Journal
EUROPEAN JOURNAL OF CELL BIOLOGY
Volume 85, Issue 6, Pages 487-500Publisher
ELSEVIER GMBH, URBAN & FISCHER VERLAG
DOI: 10.1016/j.ejcb.2006.01.014
Keywords
vinculin; talin; PIP2; focal adhesions; cell motility; interference reflection microscopy
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Funding
- Medical Research Council [G0100152, G0401026] Funding Source: researchfish
- MRC [G0401026, G0100152] Funding Source: UKRI
- Medical Research Council [G0100152, G0100152(56891), G0401026] Funding Source: Medline
- NIGMS NIH HHS [5 U54 GM64346] Funding Source: Medline
- Wellcome Trust Funding Source: Medline
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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 Delta C mutation. This mutation reduced PIP2 binding to a Vt Delta C polypeptide by > 90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin Delta C 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. (c) 2006 Published by Elsevier GmbH.
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