenotypic severity caused by a mutation in the second gene16. While most such combinations do not show any effect in terms of phenotype, some genetic interactions can either aggravate or alleviate the severity of the measured phenotype. Aggravating mutations are observed when the phenotype of the double deletion mutant is more severe than the expected phenotype seen upon combining the single deletion mutants, implying that the two genes function in parallel pathways, together affecting a given function. In contrast, alleviating mutations are observed when the phenotype of the double deletion mutant is less severe than the phenotype seen with the single deletion mutants, implying that the two genes act together as a complex or participate in the same pathway16,18. Accordingly, diverse phenotypes that can be quantified, including broad phenotypes, such as lethality, growth rates and brood size, as well as specific phenotypes, such as transcriptional reporters, have been used to identify genetic interactions. For example, Jonikas et al. relied on an ER stress reporter to examine interactions of the Saccharomyces cerevisiae ER unfolded protein response proteostasis network using pairwise gene deletion analyses19.
Genetic interaction screens involve systematically crossing pairwise deletion mutations to generate a comprehensive set of double mutants20. However, in animal models, and specifically in C. elegans, this large-scale approach is not feasible. Instead, mutant strains can be tested for their genetic interaction patterns by down-regulating gene expression using RNA interference (RNAi)21. C. elegans is a powerful system for screens based on RNAi22,23. In C. elegans, double-stranded RNA (dsRNA) delivery is achieved by bacterial feeding, leading to the spread of dsRNA molecules to numerous tissues. In this manner, the introduced dsRNA molecules impact the animal via a rapid and simple procedure21. A genetic interaction screen using RNAi can, therefore, reveal the impact of down-regulating a set of genes or most C. elegans coding genes using RNAi libraries24. In such a screen, hits that impact the behavior of the mutant of interest but not the wild type strain are potential modifiers of the phenotype being monitored25. Here, we apply a combination of mutations and RNAi screening to systematically map tissue-specific chaperone interactions in C. elegans.

<table>
	<tbody>
		<tr>
			<td>12-well-plates</td>
			<td>SPL</td>
			<td>BA3D16B</td>
			<td></td>
		</tr>
		<tr>
			<td>40 mm plates</td>
			<td>Greiner Bio-one</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm plates</td>
			<td>Greiner Bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well 2 mL 128.0/85mm</td>
			<td>Greiner Bio-one</td>
			<td>780278</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGA03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Formedium</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Sigma</td>
			<td>BO126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck</td>
			<td>1.02382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Qimaging</td>
			<td>q30548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Amresco</td>
			<td>0433-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Leica</td>
			<td>DM5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m)</td>
			<td>Sigma</td>
			<td>SCGPUO2RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Leica</td>
			<td>MZ165FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Frutarom</td>
			<td>2355519000024</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Formedium</td>
			<td>367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>1.04873.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Bio-Lab</td>
			<td>001649029100</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>22189-08-8</td>
			<td>Gift from the Morimoto laboratory</td>
		</tr>
		<tr>
			<td>Myosin MHC A (MYO-3) antibody</td>
			<td>Hybridoma Bank</td>
			<td>5-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Sigma</td>
			<td>s-0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio-Lab</td>
			<td>001903029100</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td>61930705001730</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate pouring pump</td>
			<td>Integra</td>
			<td>does it p920</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAi Chaperone library</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>VWR Life Science</td>
			<td>0837-500</td>
			<td></td>
		</tr>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Bio world</td>
			<td>41300000-1</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Duchefa Biochemie</td>
			<td>64-75-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Bio-Lab</td>
			<td>002009239100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

