Epithelial cells called enterocytes line the lumen of the small intestine and are responsible for nutrient processing and barrier maintenance. Enterocytes have highly ordered actin arrays, or brush borders, on their apical surfaces. The brush border is composed of microvilli, membrane based protrusions of parallel actin bundles. Within microvilli, myosin-1a laterally links the actin cytoskeleton to the overlying membrane, and contributes to membrane tension regulation and vesicle shedding. These physiological functions require proper localization of this motor, a process that depends on the membrane binding tail homology 1 (TH1) domain. The goal of this thesis is to provide mechanistic details as to how myosin-1a targets to microvillar membrane and how its cellular dynamics are controlled. We find that in vitro and in cells myosin-1a interacts electrostatically with phosphatidylserine through basic residues in two independent bona fide membrane binding motifs. Because membrane binding controls myosin-1a targeting and previously published solution kinetic studies show the motor/actin interaction is short lived, we hypothesized that TH1 is the master regulator of dynamics for this molecule. We used live cell single molecule total internal reflection fluorescence microscopy in combination with single particle tracking and mean squared displacement analysis to measure membrane bound lateral mobility for myosin-1a and TH1. Many myosin-1a molecules display long-lived low mobility dynamics. Similar events are absent from TH1 analysis, indicating the motor domain makes an unexpected contribution to limiting mobility of myosin-1a at the membrane/cytoskeletal interface. Structure/function analysis confirmed this result and revealed the neck region is also important to controlling myosin-1a dynamics. In the context of full length myosin-1a, the neck region also plays a role in regulating localization, perhaps through a conformational change that involves calmodulin/calcium interactions. This is the first study to examine live cell dynamics for any class I myosin at single molecule resolution. The results presented within this thesis provide novel insight as to how myosin-1a cellular targeting and dynamics are controlled, and how biochemical and biophysical properties of myosin-1a manifest in cells to help this molecule carry out physiological roles in the brush border.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04302013-172218 |
| Date | 09 May 2013 |
| Creators | Mazerik, Jessica Nicole |
| Contributors | Matthew J. Tyska, Susan R. Wente, Charles R. Sanders, James R. Goldenring, M. Shane Hutson |
| 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-04302013-172218/ |
| Rights | restrictsix, 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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