Measurements of Physiological Stress Responses in C. Elegans

Summary

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Abstract

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPR^ER), the unfolded protein response of the mitochondria (UPR^MT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Introduction

The ability of an organism to respond to changes in the intra- and extracellular environment is crucial for its survival and adaptation. This is accomplished on a cellular level through numerous protective pathways that ensure the integrity of the cell. While numerous cellular components are subject to stress-associated damage, one major involvement of cellular stress responses is to repair and protect the homeostasis of the cellular proteome. However, the compartmentalization of proteins into special structures, called organelles, poses a challenge for the cell, as it cannot rely on one centralized form of protein quality control to ensure that all the proteins withi....

Protocol

1. Standard growth conditions of temperatures & OP50 vs HT115

1. Standard growth and expansion
   1. Grow a culture of OP50 in LB (Table 1) or equivalent media of choice for 24-48 h at ambient temperature (~22-25 °C). Grow bacteria at room temperature as OP50 is an uracil auxotroph and there is a higher incidence of revertants (e.g., suppressor mutants) when grown at 37 °C. Long-term storage of OP50 cultures is not recommended (max 1 week at 4 °C).
   2. Seed a volume of ~100-200 µL of saturated OP50 culture onto a 60 mm NGM plate (Table 1) for maintenance of worms and 1 mL of saturated OP50....

Results

Using transcriptional reporters to measure activation of stress responses
Here, fluorescent transcriptional reporters are used, which serve as robust tools to measure activation of most stress responses in C. elegans. GFP expression is driven under the promoter of canonical targets of master transcriptional regulators involved in responding to compartment-specific stresses. A comprehensive list of commonly used transcriptional reporters is available in

Discussion

Here, methods to interrogate cellular stress responses in C. elegans, using fluorescent transcriptional reporters and physiological stress survival assays are described. The reporters all utilize GFP expression driven under the promoter of a downstream transcriptional target of the transcription factors involved in mounting cellular stress responses. The use of hsp-4p::GFP modulated by XBP-1s-mediated UPR^ER, hsp-6p::GFP controlled by ATFS-1-mediated UPR^MT, gst-4p::GFP<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antimycin A	Sigma-Aldrich	A8674	for mitochondrial stress
Bacto Peptone	Fisher Scientific	DF0118072	for NGM plates
BD Difco granulated agar	VWR	90000-782	for NGM plates
Calcium chloride dihydrate	VWR	97061-904	for NGM plates
Carbenicillin	BioPioneer	C0051-25	for RNAi
Cholesterol	Sigma-Aldrich	57-88-5	for NGM plates
COPAS Biosorter	Union Biometrica	350-5000-000	equipped with a 488 nm light source.
COPAS Cleaning Solution	Union Biometrica	300-5072-000	to use with COPAS
COPAS Sheath Solution	Union Biometrica	300-5070-100	to use with COPAS
DMSO	Sigma-Aldrich	472301	solvent for drugs
IPTG dioxane free	Denville Scientific	CI8280-4	for RNAi
LB Broth Miller	Fisher Scientific	BP1426500	for LB
M205FA stereoscope	Leica	10450040	equipped with a Leica DFC3000G monochromatic CCD camera, standard Leica GFP filter (ex 395-455, EM 480 LP), and LAS X software
Magnesium sulfate heptahydrate	VWR	EM-MX0070-3	for NGM plates, M9
Paraquat	Sigma-Aldrich	36541	for oxidative/mitochondrial stress
Potassium Chloride	Fisher	P217-500	for bleach soluton
Potassium phosphate dibasic	VWR	EM-PX1570-2	for NGM plates
Potassium phosphate monobasic	VWR	EM-PX1565-5	for M9
Revolve	ECHO	75990-514	equipped with an Olympus 4x Plan Fluorite NA 0.13 objective lens, standard Olympus FITC filter (ex 470/40; em 525/50; DM 560), and an iPad Pro for camera and to drive ECHO software
Sodium Azide	Sigma-Aldrich	71289-50G	for imaging
Sodium Chloride	EMD Millipore	SX0420-5	for NGM plates, M9
Sodium phosphate dibasic	VWR	71003-472	for M9
Tert-butyl hydroperoxide	Sigma-Aldrich	458139	for oxidative stress
Tetracycline hydrochloride	Sigma-Aldrich	T7660-5G	for RNAi
Tunicamycin	Sigma-Aldrich	T7765-50MG	for ER stress