Advisory Committee Chair
Alexa L Mattheyses
Advisory Committee Members
Elizabeth S Sztul
Date of Award
Degree Name by School
Doctor of Philosophy (PhD) School of Medicine
Clathrin-mediated endocytosis (CME) is an essential cellular process facilitating the internalization of a variety of cargo. Clathrin polymerization and changes in plasma membrane architecture and composition are necessary steps to mediate the formation of clathrin-coated vesicles (CCVs). However, simultaneous analysis of clathrin dynamics and membrane structure in living cells is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here we use Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy to bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape changes during vesicle assembly. Addressing this question required development of an automated MATLAB-based accelerated STAR data processing pipeline (DrSTAR) to enable the processing of multiple experimental conditions and biological replicates in a robust and reproducible environment. Moreover, DrSTAR employs a dynamic local referencing algorithm, which resolves the curvature of long-lasting CCSs. We found that de novo clathrin accumulations generate both flat and curved structures in living cells. Approximately 80% of clathrin accumulations contributed to successful endocytosis while 20% remained flat in both kidney fibroblast-like (Cos-7) cells stimulated with epidermal growth factor (EGF) and human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor (VEGF). High-throughput analysis revealed that the initiation of vesicle iv curvature does not directly correlate with clathrin accumulation. We show that clathrin accumulation drives curvature formation at shorter-lived CCVs (<20s), but clathrin undergoes a flat-to-curved transition at longer-lived CCVs (>20s). The broad spectrum of curvature initiation dynamics supports multiple productive mechanisms of vesicle formation and the flexible model of CME. The unique power of STAR microscopy has great potential to reveal the drivers of membrane curvature in the flexible model of clathrin-mediated endocytosis and to link distinct clathrin-coated structures with their impact on cellular logistics and homeostasis.
Nawara, Tomasz Jacek, "Imaging Nanoscale Plasma Membrane Dynamics Reveals Diversity in Clathrin-Coated Vesicle Formation" (2023). All ETDs from UAB. 385.