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A STUDY OF THE ROLE OF RAB27 IN LACRIMAL GLAND ACINAR CELL SECRETORY TRAFFICKING
by
Lilian Chiang
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
December 2010
Copyright 2010 Lilian Chiang
Object Description
| Title | A study of the role of Rab27 in lacrimal gland acinar cell secretory trafficking |
| Author | Chiang, Lilian |
| Author email | lilianc@usc.edu; lilian.chiang@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date submitted | 2010 |
| Restricted until | Unrestricted |
| Date published | 2010-11-01 |
| Advisor (committee chair) | Hamm-Alvarez, Sarah F. |
| Advisor (committee member) |
McDonough, Alicia Okamoto, Curtis Shen, Wei-Chiang Yu, Alan |
| Abstract | Tear proteins are supplied by the regulated fusion of secretory vesicles with the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in regulating the exocytotic pathway of these secretory vesicles. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b-/- and Rab27ash/ash/Rab27b-/- mice, but not Ashen mice deficient in Rab27a, showed significant changes in organelle morphology which included an approximate 50% decrease in abundance of secretory vesicles and decreased vesicle localization in the subapical region. Along with an apparent secretory pathway effect, knockout of Rab27b also resulted in a two-fold increase in the number of lysosomes, four-fold increase number of damaged mitochondria, two-fold increase in the formation of autophagosome-like organelles, and observed increased ER swelling and vesiculation. Confocal fluorescence microscopy analysis, also confirmed by biochemical assays, of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were highly colocalized with Rab3D (73.6±2.0% at rest) and Myosin 5C (58.3±7.4% at rest). Stimulation of cultured acinar cells with the secretagogue, carbachol, resulted in apical fusion of these secretory vesicles with the plasma membrane.; We also established live-cell protocols for examination of cultured lacrimal gland acinar cells by measuring quantifiable markers for adenovirus entry; these protocols were applied to the remainder of the live cell studies. Tagged-Rab27b constructs expressed in lacrimal gland acinar cells allowed for visualization of Rab27b dynamics in real time. Wild-type Rab27b was expressed on a large subpool of mature secretory vesicles in the subapical region of the cell. Constitutively-active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, while dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. These secretory vesicles did not colocalize with the lysosomal pathway.; Functional studies measuring release of a co-transduced secretory protein, syncollin-GFP, showed that constitutively-active Rab27b enhanced (up to ~1.5-fold of wild-type), while dominant-negative Rab27b suppressed (no significant change from resting state), stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. Rab27b–enriched secretory vesicles also required intact components of the cytoskeletal network. Rab27b colocalized highly with the dynein motor protein subunit, p150Glued. In a series of stimulation-recovery experiments, real time image series showed for the first time clear visualization of Rab27b-enriched nascent vesicles formation on the surface of a Nascent Vesicle Site (NVS) located in the basal region of the acinar cell and their gradual movement towards the subapical region to join an existing pool of secretory vesicles as they matured over a 60 min time frame. Disruption of microtubule polymerization sequesters secretory vesicles on the NVS in close proximity to the trans-Golgi network, although colocalization with specific markers was low. These data leave two possible conclusions: that the NVS as a contiguous membrane compartment of the trans-Golgi network, or that the NVS is a completely separate organelle. We also examined Rab27b colocalization with another cytoskeletal component, F-actin. Rab27b also colocalized with Myosin 5C but not directly with actin filaments. Depolymerization of actin filaments prevents mature vesicles from fusing with the apical membrane. Finally, Rab27b shows high co-localization with a truncated form of one of its binding effector proteins, melanophilin, although localization was not obviously altered by overexpression of truncated melanophilin.; Previous studies have suggested that Rab27b is a participant of the last steps of secretory trafficking in other cell types, but have not looked at its role as one in a continuous pathway. In this study, we demonstrate a chronological and multi-step role for Rab27b that is critical in the secretory pathway of lacrimal gland acinar cell. In conclusion, we have shown in this study that knockout of Rab27b results in a major secretory, as well as degradative pathway, effect on lacrimal gland acinar cell morphology. Rab27b is highly enriched on the membrane of secretory vesicles and is associated with components of the exocytotic pathway. Rab27b-enriched secretory vesicles require intact microtubules for “long distance” transport of nascent vesicles to replenish subapical Rab27b-enriched vesicle pools. Upon stimulation, actin is necessary for vesicle-to-apical membrane fusion. Overexpression of constitutively active Ra2b7b up-regulates secretion, while dominant negative Rab27b suppresses the effect of stimulation. These last functional studies correlate with in situ morphological observations of decreased secretory vesicle numbers in Rab27b knockout mice. |
| Keyword | secretory vesicles; exocytosis; lacrimal gland acinar cells; primary cell culture; intracellular trafficking; membrane |
| 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 |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3516 |
| Rights | Chiang, Lilian |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Chiang-4135 |
| Archival file | uscthesesreloadpub_Volume26/etd-Chiang-4135.pdf |
Description
| Title | Page 1 |
| Full text | A STUDY OF THE ROLE OF RAB27 IN LACRIMAL GLAND ACINAR CELL SECRETORY TRAFFICKING by Lilian Chiang A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) December 2010 Copyright 2010 Lilian Chiang |
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