The studies proposed will continue our efforts to characterize the actin- based cytoskeleton of the intestinal epithelial cell and its apical brush border (BB). These studies will be pursued with the dual goals of providing general insights into the actin based cytoskeleton of nonmuscle cells, and also to dissect the roles of actin and its associated binding proteins in the physiologic functions of the enterocyte. Our primary focus of study during this grant period will be on the family of unconventional myosins--that is, myosins other than the conventional, two-headed and tailed BB myosin which is expressed in the terminal web domain of the BB. This will include continued biochemical and functional studies on avian BB myosin I. This single-headed myosin, that has as its light chains (lc), multiple calmodulins (CM), forms the bridges which laterally tether the microvillar (MV) actin core to the membrane. Three sets of experiments are proposed. One will investigate the molecular basis for CM interaction with BB myosin heavy chain (hc), as well as the mechanism by which CM regulates the enzymatic, mechanochemical and membrane binding activities of this myosin. A second goal will be to elucidate the regulatory functions of a recently discovered kinase that phosphorylates the tail domain of BB myosin I hc. Finally, we plan to identify and characterize the MV membrane protein that serves as a receptor for BB myosin I. A second major project is based on the discovery that subclones derived from the human intestinal cell line, Caco-2, may express multiple unconventional myosins in addition to BB myosin I. Strategies for the cloning of these myosins are outlined. Once cloned, we plan to probe the function of these myosins in vivo through expression of truncate (headless) myosins in transfected Caco-2 cells. Finally, we outline a pilot study in which we hope to establish the feasibility of applying genetic and molecular genetic approaches to dissecting BB cytoskeletal protein function. To this end we propose the molecular cloning of the proteins which form the cytoskeleton of the Drosophila midgut BB--beginning with the MV core protein villin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK025387-21
Application #
2900139
Study Section
Special Emphasis Panel (NSS)
Program Officer
May, Michael K
Project Start
1979-04-01
Project End
2000-07-31
Budget Start
1999-04-15
Budget End
2000-07-31
Support Year
21
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
082359691
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
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
Collaco, Anne; Jakab, Robert; Hegan, Peter et al. (2010) Alpha-AP-2 directs myosin VI-dependent endocytosis of cystic fibrosis transmembrane conductance regulator chloride channels in the intestine. J Biol Chem 285:17177-87

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