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

Abstract

Protein and lipid clustering is an important mechanism for cell processes such as exo- and endo-cytosis and creating functional signaling complexes. It has been seen that both lipids and proteins are involved with the formation of cluster domains on cell membranes, yet little is known about the interplay between the two. In this work I began with visualizing lipid sorting to artificially induced curvature in supported lipid bilayers, with lipid tails affecting the sorting differently to curvature. These results demonstrate that lipids sort to curvature on curved, supported lipid bilayers. The sorting depends on the number of lipids in the tails with two-tailed lipids accumulating more at regions of curvature. The work then transitions to protein clustering. A plasma membrane SNAP Receptor (SNARE) protein, Syntaxin 1a (Syx1a), is known to cluster and these clusters are sites of membrane fusion in neuroendocrine cells. Syx1a was measured using dynamic measurements (FRAP and single molecule tracking), first by looking at substrate dye interactions to ensure dynamics were not altered by unintended interactions between dye and substrate. Following these studies it was determined that the commonly used poly-L-lysine substrate interacts with commonly used red fluorescent dye (Alexa Fluor 594), as a result Fibronectin is used for further studies. Single molecule dynamics of Syx1a at clusters using a novel system of nanobody interacting with enhanced green fluorescent protein (EGFP) labeled Syx1a reveals not only single molecule dynamics but cluster locations. Studies were performed to see how truncation of Syx1a affects mobility along with cholesterol depletions. Syx1a is more mobile at the center of clusters than elsewhere on the cell surface and that truncation of the Habc domain slightly slows this process. Removal of cholesterol greatly inhibited the motion at the cluster center and generally inhibited mobility thought the membrane. Overall, the work described within elucidates how clusters form on cell and synthetic membranes.

Publication Statement

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

Rights Holder

Alan Weisgerber

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

120 pgs

Discipline

Biogeochemistry

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