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

Using behavioral assays to monitor protein folding in a living organism
1. Synchronizing Animals for Behavioral Assays
<ol>
	<li>Follow standard methods for maintenance of C. elegans36, including media and plates preparation, food preparation and animal growth conditions. Carefully control cultivation conditions, including temperature (15-25 &#176;C), population size and food availability. Discard any plate that shows bacterial or fungal contamination.</li>
	<li>Pic...

The authors declare that they have no competing financial interests.

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

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

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using caenorhabditis elegans as a model system to study protein homeostasis in a multicellular organism

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

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

Watch this Scientific Journal Video about Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism at JoVE.com

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

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

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

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

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