Classical cadherins are cell adhesion proteins that regulate the actin cytoskeleton by a variety of mechanisms, that include spatially controlled actin assembly. In order to regulate changes in cell shape and motility the cadherin must coordinate high levels of cohesiveness with more dynamic states of adhesion. The cadherin associates with different states of actin organisation and these may contribute critically to the diverse functional states that the cadherin supports. However, by what mechanism the cadherin regulates the reorganisation of the cytoskeleton is unknown. In this thesis I have investigated the contribution of Ena/VASP proteins to the assembly of dynamic actin structures at cadherin-based cell adhesions, to find molecules that may function to coordinate different forms of actin regulation.
By combining analysis of native cell-cell contacts with hemophilic adhesions induced when cells bound to recombinant cadherin ligands, I found that Ena/VASP proteins were specifically recruited to newly forming adhesive contacts by cadherin hemophilic ligation. At these cadherin-based contacts Ena/VASP proteins (principally focusing on Mena) co-accumulated with E-cadherin in two distinct pools: Firstly, Mena formed punctate and linear structures that associated with the Arp2/3-mediated dendritic actin network at the leading edges of protrusive lamellipodia. Secondly, Mena staining localised in streak-like macroclusters that accumulated at the termini of actin bundles localised proximal to the leading edge. Thus Mena identified two distinct modes of actin organisation found at cadherin hemophilic adhesions: Arp2/3 dependent peripheral protrusions and actin cables. These two pools of Mena at cadherin contacts were morphologically and biochemically distinguishable as demonstrated by differences in their susceptibility to cytochalasin D and ROCK inhibition. Additionally, these two pools exhibited different dynamics when analysed by live-cell imaging.
Ena/VASP activity was essential for the formation of both of these types of cadherin-directed actin organisation. Using mouse embryo fibroblasts derived from knock-out mice, I found that depletion of Ena/VASP proteins compromised the formation of the Arp2/3-dendritic actin network and the actin bundling in cells adhering to cadherin specific substrata. Expression of Mena rescued lamellipodia formation and actin bundling, indicated that Ena/VASP activity was essential for cadherin-based contact formation. Thus Ena/VASP activity was necessary for the expression of both modes of actin organisation induced by cadherin homophilic adhesion. Ena/VASP activity was also required for junctional and perijunctional actin assembly at cell-cell contacts, further supporting a role for this family in the regulation of actin assembly and organisation at cadherin-based adhesions.
Finally, loss of Ena/VASP activity corresponded to a loss of E-cadherin clustering and a reduction in the steady-state expression of E- and N-cadherin. This was accompanied by a delay in the proteolytic processing of pro-E-cadherin to the mature form of E-cadherin. The mechanisms regulating these changes are unclear but it is likely that Ena/VASP proteins stabilise the cadherin-catenin complex through their interaction with the actin cytoskeleton. Nonetheless, these data indicate that not only can cadherins regulate the actin cytoskeleton through recruitment of Ena/VASP proteins, but these cytoskeletal changes in turn affect the lateral organisation and expression of the cadherin itself.
Taken together, my findings identify a key role for Ena/VASP proteins in the functional interplay between cadherin adhesion and the actin cytoskeleton. They demonstrate that Ena/VASP proteins are one of a cohort of actin regulatory proteins that are recruited to adhesions in response to cadherin homophilic ligation. There the Ena/VASP proteins critically determine the regulation of actin activity and organisation by cadherin adhesion and signaling. My work further highlights the concept of functional cooperativity between cadherin adhesion and the actin cytoskeleton, such that not only can adhesive interactions regulate actin activity, but the cytoskeletal response can, in turn, modify the functional state of cadherin adhesion itself. Thus the long-appreciated dependence of cadherins on the actin cytoskeleton appears to involve a functional interdependence supported by these key actin regulatory proteins.