Degree Type

Thesis

Date of Award

2019

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Yeon-Kyun Shin

Abstract

SNARE-mediated membrane fusion is a key step in the process of neurotransmission. The release of neurotransmitters into the synapse as a result of vesicle fusion, particularly when triggered by an influx of Ca2+, serves as a primary means of signal propagation and communication in cognitive processes. Much work has been done to study these SNARE-mediated fusion events and the role auxiliary proteins play in modulating the overall process. However, our understanding of certain stages in the process is comparatively lacking, and our ability to study them has so far been curtailed by the relatively primitive state of current assays. This is particularly true for the opening and expansion of the fusion pore following hemifusion, necessary for the release of neurotransmitters from the vesicle into the synapse, which has gone largely unexamined despite its importance in understanding both the initial release and the life cycles of neurotransmitter-carrying vesicles.

Despite prior difficulties, refinement of an in vitro single vesicle fusion assay to a sufficiently robust state has presented an opportunity to investigate. In this study, we develop a novel method for the study of fusion pore dynamics. Utilizing vesicles containing fluorescently labeled dextran with a vesicle-to-suspended bilayer fusion assay, we can observe the release the release of the dextran through the bilayer as the pore opens and expands beyond the hydrodynamic radius of the dextran. We further observe a halt in release as the pore shrinks beneath this radius, giving rise to multi-staged content release patterns common to many vesicle docking and fusion events in the presence of SNAREs alone.

With this assay developed, we also perform preliminary studies into the roles of SNARE accessory proteins known to impact fusion. In particular, we observe the ability of α-synuclein to influence fusion pore dilation. While still in its early stages, we believe that our single vesicle content release assay can provide valuable insight into the roles of this and other proteins in modulating the dynamics of the fusion pore, and a greater understanding of the mechanisms of SNARE-mediated fusion as a whole.

Copyright Owner

Andrew Louis Wiese

Language

en

File Format

application/pdf

File Size

61 pages

Included in

Biochemistry Commons

Share

COinS