The heat shock response is an essential cellular stress response pathway that maintains cytoplasmic proteostasis and can be characterized at the molecular, cellular, and organismal levels in sea elegans. We present a series of standardized protocols and best practices that generate robust induction of the heat shock response in sea elegans in order to help enhance reproducibility in the heat shock response field. We show that thermo tolerance, a commonly used organismal assay is not dependent on the heat shock response.
Instead, we recommend using thermal recovery, an organismal assay that is heat shock response dependent. The heat shock response become attenuated with the onset of egg laying. Therefore, developmental timing is a critical variable that must be accounted for in heat shock response experiments.
Begin by creating a synchronized worm population. Use a platinum wire pick to transfer approximately 10 gravid adult worms to a fresh plate. Make sure to remove any eggs or larvae that may have been transferred with the adults.
Allow the worms to lay eggs for one hour, then remove the adults from the plate. Maintain the synchronized worms at 20 degrees Celsius until the desired developmental stage, taking into account that developmental timing varies with each strain and the temperature at which the worms are raised. When the worms are ready for heat shock, wrap the plates with paraffin film and use a test tube rack with a lead weight to submerge them in a circulating water bath at 33 degrees Celsius for one hour.
Recover the plates from the water bath and dry them with a paper towel. Remove the paraffin film and allow the worms to recover at 20 degrees Celsius for six to 24 hours, which will be a sufficient time for GFP synthesis. To prepare slides for imaging, position a microscope slide between two other slides that have a strip of laboratory tape on them.
Make a 3%agarose solution in water and heat it in the microwave until the agaroses dissolve. Then use a one milliliter pipette to place about 150 microliters of the agarose in the center of the slide. Immediately cover the slide with a blank slide, placing it perpendicularly so that it rests on the laboratory tape, then carefully remove it.
To immobilize the worms, add a small drop of one millimolar levamisole, an M9 buffer, to the center of the agarose pad and transfer 10 worms into the drop using a platinum wire pick. Optionally, spread the levamisole off to the outside of the agarose pad and align the worms with the pick when they become paralyzed. Cover the worms with a cover slip and image them as soon as possible using a fluorescence microscope.
To perform a thermorecovery assay, synchronize the worms and maintain them at 20 degree Celsius until the desired developmental stage. Then heat shock them for six hours. Remove the plates from the water bath and allow them to recover at 20 degrees Celsius for 48 hours.
After recovery, count the number of worms that can immediately craw away after mechanical stimulation without jerky movement or paralysis. To visualize heat shock response induction at the cellular level, two fluorescent reporter sea elegans strains were analyzed. In the negative control samples without heat shock, the hsp-16.2 reporter showed normal auto fluorescence, but the hsp-70 reporter had constitutive fluorescence in the anal depressor muscle.
After one hour of heat shock, robust fluorescence was observed in both reporters when RNAi was used to knock down hsf-1 before measuring reporter induction. Fluorescence of both strains was severely reduced, indicating that these reporters are hsf-1 dependent. To quantitate induction of the heat shock response at the molecular level, two indigenous heat shock proteins were measured with rt key PCR.
It was found that a one hour 33 degree Celsius heat shock results in more than a 2, 000 fold increase in relative expression of the two heat shock genes. A thermorecovery assay demonstrated that exposure to a six hour heat shock led to a 20%decrease in worms with normal movement after a 48 hour recovery. Knockdown of hsf-1 caused a dramatic decrease in normal movement, with over 95%of worms showing jerky movement or paralysis after being prodded.
In contrast, knockdown of hsf-1 did not have a significant effect on the results of a thermo tolerance assay, in which worms are exposed to a continuous 35 degree Celsius temperature and the percentage of worms alive is measured at various time points. When performing this technique for the first time, make sure to wrap the plates with paraffin film twice to prevent water from entering the plates and ruining the experiment. Molecular assays can be extended into genomic analyses using RNA-seq.
Other organismal assays affected by the heat shock response can be included, such as life span. The unique ability to correlate molecular, cellular, and organismal heat shock response assays in sea elegans has proven invaluable for the understanding of the role of this pathway in development and disease.
