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

Abstract:

There is great interest in understanding the mechanism of action of antioxidant treatments and their effects on the cell. One major stress response pathway is the heat shock response (HSR) that 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, reductants and other chemical stresses. HSF activates expression of the Hsp70 chaperone, which helps cells deal with protein folding stress. Activation of HSR also leads to an increase in reactive oxygen species (ROS), which can damage cellular molecules. To combat this, cells utilize endogenous antioxidants to scavenge free radicals through redox reactions. Therefore, we previously examined the effect of feeding the 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. We hypothesize that one possible reason for this result is that the flies had increased HSF activity due to the resveratrol treatment. To examine this hypothesis, Drosophila larvae expressing HSF-GFP were dissected to obtain their salivary glands. These glands contain large polytene chromosomes that allow for visualization of HSF binding onto the chromosome using confocal microscopy. The major binding site that is easily visualized is a doublet of HSF binding at the Hsp70 loci on the chromosome. Salivary glands at room temperature function as a non-heat shock control and exhibit no binding of HSF-GFP at the Hsp70 loci. Salivary glands heated to 37C for 10 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 to determine if it activates HSF-GFP binding of the DNA in salivary glands under non-heat shock conditions. Future experiments may examine if the HSF-GFP is transcriptionally active when cells are treated with resveratrol.