The intestinal brush border serves as the sole site of nutrient absorption within the body, and also acts as an important barrier against luminal pathogens. The brush border is comprised of membrane protrusions called microvilli that are found on the apical surface of enterocytes. These protrusions are supported by a core bundle of 20 to 30 parallel actin filaments with the plus ends oriented towards the lumen. Our laboratory showed that protocadherin-24 and mucin-like protocadherin play a key role in the assembly and organization of the brush border. These protocadherins interact to form a trans-heterophilic adhesion complex that physically connects the distal tips of microvilli and regulates the tight packing. How these complexes are targeted to microvillar tips remains unknown. Microvillar protocadherins interact with several cytoplasmic binding partners, including the actin based motor, myosin-7b (Myo7b). Given the role of other myosin motors in building actin-based protrusions, this work focuses on elucidating the function of Myo7b in brush border assembly. We show that Myo7b is highly enriched at the tips of microvilli in both kidney and intestinal brush borders. Loss of Myo7b results in the mislocalization of the intermicrovillar adhesion links along the microvillar axis, disrupting brush border assembly. We also found that Myo7b motor domains are capable of supporting tip-directed transport. However, motor activity is supplemented by other passive targeting mechanisms, which together drive highly efficient accumulation of the adhesion complex at the tips. Additionally, we have established an in-cell reconstitution assay using filopodial protrusions that can be used to study protein interactions within the adhesion complex. Using this assay, we have begun to characterize the morphological effects of tip-enriched adhesion on actin-based protrusions. Initial experiments suggest adhesion at the distal tips regulates the stability and length of these protrusions. This work provides evidence that the actin-based motor Myo7b promotes the accumulation of adhesion complexes at microvillar tips, which may alter the morphology and dynamics of actin-based protrusions. These findings hold important implications for understanding apical morphogenesis in transporting and sensory epithelial tissues.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03262018-115455 |
| Date | 29 March 2018 |
| Creators | Weck, Meredith Lynn |
| Contributors | Dylan T. Burnette, Ph.D., Robert J. Coffey, M.D., Matthew J. Lang, Ph.D., Ryoma Ohi, Ph.D., Matthew J. Tyska, Ph.D. |
| 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-03262018-115455/ |
| 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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