Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Berro, Julien

Abstract

In this dissertation, I investigated the molecular mechanism of clathrin-mediated endocytosis (CME) in fission yeast with a sparse labeling strategy to track endocytic proteins at the single molecule level. CME is involved in a variety of biological processes, such as nutrient internalization and receptor recycling. CME is also a well-conserved biological process from yeast to mammalian cells. During clathrin-mediated endocytosis, about 60 different endocytic proteins are recruited to the endocytic site in a highly reproducible order. During the endocytic event, endocytic proteins assemble into endocytic structures, contributing to membrane invagination and endocytic vesicle formation. Based on the single molecule endocytic protein trajectories I obtained, I proved the significance of stresses within the actin meshwork. I also investigated the dwell-time distribution of single molecules of fimbrin (a protein that crosslinks actin filaments) and provide new mechanisms for fimbrin-actin binding mechanism. To study the single-molecule endocytic protein dynamics, I upgraded a two-color Total Internal Reflection Fluorescence (TIRF) microscopy system to study the single molecule dynamics of endocytic proteins. The two-color imaging system can be applied to probe relative motions between endocytic proteins in further studies.

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