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 chaperon...

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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 &amp;micro;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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</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>&amp;szlig;-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 contrac...

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

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Cancer Research

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Developmental Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 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 ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Neuroscience

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

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 ...

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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

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Basic Caenorhabditis elegans Methods: Synchronization and Observation

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Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans