Our long-term goal is to elucidate the function of the brush border (BB) cytoskeleton and its associated motor proteins in the normal physiology and pathophysiology of the intestinal epithelial cell (enterocyte). As one of the most highly-ordered F-actin assemblies observed in nature, the BB functions as the primary site of nutrient absorption in the intestinal tract. BBs are composed of tightly packed arrays of microvilli (MV) that are nearly identical in length and diameter;the plasma membrane associated with each MV is linked to a supporting actin bundle by a spiraling array of bridges composed of myosin-1a (Myo1a), a motor protein that generates force and motion directed along actin filaments. Our recent analyses of Myo1a knockout mice indicate that this motor plays a critical role in stabilizing apical membrane morphology. BBs lacking Myo1a exhibit significant vesiculation and herniation of the BB membrane, defects that are strikingly similar to symptoms observed in malabsorption enteropathies such as Celiac disease and bacterial gastroenteritis. The goal of this proposal is to investigate the molecular mechanism underlying these phenotypes by determining how the motor activity of Myo1a is exploited to regulate BB membrane structure. Our central hypothesis is that Myo1a stabilizes the BB membrane by generating mechanical forces that contribute to membrane-cytoskeleton adhesion. We will test this hypothesis by implementing a multifaceted approach, including biophysical assays based on optical trapping. Specifically, we will: (1) determine if ATP-induced membrane shedding demonstrated by isolated BBs represents a manifestation of Myo1a force generation, (2) directly measure the forces generated during interactions between Myo1 a and native BB components in a reconstituted MV motility assay, (3) measure the fraction of total BB membrane adhesion provided by Myo1a, and (4) determine how the biophysical properties of Myo1a govern its role in membrane-cytoskeleton adhesion. Intact BBs are essential for the normal absorptive function of the mucosa;MV loss is a common symptom in a number of Gl diseases that pose significant threats to human health. By elucidating factors that control MV stability, this work will develop our understanding of normal enterocyte function and help illuminate pathways and processes that may be perturbed in cases of enterocyte dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK075555-04
Application #
7788144
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
Grey, Michael J
Project Start
2007-04-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
4
Fiscal Year
2010
Total Cost
$274,686
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Li, Jianchao; He, Yunyun; Weck, Meredith L et al. (2017) Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins. Proc Natl Acad Sci U S A 114:E3776-E3785
Kim, Sun Wook; Ehrman, Jonathan; Ahn, Mok-Ryeon et al. (2017) Shear stress induces noncanonical autophagy in intestinal epithelial monolayers. Mol Biol Cell 28:3043-3056
Weck, Meredith L; Grega-Larson, Nathan E; Tyska, Matthew J (2017) MyTH4-FERM myosins in the assembly and maintenance of actin-based protrusions. Curr Opin Cell Biol 44:68-78
McKinley, Eliot T; Sui, Yunxia; Al-Kofahi, Yousef et al. (2017) Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity. JCI Insight 2:
Millis, Bryan A; Tyska, Matthew J (2017) High-Resolution Image Stitching as a Tool to Assess Tissue-Level Protein Distribution and Localization. Methods Mol Biol 1606:281-296
Feng, Qiang; Bonder, Edward M; Engevik, Amy C et al. (2017) Disruption of Rab8a and Rab11a causes formation of basolateral microvilli in neonatal enteropathy. J Cell Sci 130:2491-2505
Grega-Larson, Nathan E; Crawley, Scott W; Tyska, Matthew J (2016) Impact of cordon-bleu expression on actin cytoskeleton architecture and dynamics. Cytoskeleton (Hoboken) 73:670-679
Taneja, Nilay; Fenix, Aidan M; Rathbun, Lindsay et al. (2016) Focal adhesions control cleavage furrow shape and spindle tilt during mitosis. Sci Rep 6:29846
Tyska, Matthew J (2016) Brush Border Destruction by Enterohemorrhagic Escherichia coli (EHEC): New Insights From Organoid Culture. Cell Mol Gastroenterol Hepatol 2:7-8
Raval, Manmeet H; Quintero, Omar A; Weck, Meredith L et al. (2016) Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions. J Biol Chem 291:22781-22792

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