New insights into integrin-mediated adhesion formation

Integrin-mediated cell adhesions are highly regulated structures forming between the cell and the extracellular matrix (ECM). These structures form a bidirectional signalling platform between the cell and its environment with important functions in cell migration, shape, wound repair and tissue development. These adhesions are regulated by a complex network of proteins and lipids, allowing for an ever-expanding diversity in adhesion type and function. This network forms on a core consisting of integrin linked to actin via the large adapter protein talin. Integrins are dimeric transmembrane receptors with a large ECM binding ectodomain and two cytoplasmic tails. Talin is comprised of a FERM domain consisting of four subdomains in an atypical linear form linked to a long mechanosensitive rod domain containing thirteen helical bundle subdomains. A major pathway to activate integrin is for talin, via its F3 FERM subdomain, to bind to the β-integrin cytoplasmic tail and separate the two cytoplasmic tails. For talin-mediated integrin activation to occur, the FERM-domain containing adapter protein kindlin and the small GTPase Rap1, have both been demonstrated to be necessary. We have combined biochemical and structural approaches to elucidate novel regulatory mechanisms, which may be controlling talin-mediated integrin activation. Solving of a novel crystal structure of the talin-2 FERM domain revealed conformational plasticity in the talin FERM that alludes to a novel way of regulating the integrin activating ability of talin. Additionally, identification and characterisation of an interaction between talin and kindlin has provided a new insight into the role of kindlin in integrin activation. Furthermore, elucidation of an interaction between talin, Rap1 and the α-integrin cytoplasmic tail hints at a new twist in the integrin tail separation story and the essential function of Rap1 in adhesions, with both tails being simultaneously bound to talin. Vinculin is an important adhesion adapter protein consisting of a talin-binding head region linked to an actin binding tail domain. The talin rod contains 11 vinculin binding sites (VBS), 10 of which 13 are cryptic, which are revealed in response to mechanical force. Vinculin binding reinforces force transmission across talin, a process that is crucial for adhesion maturation. We identify a "threonine-belt" in the talin rod subdomain R8 that destabilises the domain, enabling vinculin binding in the absence of force. The accessibility of the VBS in the R8 subdomain proved essential for talin-vinculin pre-complexes to form prior to force onset, an important process in adhesion maturation. Additionally, we have identified a mechanism by which pathogenic bacteria mimic talin VBS to disrupt the talin-vinculin interaction, aiding cell entry. This multidisciplinary approach employed in this thesis has provided new insights into the complex mechanisms at play in the formation and maturation of integrin-mediated adhesions.