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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Preparation of nematode growth media plates for RNAi

  1. 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.
  2. Cool bottle to 55 °C.
  3. 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).
  4. 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.
  5. Mix the warm solution by swirling the bottle.
  6. 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.
  7. Let the plates dry overnight on the bench at room temperature, keeping the plates covered. NGM plates for RNAi can be stored at 4 °C for up to a month.

2. Growing RNAi bacteria and seeding the plates

  1. 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.
  2. In the hood or using sterile procedures, add 600 µL 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.
  3. 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 °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.
  4. Using sterile procedures, seed 75, 150 or 250 µL 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.
  5. 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 °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 °C for up to a month.

3. Non-stressful synchronization of embryos

  1. Use a worm pick to move about 100 eggs from an unsynchronized worm plate to a newly seeded NGM plate.
  2. Cultivate animals for 5 days at 15 °C, 3.5 days at 20 °C or 2.5 days at 25 °C. Animals should reach the first day of egg laying.
    NOTE: Worms are commonly cultivated at 20 °C. However, different chaperone mutant strains may require specific cultivation temperatures. For example, many temperature-sensitive strains are cultivated at 15 °C but shifted to 25 °C to expose their phenotype.
  3. 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’ cultivation temperature.
  4. Repeat step 3.3 three times or until all the animals are washed off the plate.
  5. 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.
  6. Cultivate the animals for 5 days at 15 °C, 3.5 days at 20 °C or 2.5 days at 25 °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.
  7. Add 1 mL of M9 buffer slowly and away from the bacterial lawn.
  8. 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.
  9. Repeat step 3.7 three times or until all animals are washed off the plate.
  10. Add 1 mL of M9 buffer and use a cell scraper to release the eggs from the plate.
  11. Collect the M9 buffer containing the eggs from the plates.
  12. Centrifuge the M9 buffer containing the eggs at 3,000 x g for 2 min.
  13. Remove the supernatant and add M9 buffer to reach a volume of 1 mL.
  14. Resuspend the eggs to disrupt any chunks of eggs and bacteria.
  15. 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.
  16. Remove most of the supernatant, leaving about 200 µL. Synchronized eggs can be used for RNAi screens.

4. Common phenotypic assays

  1. Cultivation of animals during experiments
    1. 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.
    2. 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 °C, 3 days at 20 °C or 2 days at 25 °C.
    3. 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 >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 ± SEM.
    4. 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.
  2. Developmental arrest/delay
    1. 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.
    2. 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.
    3. 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.
  3. Sterility or egg laying defects
    1. Cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-control bacteria begin to lay eggs.
    2. 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.
    3. 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.
  4. Embryonic lethality
    1. Cultivate age-synchronized animals as in step 4.1 until the animals begin to lay eggs.
    2. Transfer ~100 eggs to an empty plate. Spread the eggs in rows to simplify counting.
    3. 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.
  5. Paralysis assay
    1. 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.
    2. Draw a line on the back of a regular NGM agar plate using a fine marker.
    3. Place 5-10 animals on the marked line.
    4. Set a timer and wait for 10 min.
    5. 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.
  6. Thrashing assay
    1. Cultivate age-synchronized animals as in step 4.1 until animals grown on empty vector-control bacteria reach adulthood but before egg laying starts.
    2. Pipet 100 µL of M9 buffer at the animals’ cultivation temperature into a 96-well plate.
    3. Place ~15 worms, one per well, into the M9 buffer-containing wells.
    4. Let the animals adjust for 5 min.
    5. 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.

5. Validation of protein knockdown

  1. 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.
  2. For day 1 adults, cultivate animals for 4.5 days at 15 °C, 3 days at 20 °C or 2 days at 25 °C.
  3. Pick and transfer a total of 200 young adult animals into the cap of a 1.5 mL tube fill with 200 µL 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’ cultivation temperature.
  4. Close the cap carefully and centrifuge at 1,000 x g for 1 min.
  5. Add 800 µL of PBS-T (Table 1) and centrifuge at 1,000 x g for 1 min.
  6. Carefully remove the top 900 µL.
  7. Repeat steps 5.4-5.6 three times.
  8. Remove 900 µL, leaving 100 µL of solution containing the 200 worms.
  9. Add 25 µL of 5x sample buffer (Table 1).
  10. Heat the samples for 10 min at 92°C while shaking at 1000 rpm. Samples can then be frozen and kept at -20 °C.
  11. Load 20 µL of each sample and run on an SDS-PAGE gel.
  12. Perform western blot analysis using appropriate antibodies to determine the relative stability of the protein.
  13. 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.

Results

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...

Discussion

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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 & 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.

Materials

NameCompanyCatalog NumberComments
12-well-platesSPLBA3D16B
40 mm platesGreiner Bio-one627160
60 mm platesGreiner Bio-one628102
6-well platesThermo Scientific140675
96 well 2 mL 128.0/85mmGreiner Bio-one780278
AgarFormediumAGA03
AmpicillinFormedium69-52-3
bromophenol blueSigmaBO126-25G
CaCl2Merck1.02382.0500
CameraQimagingq30548
CholesterolAmresco0433-250G
ConfocalLeicaDM5500
Filter (0.22 µm)SigmaSCGPUO2RE
Fluorescent stereomicroscopeLeicaMZ165FC
GlycerolFrutarom2355519000024
IPTGFormedium367-93-1
KClMerck104936
KH2PO4Merck1.04873.1000
KOHBio-Lab001649029100
MgSO4Fisher22189-08-8Gift from the Morimoto laboratory
Myosin MHC A (MYO-3) antibodyHybridoma Bank5-6
Na2HPO4·7H2OSigmas-0751
NaClBio-Lab001903029100
PeptoneMerck61930705001730
Plate pouring pumpIntegradoes it p920
RNAi Chaperone libraryNANA
SDSVWR Life Science0837-500
ß-mercaptoethanolBio world41300000-1
stereomicroscopeLeicaMZ6
TetracyclineDuchefa Biochemie64-75-5
TrisBio-Lab002009239100
Tween-20FisherBP337-500

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