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Access Type

WSU Access

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Bhanu P. Jena

Abstract

The porosome is a lipoprotein structure, present in the plasma membrane of secretory cells, which mediates the transient fusion of secretory vesicles with the cell plasma membrane during secretion. In contrast to full fusion, where secretory vesicles fully collapse into the plasma membrane, porosome-mediated transient fusion allows vesicles to temporarily establish continuity with the cell plasma membrane to release a portion of their contents before dissociating from the membrane. The rapid vesicle recycling afforded by porosome-dependent transient fusion at the nerve terminal allows neurons to utilize their limited pool of readily-releasable synaptic vesicles in fast succession. Although the porosome is necessary for the vital process of transient fusion, much remains unknown about its detailed atomic structure. In this study, we sought to assess the protein-protein and protein-lipid interactions, primarily in the neuronal porosome complex, in physiological states including; development, secretion, and disease. Results from the study demonstrate that protein assembly within the neuronal porosome complex is incomplete during late embryonic development, however the porosome is fully assembled by 3 weeks of life. Furthermore, following a secretory stimulus, the low intracellular pH generated alters the association of SNARE proteins within the porosome complex and their dissociating molecular motor protein, n-ethylmaleimide- sensitive factor (NSF). Since porosome proteins contain lipid binding domains, we also investigated the protein-lipid interactions of the porosome; demonstrating numerous lipid families to be specifically enriched in the neuronal porosome. In support of a transient fusion mechanism, analysis of lipid membranes within the synapse revealed that synaptic vesicle lipids differ greatly from those present at the plasma membrane. Our studies additionally demonstrate changes in the porosome chemistry in disease states, such as cystic fibrosis and post-traumatic stress disorder (PTSD). In both cases, changes in composition of porosome proteins observed suggests altered neurotransmitter release. Distribution of the porosome proteins is also impacted in different brain regions in PTSD, which is likely associated with the pathology of the disease. Together, these results demonstrate the importance of the porosome secretory machinery in cells, including in neurons.

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