Date of Award

2022

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natural Science and Mathematics, Chemistry and Biochemistry

First Advisor

Michelle K. Knowles

Second Advisor

Daniel Linseman

Third Advisor

Scott Horowitz

Fourth Advisor

Erich G. Chapman

Keywords

Biochemistry, Exosomes

Abstract

Exosomes are small lipid-based vesicles that can carry biomolecules from one cell to another. While exosomes are crucial to maintain homeostasis in healthy cells, they are exploited by unhealthy cells to aid disease progression. Exosomes likely facilitate disease progression via the transfer of disease-causing biomolecules from unhealthy to healthy cells. Exosomes are generated in Multivesicular endosomes (MVEs) and are then secreted into the extracellular space to travel to other cells. Despite being a crucial step, very little is known about exosomes release mechanism and dynamics. To further our understanding of exosomes, specifically their secretion, my work has focused on investigating spatiotemporal dynamics and kinetics of both MVEs and MVE interacting proteins. We visualized and characterized single MVE fusion events in lung cancer cells (A549) using CD63 fluorescent probes and total internal reflection fluorescence (TIRF) microscopy. The kinetics of release, or loss of fluorescence post-fusion, can relay information about the fate of exosomes. Using kinetic analysis we determined that a portion of exosomes are free to diffuse away from the fusion site, but some exosomes remain attached to the surface of the secreting cell. One challenge of measuring constitutive fusion events in a single vesicle fusion assay is the tedious process of manually finding and analyzing fusion events that occur at random points in time and in a relatively slow fashion (~1-3 events per minute). To overcome this, we developed a fast and fully automated algorithm to detect and analyze fusion events. To uncover some of the protein regulators we investigated the involvement of different SNAREs, and SNARE interacting proteins such as Syntaxins, SNAPs, and VAMP7 using in single vesicle fusion assays. Studying MVE fusion, exosome release, and potential fusion regulatory proteins provides insights into the exosome release mechanism, expands our understanding of the fusion process, and gives direction for future studies to identify potential therapeutic targets that can be used to modulate exosome secretion.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Anarkali Mahmood

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

126 pgs

Discipline

Chemistry

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