The interplay between cell-cell adhesions and their cytoskeleton demonstrates the complexity in generating and maintaining multicellular organisms. These interactions, however, are not static. Rather, they produce dynamic structures that are influenced by an array of both internal and external factors, including mechanical force. An example of a mechanically active zone within polarised epithelial cells is the zonula adherens (ZA), found at the apical-lateral interface between cells. Epithelial (E-) cadherin is a transmembrane protein that concentrates to support contractile tension at junctions by functionally coupling to the dynamic actin cytoskeleton. With both actin binding and bundling properties, vinculin is emerging as a key regulator in adhesion junctions. At the ZA, vinculin is a cytoplasmic component of the cadherin-catenin complex, however, unlike other junctional components, this protein is also enriched within cell-matrix adhesions. This project set out to further our understanding of vinculin’s role at E-cadherin-based cell-cell adhesions.
The first part of this thesis sets the foundation for my research. Using an shRNA-knockdown approach I demonstrate the importance of vinculin within the zonula adherens and define a role for vinculin in the regulation of junctional F-actin homeostasis.
The second part of this thesis focused on different actin nucleators that interact directly with vinculin and found that junctional depletion of vinculin affects junctional localisation of Mena and VASP. I developed vinculin mutants that uncoupled Mena/VASP and found that junctional vinculin can regulate F-actin homeostasis through recruitment of Ena/VASP proteins.
Finally, I demonstrated that vinculin mediates a tension-sensitive actin regulatory pathway at the epithelial ZA. Using the vinculin mutants and constructs described in Chapters 3 and 4, the experiments demonstrate that vinculin is a target of contractile tension and recruits Mena/VASP at the ZA to mediate tension-sensitive actin assembly in this location. Functionally, I show that this mechanism supports E-cadherin accumulation and junctional tension.
The work documented in this thesis has served to emphasise the complex interplay between mechanical force, cell-cell adhesions and their cytoskeleton, highlighting two manifestations of force on cell biology: 1) the capacity for mechanical forces to regulate protein recruitment and localisation; and 2) the capacity for cellular force generation to lead to potential design incompatibilities that cells must solve to effectively generate force.