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Title page for ETD etd-03192010-154740

Type of Document Dissertation
Author McConnell, Russell E.
URN etd-03192010-154740
Title Microvillar membrane shedding; a novel aspect of brush border function regulated by myosin-1a
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
James R. Goldenring Committee Chair
Alissa M. Weaver Committee Member
Anne K. Kenworthy Committee Member
Matthew J. Tyska Committee Member
  • myosin
  • microvillus
  • intestine
  • epithelium
  • vesicle
  • brush border
Date of Defense 2010-03-03
Availability unrestricted
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 end–directed 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.
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  01titlepage.pdf 23.93 Kb 00:00:06 00:00:03 00:00:02 00:00:01 < 00:00:01
  02acknowledgements.pdf 28.76 Kb 00:00:07 00:00:04 00:00:03 00:00:01 < 00:00:01
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  04references.pdf 76.77 Kb 00:00:21 00:00:10 00:00:09 00:00:04 < 00:00:01

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