Project Grant F31ES037550

INVESTIGATING THE MOLECULAR BASIS OF CHAPERONE-MEDIATED CONDENSATE DISPERSAL IN RESPONSE TO ENVIRONMENTAL STRESS - PROJECT SUMMARY BACKGROUND: CELLS RESPOND TO SUDDEN CHANGES IN THEIR ENVIRONMENT (E.G. HEAT SHOCK) BY FORMING BIOMOLECULAR CONDENSATES AND OVEREXPRESSING MOLECULAR CHAPERONES. AFTER THE STRESS SUBSIDES, THE MOLECULAR CHAPERONES HSP104, HSP70, AND SIS1 DISPERSE CONDENSATES. HOWEVER, THE MOLECULAR MECHANISMS FOR HOW CHAPERONES ENGAGE CONDENSATES TO ACCOMPLISH THIS REACTION REMAIN UNCLEAR. WHILE WE DO NOT YET UNDERSTAND HOW CHAPERONES DISPERSE CONDENSATES, WE KNOW HOW THEY BEHAVE WITH MODEL (BUT NOT COGNATE) SUBSTRATE, LUCIFERASE. INTERESTINGLY, TO TAKE APART LUCIFERASE AGGREGATES REQUIRES HSP104, HSP70, SIS1, AND YDJ1. HOWEVER, FOR THE DISPERSAL OF PAB1 CONDENSATES, YDJ1 INHIBITS DISPERSAL. THIS INHIBITORY BEHAVIOR IS PUZZLING BECAUSE YDJ1 AND SIS1 ARE BOTH MEMBERS OF THE SAME J-DOMAIN FAMILY AND ARE CO-CHAPERONES FOR HSP70. THEREFORE, EXPLORING THE DISTINCT EFFECTS OF YDJ1 AND SIS1 WILL CLARIFY THE BEHAVIORS AND INTERACTIONS NECESSARY FOR EFFECTIVE DISPERSAL OF COGNATE SUBSTRATES. I HYPOTHESIZE THE J-DOMAIN PLAYS A CRUCIAL ROLE IN REGULATING CONDENSATE DISPERSAL AND THAT TEMPERATURE-INDUCED UNFOLDED REGIONS OF CONDENSATES ACT AS SIGNALS FOR CHAPERONES. MY PRELIMINARY DATA INDICATES THAT THE INHIBITION STATE OF THE J-DOMAIN MAY PLAY A SIGNIFICANT ROLE IN REGULATING CONDENSATE DISPERSAL. OUR EVIDENCE SUGGESTS THAT WHEN THE J-DOMAIN IS UNINHIBITED, OR "FREE", IT LEADS TO INHIBITION OF CONDENSATE DISPERSAL THAT CAN SOMETIMES BE RESCUED BY INCREASING THE CONCENTRATION OF HSP70 IN THE REACTION. SPECIFIC AIMS: TO BUILD A STRONG MOLECULAR FRAMEWORK FOR HOW CELLS RECOVER AFTER AN ENVIRONMENTAL CHANGE, I PROPOSE TO (1) DETERMINE WHY WE OBSERVE DIFFERENT BEHAVIOR AMONG TWO JDPS IN PAB1 CONDENSATE DISPERSAL AND (2) IDENTIFY CRITICAL INTERACTIONS AMONG CHAPERONES THEMSELVES AS WELL AS WITH CONDENSATES. STUDY DESIGN: HERE, WE PROPOSE TO COMBINE CELLULAR, BIOCHEMICAL, AND BIOPHYSICAL TECHNIQUES TO ANSWER THESE QUESTIONS. TO PROBE THE BEHAVIORAL DIFFERENCES OF YDJ1 AND SIS1, I USED A COMBINATION OF MUTATIONAL CONSTRUCTS AND FLUORESCENCE ANISOTROPY. IN ADDITION, TO COMPLEMENT THE ANISOTROPY EXPERIMENTS, I WILL MEASURE HOW WELL EACH JDP STIMULATES ATP HYDROLYSIS OF HSP70. TO VALIDATE THE J-DOMAIN EFFECTS OBSERVED IN VITRO, I WILL USE CRISPR TO INTEGRATE MY MUTANTS IN YEAST AND MONITOR THEIR EFFECT ON FITNESS AND DISPERSAL. NOTABLY, THIS PROPOSAL INCLUDES THE USE OF HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY (HDX-MS), A POWERFUL BIOPHYSICAL TECHNIQUE THAT REPORTS ON THE CONFORMATIONAL CHANGES IN PROTEIN SYSTEMS. WITH HDX-MS, WE CAN MONITOR THE CHAPERONE DYNAMICS AND IDENTIFY CRITICAL CHAPERONE-CONDENSATE INTERACTIONS. TOGETHER WITH HDX-MS, I WILL USE CROSSLINKING MASS SPECTROMETRY TO SUPPLEMENT THE INTERACTIONS SITES OBSERVED FROM HDX-MS. TRAINING: THIS CO-MENTORED TRAINING PLAN IS DONE UNDER EQUAL SUPERVISION OF DR. TOBIN R. SOSNICK AND DR. D. ALLAN DRUMMOND, LONG-TIME COLLABORATORS AT THE UNIVERSITY OF CHICAGO WHERE I WILL ADVANCE MY EXPERIMENTAL SKILLS AND INTERPERSONAL SKILLS TO BECOME AN INDEPENDENT RESEARCHER.