USING VINCULIN MUTANTS TO ASCERTAIN THE ROLE OF VINCULIN IN CELLULAR MOTILITY AND MECHANOTRANSDUCTION
Sharpe, Leilani Marie
MetadataShow full item record
Vinculin is a 117kDa soluble cytoplasmic protein that localizes to focal adhesions. Biochemical studies show that vinculin tail binds actin and vinculin head binds talin. These ligand binding interactions imply a role for vinculin as a mechanical linker between the actin cytoskeleton and the extracellular matrix. The consensus within the field is that expression of vinculin increases the adhesion of cells to the extracellular matrix and slows cellular migration speed. Vinculin is also implicated in the cellular mechanotransduction response because focal adhesion size is positively correlated with increased traction force between a cell and its substrate and because vinculin is recruited to focal adhesions with the application external force to a cell. The binding sites for many of vinculin’s ligands are regulated by an autoinhibition mechanism inherent to the structure of vinculin. However, it is unknown how vinculin’s autoinhibition and vinculin’s interactions with its ligands regulate vinculin’s role in cellular migration and mechanotransduction. To study vinculin’s role in these processes, a panel of vinculin ligand binding and autoinhibition mutants were expressed in an embryonic fibroblast line isolated from a Vcl-null mouse. I used timelapse microscopy to quantify the effect of vinculin and vinculin mutants on the speed of randomly migrating cells. My data shows that the presence of vinculin has no significant effect on cell speed. This result was unexpected, but it is in agreement with one other publication on the random migration of Vcl-null versus vinculin-expressing cells. To investigate another phenotype of vinculin-expressing cells, I developed a device capable of introducing uniaxial stretch to silicone-based cell culture chambers. This device was used to apply stretch to live cells in order to quantify mechanoresponsive change in mature vinculin-containing focal adhesions. My data shows that mature focal adhesions do not increase in size or number with greater frequency than unpaired, timelapse controls. This lack of force-dependent change in mature focal adhesions suggests the hypothesis that effects of external forces are restricted to a different subpopulation of focal adhesions, possibly nascent focal adhesions. Research Advisor: Susan W. Craig, Ph.D. Thesis Reader: Douglas Robinson, Ph.D.