using caenorhabditis elegans to screen for tissue-specific chaperone interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using temperature-sensitive mutations in UNC-45 to screen for aggravating or alleviating interactions under permissive or restrictive conditions, respectively 
Muscle assembly and maintenance offer an effective system to study tissue-specific chaperone interactions. The functional unit of contractile muscles, the sarcomere, presents a crystalline-like arrangement of structural and regulatory proteins. The stability of the motor protein myosin and its incorporation into the thick filaments of contractile muscle sarcomeres depends on cooperation of chaperones and UPS components30. An example of one such chaperone is the conserved and specialized myosin chaperone UNC-45 that is mainly expressed in body wall muscle31,32,33,34,35. Mutations in UNC-45 have been shown to induce myosin disorganization and severe motility defects in C. elegans31,36. UNC-45 tandem modules assemble into a multi-site docking platform37 that enforces collaboration between UNC-45, HSP-90 and HSP-1 and likely other chaperones and co-chaperones in myosin filament assembly25,36,37,38. To confirm known UNC-45 interactions and identify novel genetic interactions in muscle proteostasis, we established a strategy using C. elegans temperature-sensitive unc-45 mutations as a sensitized genetic background for tissue-specific chaperone interaction screening19,25.
Single amino acid substitutions in C. elegans UNC-45 (L822F and E781K, corresponding to the e286 and m94 alleles, respectively; unc-45(ts)) are responsible for temperature-dependent motility defects and myosin disorganization phenotypes when affected animals are grown under restrictive conditions (&#62;22 &#176;C). In contrast, these unc-45(ts) mutants show no movement or myosin organization defects at the permissive temperature (15 &#176;C)31. In the proposed approach, age-synchronized unc-45(e286) animals at the first larval stage (L1) were depleted of different molecular chaperones by RNAi (97 genes) and then monitored for motility defects under permissive conditions (15 &#176;C) (Figure 1). We confirmed the known interaction of UNC-45 and HSP-90 proteins in our genetic interactions screen and identified three hsp-90 co-chaperones, sti-1, ahsa-1, and daf-41, as specifically causing a synthetic movement defect in unc-45(ts) mutant animals but not in wild-type animals25. We went on to examine whether sti-1-, ahsa-1- or daf-41-associated synthetic phenotypes were caused by myosin disorganization by monitoring the subcellular arrangement of myosin heavy chain A (MYO-3), using established immuno-staining techniques39. Whereas treatment of wild type animals with sti-1, ahsa-1 or daf-41 RNAi did not affect myofilament organization, depletion of these genes in unc-45(e286) mutant animals resulted in complete disruption of sarcomeric structures and MYO-3 mislocalization, even under permissive conditions (15 &#176;C). This effect was comparable to what was seen with unc-45(e286) single mutants grown at the restrictive temperature (25 &#176;C)25. These results were confirmed using another unc-45(ts)-allele, namely unc-45(m94) mutant animals25.
To identify chaperones that destabilize UNC-45, we next screened for chaperones that improved the motility of unc-45(286) animals at 25 &#176;C, relying on gene knockdown by RNAi. While unc-45(e286) mutant animals displayed severe movement defects at 25 &#176;C, the motility of animals treated with RNAi against genes encoding four of the 97 chaperones screened was significantly improved. Here too, the results were confirmed using unc-45(m94) mutant animals. Thus, the use of temperature-sensitive mutations allows for establishing both aggravating and alleviating screens, depending on the temperature at which the screen is conducted.
Using tissue-specific over-expression of a chaperone to screen for aggravating interactions 
We next utilized tissue-specific over-expression of a single chaperone as a mild perturbation of the muscle chaperone network for screening purposes. Specifically, we utilized animals over-expressing wild type dnj-24, encoding the C. elegans homolog of the Hsp40 protein DNAJB6 in the C. elegans body wall muscle (DNJ-24M). As above, L1 DNJ-24M animals were treated with RNAi for different chaperones and monitored for motility defects (20 &#176;C) (Figure 1). While DNJ-24M animals showed no notable motility defects, three genes (of the 48 chaperone-encoding genes examined; 6%), namely hsp-1, rme-8, and dnj-8, specifically affected the motility of DNJ-24M-expressing animals but not wild type animals. It is of note that testing the specificity of the hits using animals over-expressing a different chaperone, namely HSP-90, in muscle (HSP90M), showed no effect on HSP90M motility upon hsp-1, rme-8, or dnj-8 RNAi treatment40. Taken together, the screening platform, employing mild perturbation to the chaperone network, such as the expression of metastable mutant proteins or tissue-specific over-expression, resulted in a highly specific hit rate (normally ~5%).
