The overall goal of this proposed research is to understand the molecular basis of membrane-cytoskeletal interactions in vertebrate cells. The extensive plasma membrane formed by microvilli on the apical surface of the intestinal epithelial cell interacts along the length of the microvilli with the actin cytoskeleton through periodically spaced, ATP-sensitive cross bridges composed of a 110,000-dalton protein-calmodulin complex. This complex also has calcium-activated Mg2+ ATPase activity. To define and characterize the molecular basis of the association of the complex with both the membrane and cytoskeleton and to define the function of the ATPase, the ATPase activity and actin and membrane binding of the complex will be studied under well defined conditions by reconstitution with proteins purified from the microvillus, isolated microvilli and microvillar cytoskeletons and liposomes. Biochemical, biophysical, electron microscopic and immunochemical approaches will be used to study both native and reconstituted systems. The possible function of the ATPase in microvillar motility, in regulation of the cytoskeletal and membrane binding of the complex and in calcium ion transport will be determined. The possible association of the complex with the lipid bilayer of the membrane and with other membrane proteins will be studied. In addition, regulation of these events by calcium ion binding to the complex and by phosphorylation of the 110,000-dalton protein will also be characterized. These studies should enhance our understanding of cytoskeletal-membrane interactions and their regulation in intestinal epithelial and other cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035448-03
Application #
3288227
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1985-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1989-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Swanljung-Collins, H; Collins, J H (1992) Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation. J Biol Chem 267:3445-54
Swanljung-Collins, H; Collins, J H (1991) Rapid, high-yield purification of intestinal brush border myosin I. Methods Enzymol 196:3-11
Swanljung-Collins, H; Collins, J H (1991) Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound. J Biol Chem 266:1312-9
Borysenko, C; Rieker, J P; Swanljung-Collins, H et al. (1988) Phosphorylation of brush border myosin at threonine on its 20 kDa light chains by a calmodulin-independent kinase activates its ATPase. FEBS Lett 235:149-52
Rieker, J P; Swanljung-Collins, H; Collins, J H (1987) Purification and characterization of a calmodulin-dependent myosin heavy chain kinase from intestinal brush border. J Biol Chem 262:15262-8
Swanljung-Collins, H; Montibeller, J; Collins, J H (1987) Purification and characterization of the 110-kDa actin- and calmodulin-binding protein from intestinal brush border: a myosin-like ATPase. Methods Enzymol 139:137-48
Rieker, J P; Collins, J H (1987) Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases. FEBS Lett 223:262-6
Rieker, J P; Swanljung-Collins, H; Montibeller, J et al. (1987) Isolation and characterization of calmodulin-dependent myosin heavy chain kinase from intestinal brush border. Methods Enzymol 139:105-14