Utilizing Live Cell Imaging in Drosophila melanogaster Salivary Glands to Determine if Resveratrol Treatment Activates Heat Shock Factor DNA Binding

Abstract:

One major stress response pathway is the heat shock response (HSR), which is mediated by the transcription factor, heat shock factor (HSF). The HSR is activated in cells exposed to conditions that induce protein misfolding, such as: high heat, oxidants, and other chemical stresses. Under such stressors, HSF activates expression of the Hsp70 chaperone, which helps cells deal with protein folding stress. However, HSR activation also leads to an increase in reactive oxygen species (ROS), which can damage cellular molecules. To combat this, cells are known to utilize endogenous antioxidants to scavenge free radicals through redox reactions. Therefore, we previously examined the effect of feeding an exogenous antioxidant, resveratrol, on the ability of wildtype Drosophila to withstand heat stress. Treatment with 100uM and 400uM resveratrol increased the ability of the flies to withstand heat stress-induced paralysis. We hypothesize that this result occurred because the flies had increased HSF activity due to the resveratrol treatment. To examine this hypothesis, Drosophila larvae expressing HSF-GFP were dissected to obtain salivary glands. These glands contain large polytene chromosomes that allow for visualization of HSF chromosomal binding using confocal microscopy. The most easily visible binding site is an HSF doublet binding at the Hsp70 loci. Salivary glands at room temperature function as a non-heat shock (NHS) control and exhibit no binding of HSF-GFP at the Hsp70 loci. Salivary glands heated to 37C for 10, 20, 40 minutes function as the positive control and exhibit the expected Hsp70 doublet from HSF-GFP binding of the DNA. We are testing variable concentrations (100uM, 200uM, and 400uM) of resveratrol dissolved in 0.5% DMSO to determine if it activates HSF-GFP binding of the DNA in salivary glands under non-heat shock conditions. Our preliminary data indicates resveratrol treatment does not lead to the recruitment of HSF at HSP70 loci in living polytene nuclei. Follow-up experiments are currently underway to examine the levels of Hsp70 protein in cells treated with resveratrol.