Using tissue-specific RNAi to screen for tissue-specific genetic interactions 
Tissue-specific RNAi-sensitive strains allow for tissue-specific knockdown of genes while still using bacterial feeding for dsRNA delivery. These strains are mutant for the RDE-1 argonaute protein, a major component in the RNAi pathway required for effective gene silencing21. However, expressing wild type RDE-1 under the control of a tissue-specific promoter led to effective tissue-specific gene knockdown41,42. This tool thus allows for genetic interaction screens without using tissue-specific chaperones, such as UNC-45 or DNAJ-24M. For example, hsp-6 (mortalin) knockdown in wild type animals during development resulted in a strong developmental arrest (96&#177;1% of the RNAi-treated animals). At the same time, hsp-6 knockdown in a strain expressing wild type RDE-1 in muscle did not cause developmental arrest, whereas expressing wild type RDE-1 in intestinal cells resulted in a strong developmental arrest phenotype (90&#177;3%; Figure 2). Thus, HSP-6 function in intestinal cells is required for normal development. A mutation in hsp-6(mg585) that causes a mild growth delay can, therefore, be used to screen for aggravating or alleviating chaperone interactions by crossing that mutated gene into an intestinal-specific RNAi strain and screening the chaperone RNAi library.
Monitoring age-dependent changes in the folding environment using genetic interactions 
Animals show an age-dependent decline in motility that is associated with sarcomeric disorganization43,44,45. Changes in protein folding capacity coincide with altered regulation and composition of the cellular proteostasis machinery28, including altered levels of UNC-45, CHN-1, and UFD-2, proteins of the muscle quality control machinery46. In agreement, myosin folding and degradation are affected by alterations in proteostatic capacity at the transition to adulthood47. We, therefore, asked whether such changes could impact chaperone interactions. For example, we monitored the impact of sti-1, ahsa-1 and daf-41 knockdown on motility over time. We found that although sti-1-, ahsa-1- and daf-41-RNAi treated animals showed reduced motility during larval development, motility strongly declined in unc-45(ts) adult worms. Moreover, MYO-3 organization in unc-45(ts) mutant animals treated with sti-1, ahsa-1 or daf-41 RNAi was similar to that of wild type worms at the fourth larval stage (L4), although the mutants exhibited disrupted sarcomeres after the animals reached adulthood (Figure 3). In contrast, both unc-45(ts) mutant animals treated with an empty vector control and wild type animals remained unaffected (Figure 3). Thus, proteostasis dynamics as a function of age or environmental conditions27,45,46,48,49 could critically impact chaperone interactions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61140/61140fig01.jpg" /> 
Figure 1: Setup of RNAi synthetic interaction screens using C. elegans carrying a mutation in a gene encoding a chaperone of interest.&#160;(A) Schematic representation of the basic setup of targeted chaperone interaction screens. (B) Hit validation requires confirmation of the genetic interaction using another chaperone mutant, as well as validating the specify of RNAi knockdown. (C-D) Simple readouts, such as motility defects, can be quantified to determine aggravating or alleviating interactions, using paralysis or thrashing assays, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61140/61140fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61140/61140fig2v3.jpg" /> 
Figure 2: Tissue-specific RNAi of the mitochondrial chaperone hsp-6 can be used to examine genetic interactions in one tissue.&#160;Wild type intestine- and muscle-specific RNAi strains were treated with hsp-6 RNAi and (A) developmental delay was scored or (B) images were taken on the first day of adulthood. Data are mean &#177; SEM, N=6. Scale bar is 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61140/61140fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61140/61140fig3v4.jpg" /> 
