Poster #070, Sanford Burnham Prebys
Identification of host factors involved in heat stress-induced TFEB/HLH-30 nuclear translocation via reverse genetic screening in Caenorhabditis elegans.
Mentors: Cheng-ju Kuo, phD, Diego Hernandez, Research Assistant. Caroline Kumsta, Ph.D.
Autophagy is a conserved cellular degradation process that clears damaged intercellular components to maintain protein homeostasis (proteostasis). A key regulator of autophagy is the master transcription factor TFEB (HLH-30 in C. elegans), which translocates from the cytoplasm to the nucleus in response to heat stress (HS). Because both autophagy and TFEB/HLH-30 are required for heat stress tolerance, we hypothesize that HS-induced nuclear translocation of TFEB/HLH-30 is a critical step in mediating the protective heat stress response. To identify the factors that regulate HS-induced TFEB/HLH-30 nuclear localization, I joined the lab to perform a reverse genetic RNA interference (RNAi) screen.
By feeding C. elegans with RNA-producing bacteria, I knocked down candidate genes and examined TFEB/HLH-30 nuclear localization before and after HS. If knockdown of a gene prevented TFEB/HLH-30 from translocating to the nucleus after HS, that gene was considered a potential positive regulator of nuclear translocation. Conversely, if gene knockdown increased nuclear localization of TFEB/HLH-30 under basal, non-stress condition, the gene was considered a potential negative regulator. Using a transgenic C. elegans strain expressing GFP-tagged HLH-30, I monitored TFEB/HLH-30 localization by fluorescence microscopy before and after heat shock. While the initial screen of 14 genes didn’t yield any targets, I am currently screening an additional batch of 11 genes.
Through this project, I gained extensive hands-on experience with C. elegans research techniques. I learned how RNAi functions and knew how to perform RNAi in worms. I also learned to use fluorescence microscopy to visualize GFP-labeled worms and image TFEB/HLH-30 nuclear localization. By screening the HS-induced TFEB/HLH-30 nuclear translocation factors, this research has the potential to 1. uncover novel, heat-stress specific, signaling pathways 2. reveal nuclear transport dynamics under heat stress 3. elucidate the connection between thermal adaptation and longevity 4. discover therapeutic targets for TFEB/HLH-30.