Thursday, December 26, 2024
12/26/2024
PROJECT SUMMARY The aim of this proposal is to elucidate how ESCRT (Endosomal Sorting Complex Required for Transport) machinery is regulated and deployed in neurons. ESCRT is a set of conserved and sequentially-acting protein complexes that play crucial roles in cellular processes such as endosomal traffic, cytokinesis, and membrane repair. One important function of ESCRT in neurons is the formation of multivesicular endosomes, which are critical for proteostasis, signaling, and the formation of extracellular vesicles that carry cargoes critical for signaling and disease. Despite their central importance, the regulation of neuronal ESCRT complexes remains poorly understood for two main reasons: First, we lack the tools to visualize their activities in vivo at high resolution. Second, chronic disruption of ESCRT causes many pleiotropic and secondary phenotypes in membrane traffic, making it difficult to decipher its mechanisms of action and to test the physiological importance of ESCRT-dependent events. As a result, it remains unclear how cellular processes like extracellular vesicle cargo traffic are spatially and temporally regulated within the polarized and complex morphology of neurons. Our research seeks to fill this gap by developing new visualization and manipulation tools specifically tailored for studying ESCRT dynamics, using Drosophila neurons as a model. In Aim 1, we will investigate the dynamics of synaptic ESCRT machinery at endosomes, and explore how neuronal activity influences ESCRT dynamics. Utilizing quantitative analyses of high-resolution microscopy, we aim to measure ESCRT recruitment to neuronal endosomes and examine its relation to endosomal and extracellular vesicle cargoes. Aim 2 focuses on devising new tools for temporal and spatial control of ESCRT and testing the kinetics and spatial organization of multivesicular endosome biogenesis and the resultant release of extracellular vesicles. These tools will enable us to engineer inducible systems for controlling ESCRT activities, investigate the temporal sequence of chronic ESCRT phenotypes, and answer questions about the dynamics and regulation of extracellular vesicle release. Overall, our research aims to provide mechanistic insights into synapse-specific regulation of ESCRT machinery, shedding light on its role in synaptic function and disease. Furthermore, our tools will have broad applicability to the field for studying ESCRT-related questions across different experimental systems, offering opportunities for manipulating extracellular vesicle release and other ESCRT-dependent processes in diverse cell types or organisms.
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