Role of vinculin in regulating focal adhesion turnover.
Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
Ruth M
RM
Saunders
Mark R
MR
Holt
Lisa
L
Jennings
Deborah H
DH
Sutton
Igor L
IL
Barsukov
Andrey
A
Bobkov
Robert C
RC
Liddington
Eileen A
EA
Adamson
Graham A
GA
Dunn
David R
DR
Critchley
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002465:Cell Movement / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology; D022001:Focal Adhesions / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016596:Vinculin / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*
D019269:Phosphatidylinositol 4,5-Diphosphate; D016596:Vinculin