Project Grant R35GM155253

MECHANISMS OF MEMBRANE TETHERING IN AUTOPHAGY - PROJECT SUMMARY/ABSTRACT CELLS ARE IN A CONSTANT BATTLE TO MAINTAIN HOMEOSTASIS AND RESPOND TO STRESS. AUTOPHAGY IS A CONSERVED EUKARYOTIC PATHWAY THAT RESPONDS TO CELLULAR STRESSES. AUTOPHAGY IDENTIFIES AND ENCAPSULATES CELLULAR DEBRIS IN AN AUTOPHAGOSOME, WHICH IS ULTIMATELY FUSED WITH THE LYSOSOME FOR DEGRADATION. STUDIES HAVE SHOWN THAT THE FINAL STEP OF AUTOPHAGY, TERMED AUTOLYSOSOMAL FUSION, REQUIRES SEVERAL FACTORS. THIS INCLUDES MEMBRANE EFFECTOR PROTEINS, RABS AND ATG8 HOMOLOGS, MULTIFUNCTIONAL SCAFFOLDING PROTEINS AND SPECIALIZED LIPID HEADGROUPS (E.G. PI3P). THE ASSEMBLY OF PROTEINS 'HUBS' OFTEN PROMOTE CELLULAR PROCESSES AND ARE REQUIRED FOR FUNCTION. THE HOMOTYPIC FUSION AND VACUOLE PROTEIN SORTING (HOPS) COMPLEX, ECTOPIC P-GRANULES 5 AUTOPHAGY TETHERING FACTOR (EPG-5), AND PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING FAMILY M MEMBER 1 (PLEKHM1) ARE SCAFFOLDING PROTEINS PRESENT AT THE FINAL STEPS OF AUTOPHAGY. MUTATIONS IN THESE MULTIFUNCTIONAL SCAFFOLDS LEAD TO POOR CELLULAR HEALTH AND HAVE BEEN IMPLICATED IN SEVERAL HUMAN DISEASES, SPECIFICALLY DEFECTS IN THESE PROTEINS LEAD TO NEURODEGENERATIVE DISEASES. DESPITE THE IMPORTANCE OF THE FINAL STAGES OF AUTOPHAGY, WE CURRENTLY LACK FUNDAMENTAL INFORMATION ON HOW THESE INTERACTION HUBS TETHER AND ORGANIZE SITES OF AUTOLYSOSOMAL FUSION. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO RESOLVE THE PROTEIN INTERACTION NETWORK THAT DRIVE AUTOLYSOSOMAL TETHERING. WE ARE INITIATING BOTH IN VITRO AND IN VIVO TECHNIQUES TO DISCOVERY THE MOLECULAR INTERACTIONS THAT DRIVE AUTOLYSOSOMAL TETHERING. SINGLE PARTICLE CRYO-ELECTRON MICROSCOPY ANALYSIS WILL SERVE AS OUR MAIN TOOL TO DETERMINE HOW THE HUMAN HOPS COMPLEX ENGAGES WITH AUTOPHAGY ADAPTOR PLEKHM1 AT THE MEMBRANE SURFACE. THESE STUDIES HAVE THE POTENTIAL TO REVEAL THE MOLECULAR INTERACTIONS WHICH DRIVE THE FORMATION OF THE AUTOLYSOSOMAL INTERFACE AND GENERATE SPECIFICITY WITHIN THE AUTOPHAGY PATHWAY. IN TANDEM, WE WILL UTILIZE CRYO- FOCUSED ION BEAM (CRYO-FIB) MILLING ALONG WITH IN SITU CRYO-ELECTRON TOMOGRAPHY METHODS TO EXAMINE AUTOLYSOSOMAL TETHERING IN THE NATIVE CELLULAR ENVIRONMENT. TO ACCOMPLISH THIS, WE WILL FOCUS ON EPG-5, A SCAFFOLD THAT BINDS TO BOTH LYSOSOME AND AUTOPHAGOSOME DIRECTLY (VIA PROTEIN-PROTEIN INTERACTIONS WITH RAB7 AND ATG8 HOMOLOGS, RESPECTIVELY). EPG-5 IS AN IDEAL TARGET FOR IN SITU STUDIES GIVEN ITS LARGE SIZE (300 KDA) AND DISTINCT SHAPE. BY USING CELL BIOLOGICAL TECHNIQUES, WE WILL ENRICH AUTOLYSOSOMAL TETHERING EVENTS AND PERFORM CRYO-FIB MILLING. THESE INNOVATIVE APPROACHES HAVE THE POTENTIAL TO DISCOVER THE CELLULAR CONTEXT OF AUTOLYSOSOMAL FUSION AT RESOLUTIONS (