Dissecting a Novel Pathway for Degradative Endosome Generation during Rapid Synaptic Vesicle Recycling Pathway - Project Summary/Abstract Late endosomes (multivesicular bodies, MVBs) are basic endocytic organelles for protein homeostasis. Rapid MVB formation is essential for the emergence of cellular responses such as nutrient sensing, signal desensitization, and synaptic vesicle recycling. Additionally, MVBs serve as a frontline defense against pathogens and toxins. Their dysfunctions contribute to disorders like tumor cell invasion, neurological diseases and chronic infections. Despite their physiological significance in health and disease, the molecular mechanisms underlying rapid MVB formation remain poorly understood. The challenges arise from 1) MVB formation processes occurring on hundred milliseconds to a few seconds time scale, and 2) the spatial coexistence of MVBs with other endocytic organelles within diffraction-limited spaces. Thus, traditional methods struggle to capture the rapid membrane remodeling process and to visualize specific molecular players involved. I will push the boundaries of our mechanistic understanding of rapid MVB formation by developing new experimental paradigms that integrate time-resolved electron and super-resolution microscopy imaging techniques. My long-term goal is to reveal the millisecond-to-second progression of MVB formation during fastest clathrin-independent synaptic recycling, called ultrafast endocytic pathway, and understand how proteins coordinate rapid membrane remodeling at the structural level. Towards this goal, I will focus on the following two aspects in this study: 1) Capturing the rapid membrane dynamics during MVB formation and identifying the molecular components driving this process. 2) Developing approaches to visualize these proteins at nanometer resolution using electron microscopy and super-resolution imaging, and study how they rapidly remodel membranes to form MVBs. These experiments will lay the foundations for future research into the general mechanisms of rapid MVB formation. Furthermore, they may also lead to novel strategies to dissect infectious diseases and neurodegenerative diseases driven by defective rapid membrane trafficking.
