Authors: Wong, Timothy

Institution: University of British Columbia

Publishing Date: 14 Apr 2025

Summary: Clathrin vesicles and caveolae are plasma membrane invaginations involved in endocytosis. Clathrin oligomers combine to form clathrin pits that mature to become vesicles. Caveolae flatten and disassemble into smaller oligomers, or scaffolds, containing the major caveolae protein, caveolin-1 (CAV1). Super-resolution microscopy, including single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED) microscopy, provide high-resolution views of fluorescently labeled molecules in situ within the cell. Here, I applied SuperResNET network analysis software to SMLM data to study the impact of small molecule inhibitors on clathrin structure and of point mutations on CAV1 structure and used STED to define a novel endocytic route for CAV1 in response to mechanical stress. Clathrin-targeted pitstop 2, dynamin-targeted dynasore and actin depolymerizing agent latrunculin A inhibit clathrin endocytosis but their impact on clathrin structures is poorly understood. SuperResNET-SMLM analysis reveals that smaller, more elongated pitstop 2 blobs correspond to clathrin pits, control and dynasore-induced blobs resemble vesicles while actin depolymerization induces the formation of larger heterogeneous structures. SuperResNET analysis therefore detects the differential impact of these three small molecule clathrin inhibitors on their target structures within the cell. Point mutations in the caveolin scaffolding domain (CSD) impede CAV1 interactions with effector proteins, but their impact on caveolae or scaffold structure is unclear. SuperResNET-SMLM convex hull analysis shows a size reduction effect of the CSD mutant on caveolae and intermediate scaffolds, that may reflect N-terminus folding toward the membrane preventing CSD interaction with effector proteins. SuperResNET analysis therefore detects the impact of a point mutation on CAV1 domain structure in the cell. CAV1 fate after caveolae disassembly in response to osmotic shock is unknown. Here, STED imaging shows that CAV1 scaffolds localize to spherical, non-acidic endocytic structures positive for Lysosomal-associated membrane protein 1 (LAMP1)-GFP. CAV1 endocytosis is not due to cell expansion but rather occurs upon reduction in cell volume following extended hypotonic shock. Upon isotonic recovery, CAV1 traffics back to the cell surface. These results describe a novel trafficking route for non-caveolar CAV1 endocytosis in response to osmotic shock. My dissertation demonstrates the use of super-resolution microscopy to study endocytic structures in their natural context, shedding light on caveolin and clathrin-mediated endocytosis.