Access Type

Open Access Thesis

Date of Award

January 2017

Degree Type

Thesis

Degree Name

M.S.

Department

Biological Sciences

First Advisor

Karen A. Beningo

Second Advisor

Arun Anantharam

Abstract

Regulated exocytosis from chromaffin cells in the adrenal medulla plays a critical role in maintaining organismal homeostasis. In the absence of stress, these cells release physiologically relevant substances into the blood stream only in limited quantities, whereas stressful conditions result in a rapid deluge of signaling molecules used, for example, to increase heart rate and pain tolerance. Although the cellular mechanisms governing the switch from low-level to stress-induced secretion are not well understood, recent evidence has implicated the exocytotic Ca2+-sensing protein Synaptotagmin (Syt) in this role.

Two isoforms of Syt are expressed in chromaffin cells (Syt-1 and Syt-7), and each is sorted to a different secretory vesicle population. Fusion events mediated by Syt-7 occur under milder stimulation conditions and result in slower release of vesicle cargo through a narrow fusion pore that often reseals (“kiss-and-run” exocytosis). Conversely, Syt-1 events require stronger cellular stimulation (characteristic of a stress response) and typically result in vesicles fully collapsing into the plasma membrane to rapidly release all cargo. Furthermore, Syt-7 remains clustered at fusion sites while Syt-1 rapidly diffuses away.

Although the distinct phenotypes of Syt-1 and Syt-7 may explain how chromaffin cells tune their secretory response to deal with intermittent periods of stress, a significant gap in this field is understanding the intermolecular basis for these phenotypic differences. The current study aimed to address this gap by using polarized Total Internal Reflection Fluorescence (pTIRF) microscopy to compare the phenotypes of various Syt chimeras, containing portions of both Syt-1 and Syt-7, with the behavior of the wild-type (WT) proteins. The Ca2+-binding loops of the Syt C2B domain were a prime candidate to target based on this domain’s established role in exocytosis and Ca2+-mediated interactions with other molecules. Chimeras composed of a Syt-1 background with individual C2B loops converted to match Syt-7 showed a mild increase in Syt persistence at fusion sites and slight inhibition of fusion pore expansion when compared to WT Syt-1. These effects were enhanced substantially when all three C2B loops were converted to the Syt-7 genotype, suggesting that the coordinated action of these loops serves as a major determinant of the exocytotic fusion mode.

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