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CHARACTERIZATION OF THE CLASS V MYOSIN
MOTOR, MYOSIN 5C, IN LACRIMAL ACINI
by
Ronald Marchelletta
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)
December 2007
Copyright 2007 Ronald Marchelletta
Object Description
| Title | Characterization of the class v myosin motor, Myosin 5c, in lacrimal acini |
| Author | Marchelletta, Ronald |
| Author email | ronmarchelletta@hotmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2007-10-15 |
| Date submitted | 2007 |
| Restricted until | Restricted until 2 Nov. 2009. |
| Date published | 2009-11-02 |
| Advisor (committee chair) | Hamm-Alvarez, Sarah |
| Advisor (committee member) |
Okamoto, Curtis Haworth, Ian Mircheff, Austin |
| Abstract | Lacrimal gland acinar cells (LGACs) are the major contributor to ocular surface proteins extruded into the tear film. It has been suggested that a reduction in LGAC output is attributed to onset of dry eye syndromes ranging from mild dye eye to the more severe Sjögren's Syndrome. We investigated the role of the actin-based myosin motor, Myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles (SVs) in primary rabbit LGAC. Upon stimulation with carbachol (CCh), Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of GFP fused to the tail domain of Myo5c (Ad GFP-Myo5c-tail) showed that this protein was localized to SVs. Furthermore, its expression induced a significant (p less than or equal to 0.05) decrease in CCh-stimulated release of two SV content markers, secretory component (SC) and syncollin-GFP (Syn-GFP) of 58.14% and 58.5%, respectively. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGACs to compare various parameters of SVs labeled with either GFP-labeled protein in resting and stimulated LGACs. These studies revealed that carbachol-stimulated actin remodeling and compound fusion were exhibited by SVs labeled with GFP-Myo5c-full and was impaired in SVs labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-MyoVc-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.; In addition, Myo5c and dynein motor complex constituents, p150Glued and dynein intermediate chain (DIC 74) are observed to be co-localized beneath the apical plasma membrane (APM) in LGACs. This association of Myo5c and the dynein motor complex can be shown biochemically by density gradient analysis. Density gradient analysis also revealed another population of SVs that are enriched in Myo5c and the mature secretory vesicle marker, Rab3D. The p150Glued/ Myo5cenriched SVs may represent an immature SV population (iSVs). Taken together these results indicate that Myo5c may have a role in the maturation and the regulated exocytosis of SVs in LGACs. |
| Keyword | myosin; unconventional motor; lacrimal acini; intracellular traffic; Myosin 5; actin reorganization |
| 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-m903 |
| Rights | Marchelletta, Ronald |
| 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-Marchelletta-20071102 |
| Archival file | uscthesesreloadpub_Volume23/etd-Marchelletta-20071102.pdf |
Description
| Title | Page 1 |
| Full text | CHARACTERIZATION OF THE CLASS V MYOSIN MOTOR, MYOSIN 5C, IN LACRIMAL ACINI by Ronald Marchelletta 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) December 2007 Copyright 2007 Ronald Marchelletta |
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