Studies on the cytoskeletal proteins vinculin and talin.
thesisposted on 19.11.2015, 09:07 by Sarah J. Bolton
The role of vinculin in cell adhesion was investigated by isolating NIH3T3 and Balb/c 3T3 cells containing a plasmid vector expressing a vinculin antisense RNA under the control of the MMTV promoter. Stable cell lines of NIH3T3 cells were cultured with dexamethasone but none showed any reduction in vinculin protein levels. Two Balb/c 3T3 cell lines, when cultured with dexamethasone displayed a marked reduction in vinculin levels and displayed an altered cell shape from a spread to a more spindle-shaped morphology. These phenotypic changes were reversible upon removal of the dexamethasone. The reduced adhesion corresponded with a reduction in the growth rate of the clones. The cells were also unable to spread on fibronectin and the ususal increase in the phosphotyrosine content of two signalling proteins, paxillin and pp125FAK, normally associated with cell adhesion, was not observed. The results establish that vinculin is a key component of integrin-mediated cell adhesion. To explore the structure-functional relationship of talin, six anti-talin monoclonal antibodies were microinjected into human fibroblasts and the effect upon the actin cytoskeleton was assessed. Two of the antibodies, TA205 and TD77 resulted in the disintegration of actin stress fibres, and migration of CEF was also severely impaired following microinjection of either antibody. The epitopes recognised by TA205 and TD77 had been mapped to talin residues 102-497 and 2269-2541 respectively. Microinjection of CEF with a polypeptide containing residues 102-497 demonstrated that they were mainly associated with the detergent-soluble cytoplasmic portion of the cell and were also able to disrupt stress fibre and focal adhesion integrity. Residues 2269-2541 were associated with the detergent-insoluble cytoskeletal fraction of the cell but did not affect stress fibre or focal adhesion integrity. Attempts to identify proteins that interacted with residues 102-497 of talin were unsuccessful but an actin-binding site was identified within residues 2269-2541. The results indicate the presence of domains within the N- and C-terminus of talin that are essential to the involvement of talin in the formation of focal adhesions.