Collections > Electronic Theses and Dissertations > Characterizing the Role of Actin Binding, Bundling, and Tyrosine Phosphorylation in Modulating Vinculin Function

Vinculin is an essential adhesion protein involved in controlling motility and force transduction, in part by coupling the actin cytoskeleton to the extracellular matrix. Vinculin is regulated by intramolecular interactions between the N-terminal head domain (Vh) and the C-terminal tail domain (Vt) which are linked by a proline-rich region. Upon binding to F-actin, Vt undergoes a conformational change that exposes a cryptic dimerization site that enables it to self-associate and bundle F-actin.Previous publications have investigated vinculin variants that are deficient in F-actin binding and have found a number of cellular defects; however, these constructs either lack Vt or contain large deletions that significantly destabilize Vt, and it is likely that the observed phenotypes are due to a number of disrupted interactions. In an effort to improve the vinculin/F-actin model using select mutations with little structural consequences, we have identified residues that selectively bind and/or bundle F-actin. Deletion of the C-terminal hairpin (Vt DC5) maintains a normal structure and binding to other interaction partners in comparison to wildtype (WT) Vt. However, Vt DC5 is unable to bundle F-actin and forms a non-functional dimeric species. To disrupt the vinculin/F-actin interaction, we characterized two variants: I997A and V1001A. When these variants are expressed in cells, cells are smaller and have fewer with larger adhesions when allowed to spread on FN. Cells expressing these variants are unable to reinforce when pulses of force are applied via integrins. These data suggest that vinculin-mediated F-actin binding and bundling plays a role in cell spreading and cellular reinforcement. The C-terminal hairpin contains the only tyrosine phosphorylation site in Vt (Y1065). However, the role of tyrosine phosphorylation in Vt and its full impact on regulating vinculin have not been elucidated. I provide evidence that Src-mediated phosphorylation of Vt modulates F-actin bundling. I have structurally characterized both phosphorylated Vt and Vt variants that either mimic or prevent phosphorylation to understand its impact on vinculin function. Furthermore, I utilize the variants to study the cellular role of Y1065 phosphorylation on cell spreading and cellular reinforcement. With the results of these studies, we propose an interface the enables actin-induced vinculin dimerization.