Adhesion and motility are fundamental processes that gate cell metastasis. Moderate over-expression (10-30% above endogenous levels) of the adhesion plaque protein, vinculin, inhibits cell motility and increased cell adhesion, whereas down-regulation of vinculin increase cell motility and inhibits adhesion. The long-term goal of this research is to understand how vinculin regulates cell adhesion and motility. The hypothesis to tested is that the interactions of vinculin's head (Vh) and tail domain (Vt) are mechanistically important to the role of vinculin in regulating cell adhesion and motility in living cells. A tetracycline-regulated expression vector will be used to over-express 6xHisEGFP-tagged vinculin and functional mutants of vinculin in Chinese hamster ovary (CHO) cells. The relationship between the intracellular concentration of vinculin and inhibition of fibronectin (FN)- mediated cell motility (maximum migration speed at the optimal receptor occupancy), increased adhesion (mean detachment strength as a function of receptor occupancy), and rate and extent of cell spreading will be established. The effects of vinculin over-expression on stabilization of actin filaments, molecular composition of focal adhesion plaques, dynamics of adhesion plaques, filopodia, and lamellipodia, localization of vinculin, and vinculin's association with cellular proteins will be assayed by cytological analysis of fixed and living cells and by biochemical methods. The contribution of vinculin's ligand-binding activities to the over-expression phenotype will be probed through site-directed mutagenesis to knock-out individual activities. Mutants in the actin, acidic phospholipid, and Vh binding functions of Vt will be made by systematically changing all clustered charges in a window of 5 amino acids to alanine (20 mutations). Mutants will be characterize to identify Vt mutants that retain """"""""native"""""""" structure but lose at least one of the 3 measured functions of Vt. Mutations in the VASP- and talin- binding functions will be based on known point mutations that knock out these activities in vitro. Mutations will be expressed in CHO cells in the context of individual vinculin subdomains and in the context of intact vinculin. The properties of these cells in the cell adhesion and locomotion assays will be assayed as a function of the amount of over- expressed mutant protein, and the phenotypes will be analyzed cytologically and biochemically as described, to connect phenotype with a particular ligand-binding function. New therapeutic targets for control of metastasis may be defined by this basic research on motility and adhesion.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041605-22
Application #
6018758
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1977-08-01
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
22
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Nanda, Suman Yadav; Hoang, Thuy; Patel, Priya et al. (2014) Vinculin regulates assembly of talin: ?3 integrin complexes. J Cell Biochem 115:1206-16
Dumbauld, David W; Lee, Ted T; Singh, Ankur et al. (2013) How vinculin regulates force transmission. Proc Natl Acad Sci U S A 110:9788-93
Coyer, Sean R; Singh, Ankur; Dumbauld, David W et al. (2012) Nanopatterning reveals an ECM area threshold for focal adhesion assembly and force transmission that is regulated by integrin activation and cytoskeleton tension. J Cell Sci 125:5110-23
Peng, Xiao; Maiers, Jessica L; Choudhury, Dilshad et al. (2012) ?-Catenin uses a novel mechanism to activate vinculin. J Biol Chem 287:7728-37
Chen, Hui; Choudhury, Dilshad M; Craig, Susan W (2006) Coincidence of actin filaments and talin is required to activate vinculin. J Biol Chem 281:40389-98
Cohen, Daniel M; Kutscher, Brett; Chen, Hui et al. (2006) A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions. J Biol Chem 281:16006-15
Chen, Hui; Cohen, Daniel M; Choudhury, Dilshad M et al. (2005) Spatial distribution and functional significance of activated vinculin in living cells. J Cell Biol 169:459-70
Cohen, Daniel M; Chen, Hui; Johnson, Robert P et al. (2005) Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin. J Biol Chem 280:17109-17
Johnson, R P; Craig, S W (2000) Actin activates a cryptic dimerization potential of the vinculin tail domain. J Biol Chem 275:95-105
Steimle, P A; Hoffert, J D; Adey, N B et al. (1999) Polyphosphoinositides inhibit the interaction of vinculin with actin filaments. J Biol Chem 274:18414-20

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