Standardized Methods for Measuring Induction of the Heat Shock Response in <em>Caenorhabditis elegans</em>

Abstract

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level.While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay.Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Digital Microfluidics Supported Microproteomics for Quantitative Proteome Analysis of Single Caenorhabditis elegans Nematodes

“…L1 larvae were transferred to NGM-OP50 plates and incubated at 20 °C until they reached the L4 stage, at which point they were split to create two biological conditions. One synchronized L4 population was subjected to a heat treatment by incubation at 33 °C for 1 h based on a procedure described recently, 22 whereas the other was kept at default conditions (20 °C) to serve as control.…”

The CBP-1/p300 Lysine Acetyltransferase Regulates the Heat Shock Response in C. elegans

“…To identify lysine acetyltransferases that may regulate the HSR, we performed an RNA interference (RNAi) screen by targeting ∼ 75% of all putative lysine acetyltransferases (KATs) in C. elegans using RNAi knockdown ( Figure 1A ) C. elegans KATs were identified by searching the C. elegans protein database for proteins containing conserved acetyltransferase domains to those of known human KATs ( Sheikh and Akhtar, 2019 ). We utilized quantitative RT-PCR to assess the heat shock inducibility of hsp-16.2 mRNA expression, a widely used marker of HSR activity, after KAT RNAi knockdown ( Golden et al, 2020 ). We induced RNAi 19 h after the L1 stage until Day 1 of adulthood, when we extracted RNA before and immediately after a 1-h heat shock at 33°C to assess hsp-16.2 expression ( Figure 1B ).…”

Geranylgeranylacetone Ameliorates Beta-Amyloid Toxicity and Extends Lifespan via the Heat Shock Response in Caenorhabditis elegans

“…N2 worms were grown at 20°C on NGM control or 10 μM GGA plates starting at the L1 larval stage until day one of adulthood. RNA was isolated using Trizol Reagent (Thermo Fisher) as previously described with the addition of a freeze-thaw in liquid nitrogen and use of a Direct-zol RNA kit (Zymo Research) ( Golden et al, 2020 ). RNA was reverse transcribed with an iScript cDNA Synthesis Kit (Bio-Rad) and RT-qPCR was performed using an iTaq Universal SYBR Green Supermix (Bio-Rad) and the CFX Connect cycler (Bio-Rad).…”