Epithelial cells lining the lumen of the small intestine, called enterocytes, possess an exquisitely ordered array of microvilli collectively referred to as the brush border (BB). Microvilli are actin-rich membrane protrusions that cover the apical surface of most epithelial cell types; within microvilli of the enterocyte BB, the motor protein myosin-1a forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite being one of the most abundant proteins in the enterocyte BB, it is unclear if myosin-1a demonstrates motor activity in the context of the microvillus, and if so, what roles this activity would play. Here, we show in vitro that addition of ATP to isolated BBs induces a plus enddirected translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is "shed" into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, we conclude that Myo1a powers this novel form of motility. In vivo, we find a morphologically similar type of vesicle release from the tips of enterocyte microvilli; these vesicles retain the right-side-out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myosin-1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the regulation of this novel activity. Intriguingly, we find that vesicles interact with bacteria present in the intestinal lumen, suggesting a role for vesicle production in mucosal barrier function. Furthermore, vesicles are able to detoxify the bacterial compound lipopolysaccharide, protecting cultured epithelial cells from its potent pro-inflammatory effects. In combination, these data show that myosin-1a is mechanically active in the context of enterocyte microvilli, that this activity is required for the proper regulation of vesicle production from microvilli, and that vesicles released from enterocyte microvilli may help to protect the epithelium from the intestinal microbiota.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03192010-154740 |
| Date | 22 March 2010 |
| Creators | McConnell, Russell E. |
| Contributors | James R. Goldenring, Anne K. Kenworthy, Alissa M. Weaver, Matthew J. Tyska |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03192010-154740/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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