The actin-based cytoskeleton plays essential roles in a wide range of cellular phenomena including generation of cell shape, polarity , motility, cell signal transduction mechanisms, transport and localization of organelles and mRNAs, and modulation of the mechanical and physiological properties of cell membranes. The actin-based cytoskeletal apparatus that underlies the apical, microvillous or brush border (BB) of the intestinal epithelial cell is among the most high ordered cytoskeletal arrays in nature. The BB cytoskeleton has served for decades as a model system to investigate the organization, composition and functional properties of the actin-based cytoskeleton. The proposed studies will focus on several actin-based molecular motors (myosins) that are major components of the BB cytoskeleton in both vertebrates and Drosophila. The myosins to be investigated are class I myosins, one of fifteen known structurally microvillous by bridges composed of BB myosin-I (BBMI), which contains several bound calmodulin light chains, is among the best biochemically characterized myosins, yet nothing is known regarding its functions in vivo. Proposed functions include a role for this myosin in maintaining apical membrane polarity through vesicle transport or facilitation of nutrient absorption either by generation of microvillar movements or through mechanochemical regulation of membrane transporters in the BB membrane. These and other hypotheses will be tested through the phenotypic characterization of mice lacking the gene for BBMI (mice were made by collaborators). Studies will include phenotypic characterization BBMI-/- mice with respect to eneterocyte morphology, polarity, cytoskeletal composition and stability. Effects on intestinal functions including nutrient transport and response to intestinal pathogens and mucosal injury will be examined. In vivo dynamics of BBMI will be determined through visualization of GFP-tagged BMIO in the intestinal epithelial cell line, Caco2BBe. In Drosophila, the intestinal BB contains two class-I myosins, myosin-Ib (MIb) and myosin-Ia (MIa). Preliminary analysis suggests that both of these myosins are essential. Animals lacking MIb, which like BBMI tethers the BB membrane to underlying cytoskeleton, die as growth arrested first instar larvae, a phenocopy of starvation. This suggests a critical role for MIb in nutrient absorption. Characterization of MIb and MIa will include generation of multiple mutant alleles and phenotypic assessment of effects on epithelial cell organization, polarity, BB structure/composition and assessment of effects on gut physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK025387-25
Application #
6607619
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
May, Michael K
Project Start
1979-04-01
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
25
Fiscal Year
2003
Total Cost
$407,396
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Hegan, Peter S; Chandhoke, Surjit K; Barone, Christina et al. (2016) Mice lacking myosin IXb, an inflammatory bowel disease susceptibility gene, have impaired intestinal barrier function and superficial ulceration in the ileum. Cytoskeleton (Hoboken) 73:163-79
Hegan, Peter S; Ostertag, Eric; Geurts, Aron M et al. (2015) Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells. Cytoskeleton (Hoboken) 72:503-16
Hegan, Peter S; Kravtsov, Dmitri V; Caputo, Christina et al. (2015) Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI. Cytoskeleton (Hoboken) 72:455-76
Hegan, Peter S; Lanahan, Anthony A; Simons, Michael et al. (2015) Myosin VI and cardiomyopathy: Left ventricular hypertrophy, fibrosis, and both cardiac and pulmonary vascular endothelial cell defects in the Snell's waltzer mouse. Cytoskeleton (Hoboken) 72:373-87
Mazzolini, Rocco; Rodrigues, Paulo; Bazzocco, Sarah et al. (2013) Brush border myosin Ia inactivation in gastric but not endometrial tumors. Int J Cancer 132:1790-9
Hegan, Peter S; Giral, Hector; Levi, Moshe et al. (2012) Myosin VI is required for maintenance of brush border structure, composition, and membrane trafficking functions in the intestinal epithelial cell. Cytoskeleton (Hoboken) 69:235-51
Mazzolini, Rocco; Dopeso, Higinio; Mateo-Lozano, Silvia et al. (2012) Brush border myosin Ia has tumor suppressor activity in the intestine. Proc Natl Acad Sci U S A 109:1530-5
Kravtsov, Dmitri V; Caputo, Christina; Collaco, Anne et al. (2012) Myosin Ia is required for CFTR brush border membrane trafficking and ion transport in the mouse small intestine. Traffic 13:1072-82
Chandhoke, Surjit K; Mooseker, Mark S (2012) A role for myosin IXb, a motor-RhoGAP chimera, in epithelial wound healing and tight junction regulation. Mol Biol Cell 23:2468-80
Ramamurthy, Bhagavathi; Cao, Wenxiang; De la Cruz, Enrique M et al. (2012) Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VI. Cytoskeleton (Hoboken) 69:59-69

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