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THE CHARACTERIZATION OF SNARES AND RABS
IN LACRIMAL GLAND ACINAR CELLS
by
Eunbyul Evans
____________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2008
Copyright 2008 Eunbyul Evans
Object Description
| Title | The characterization of SNAREs and rabs in lacrimal gland acinar cells |
| Author | Evans, Eunbyul |
| Author email | esou@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2008-03-27 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-07-28 |
| Advisor (committee chair) | Hamm-Alvarez, Sarah F. |
| Advisor (committee member) |
Okamoto, Curtis Toshio Wang, Clay C. C. Chow, Robert HP |
| Abstract | The lacrimal gland secretes tear proteins and fluid to help maintain a healthy ocular surface. Dry eye diseases are often characterized by decreased secretory function of the lacrimal gland. Since the mechanisms of lacrimal gland secretion are poorly understood, the molecular mechanisms involved in the exocytosis of secretory vesicles in the lacrimal gland acinar cells (LGACs), the main secretory cells of the lacrimal gland, were investigated. The first part of my research explored the role of soluble Nethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) in apical exocytosis in LGACs. Immunofluorescence studies revealed the subcellular localization of VAMP2 and VAMP8 on potential secretory vesicles in the subapical region in resting and/or carbachol (CCH)-stimulated acini. Syntaxin 3 and SNAP-23 were localized to the apical plasma membrane (APM). Binding studies revealed VAMP2/syntaxin 3/SNAP-23 and VAMP8/syntaxin 3/SNAP-23 form a SNARE complex, presumably at the APM. Immunofluorescence studies corroborated the data, showing the colocalization of GFPVAMP2, expressed utilizing an adenoviral construct, with syntaxin 3 and SNAP-23. These studies identified the SNARE complexes that may be involved in secretory vesicle exocytosis at the APM in LGACs.; The second part of my research explored the role of rab3D in the trafficking of polymeric IgA receptor (pIgR) through the regulated secretory pathway in LGACs. Immunofluorescence studies showed pIgR, characterized as mediating transcytosis of polymeric immunoglobulins in epithelial cells, colocalizing with rab3D, a regulated secretory vesicle marker, in LGACs. The release rate of secretory component (SC), the proteolytically cleaved fragment of the extracellular region of the pIgR, was shown to be very similar to syncollin, a regulated secretory protein marker. In pull-down assays, recombinant wild-type rab3D (rab3DWT) and GDP-locked rab3DT36N pull-downed pIgR from resting acini, however, the GTP-locked mutant rab3DQ81L did not. Adenoviral-mediated expression of rab3DT36N in LGACs inhibited SC release with CCH-stimulation. The pull-down of pIgR by rab3DWT and the colocalizaton of pIgR with endogenous rab3D decreased upon CCH-stimulation, indicating that secretogogues can modulate the interaction between rab3d and pIgR. These studies revealed a novel localization of pIgR to regulated secretory vesicles in LGACs, and SC release from these vesicles appears to be affected by its interaction with rab3D. |
| Keyword | SNAREs; rab gtpases; pIgR; rab3D; vesicle trafficking; acinar cells |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m1426 |
| Contributing entity | University of Southern California |
| Rights | Evans, Eunbyul |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@dots.usc.edu |
| Filename | etd-Evans-20080728 |
| Archival file | uscthesesreloadpub_Volume40/etd-Evans-20080728.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@dots.usc.edu |
| Full text | THE CHARACTERIZATION OF SNARES AND RABS IN LACRIMAL GLAND ACINAR CELLS by Eunbyul Evans ____________________________________________________________________ A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) August 2008 Copyright 2008 Eunbyul Evans |
