Off-campus WSU users: To download campus access dissertations, please use the following link to log into our proxy server with your WSU access ID and password, then click the "Off-campus Download" button below.

Non-WSU users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Access Type

WSU Access

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Bhanu P. Jena

Abstract

For nearly half a century, it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the `all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess an `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the `Porosome', the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nanometer resolution and in real time, and its functional reconstitution into artificial lipid membrane, have been elucidated. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors called SNAREs, have been implicated in membrane fusion in cells. For example in neurons, target membrane proteins SNAP-25 and syntaxin (t-SNARE) present at the porosome base, and a synaptic vesicle-associated membrane protein (v-SNARE), are part of the conserved protein complex involved in fusion of synaptic vesicle membrane at the porosome. Studies demonstrate that t-SNAREs and v-SNAREs, when present in opposing lipid membrane, interact in a circular array, and in the presence of calcium, form conducting channels. The interaction of t-SNARE and v-SNARE proteins to form conducting channels is strictly dependent on the presence of these proteins in opposing membrane. Following stimulation of cell secretion, it has been demonstrated that secretory vesicles swell via rapid transport of water and ions, and the intravesicular pressure thus created enables the expulsion of vesicular contents from the cell via the SNARE channel and the porosome. The focus of the present study was to understand at the molecular level, membrane-associated t-/v-SNARE assembly and secretory vesicle swelling involved in cell secretion. Using t- and v-SNARE reconstituted proteoliposomes, and isolated secretory vesicles, the present study was conducted employing various biochemical and biophysical approaches. The results from this work were published in seven research papers, and provide a molecular understanding of both t-/v-SNARE assembly and secretory vesicle swelling during cell secretion.