Figure 3: Age-dependent effects of RNAi.&#160;(A) Motility with age. Wild type or unc-45(e286) embryos were placed on sti-1, ahsa-1 or daf-41 RNAi-seeded plates at 15 &#176;C and scored for motility using a thrashing assay at each developmental stage, L1-L4, young adult and day 1 of adulthood. Data are mean &#177; SEM, N=15. (B) Confocal images of body wall muscle. Animals were treated as in A and fixed at the L4, young adult and day 1 of adulthood stages and immuno-stained with anti-MYO-3 antibodies. Scale bar is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61140/61140fig3v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Preparation instructions</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">1 M CaCl2 (1 L)</td>
			<td>Add 147.01 g CaCl2&#183;2H2O</td>
			<td rowspan="3">Store at RT</td>
		</tr>
		<tr>
			<td>Add dH2O to 1 L</td>
		</tr>
		<tr>
			<td>Autoclave or filter (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td rowspan="6">1 M KH2PO4, pH 6.0 (1 L)</td>
			<td>Add 136.09 g KH2PO4</td>
			<td rowspan="6">Store at RT</td>
		</tr>
		<tr>
			<td>Add 800 mL dH2O</td>
		</tr>
		<tr>
			<td>Mix using magnetic stirrer until dissolved</td>
		</tr>
		<tr>
			<td>Titrate pH using KOH</td>
		</tr>
		<tr>
			<td>Add dH2O to 1 L</td>
		</tr>
		<tr>
			<td>Autoclave or filter (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td rowspan="3">1 M MgSO4 (1 L)</td>
			<td>Add 248.58 g MgSO4&#183;7H2O</td>
			<td rowspan="3">Store at RT</td>
		</tr>
		<tr>
			<td>Add dH2O to 1 L</td>
		</tr>
		<tr>
			<td>Autoclave or filter (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td rowspan="3">Cholesterol solution (50 mL)</td>
			<td>Add 250 mg cholesterol to a 50 mL Falcon tube</td>
			<td rowspan="3">Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Completely dissolve in 40 mL ethanol</td>
		</tr>
		<tr>
			<td>Add ethanol to 50 mL</td>
		</tr>
		<tr>
			<td rowspan="4">1 M IPTG (50 mL)</td>
			<td>Add 11.9 g IPTG (isopropyl-&#946;-D-thiogalactopyranoside) to a 50 mL Falcon tube</td>
			<td rowspan="4">Store in the dark at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Completely dissolve in 40 mL dH2O</td>
		</tr>
		<tr>
			<td>Add dH2O to 50 mL</td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m), aliquot 1mL tubes</td>
		</tr>
		<tr>
			<td rowspan="4">Ampicillin stock (50 mL)</td>
			<td>Add 5 g ampicillin to a 50 mL Falcon tube</td>
			<td rowspan="4">Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Completely dissolve in 40 mL dH2O</td>
		</tr>
		<tr>
			<td>Add dH2O to 50 mL</td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m), distribute 1 mL aliquot into Eppendorf tubes</td>
		</tr>
		<tr>
			<td rowspan="4">Tetracycline stock (50 mL)</td>
			<td>Add 250 mg tetracycline to a 50 mL Falcon tube</td>
			<td rowspan="4">Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Completely dissolve in 40 mL ethanol</td>
		</tr>
		<tr>
			<td>Add ethanol to 50 mL</td>
		</tr>
		<tr>
			<td>Aliquot 1 mL tubes</td>
		</tr>
		<tr>
			<td rowspan="6">M9 buffer (1 L)</td>
			<td>Add 5.8 g Na2HPO4&#183;7H2O</td>
			<td rowspan="6">Store at RT</td>
		</tr>
		<tr>
			<td>Add 3 g KH2PO4</td>
		</tr>
		<tr>
			<td>Add 5 g NaCl</td>
		</tr>
		<tr>
			<td>Add 0.25 g MgSO4&#183;7H2O</td>
		</tr>
		<tr>
			<td>Add dH2O to 1 L</td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td rowspan="8">1X PBS-T (pH 7.4) ( 1 L)</td>
			<td>Add 8 g of NaCl</td>
			<td rowspan="8">Store at RT</td>
		</tr>
		<tr>
			<td>Add 200 mg of KCl</td>
		</tr>
		<tr>
			<td>Add 1.44 g Na2HPO4&#183;7H2O</td>
		</tr>
		<tr>
			<td>Add 240 mg KH2PO4</td>
		</tr>
		<tr>
			<td>Completely dissolve in 800 mL dH2O</td>
		</tr>
		<tr>
			<td>Titrate pH using KOH tp pH 7.4</td>
		</tr>
		<tr>
			<td>Add 500 &#181;L Tween-20</td>
		</tr>
		<tr>
			<td>Add dH2O to 1 L</td>
		</tr>
		<tr>
			<td rowspan="6">5x sample buffer</td>
			<td>Add 6.8 mL dH2O</td>
			<td rowspan="6">Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Add 2 mL 0.5M Tris pH 6.8</td>
		</tr>
		<tr>
			<td>Add 3.2 mL Glycerol</td>
		</tr>
		<tr>
			<td>Add 1.6 mL 20% SDS</td>
		</tr>
		<tr>
			<td>Add 0.8 mL &#223;-mercaptoethanol</td>
		</tr>
		<tr>
			<td>1% bromophenol blue</td>
		</tr>
	</tbody>
</table>
Table 1: Solution Recipes

Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

using-caenorhabditis-elegans-to-screen-for-tissue-specific-chaperone

Nanyang Technological University

Research

JoVE Journal

Biology

2.8K Views.  Ben-Gurion University of the Negev. To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Video: Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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.

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.

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 ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...