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.