Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells

Abstract

Cellular locomotion is driven by repeated cycles of protrusion of the leading edge, formation of new matrix adhesions and retraction of the trailing edge. In this study we addressed the molecular composition and dynamics of focal complexes, formed under the leading lamellae of motile cells, and their maturation into focal adhesions. We combined phase-contrast and fluorescence microscopy approaches to monitor the incorporation of phosphotyrosine and nine different focal adhesion proteins into focal complexes in endothelial cells, migrating into an in vitro `wound'. We show that newly formed complexes are located posterior to an actin-, VASP- and α-actinin-rich region in the lammelipodium. They are highly tyrosine phosphorylated, contain β3-integrin, talin, paxillin and low levels of vinculin and FAK, but are apparently devoid of zyxin and tensin. The recruitment of these proteins into focal complexes occurs sequentially, so that their specific protein composition depends on their age. Interestingly, double color, time-lapse movies visualizing both paxillin and zyxin, indicated that the transition from paxillin-rich focal complexes to definitive, zyxin-containing focal adhesions, takes place only after the leading edge stops advancing or retracts. These observations illuminate, for the first time, early stages in focal complex assembly and the dynamic process associated with its transformation into focal adhesion.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…The focal adhesion complex is defined as the sum of the interactions of more than 150 diverse, signaling, scaffolding and cytoskeletal proteins and their corresponding downstream signaling cascades [22,47]. The composition of focal adhesions has been shown to vary over time depending on its acquired level of maturity.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Focal adhesions contain more than fifty proteins, many of which are tyrosine phosphorylated and/or themselves tyrosine kinases. Tyrosine phosphorylation at adhesion sites occurs after the initial recruitment of talin and paxillin apparently followed by FAK and vinculin to focal sites [74][75][76][77]. Tensin and zyxin are generally absent from nascent adhesions [74].…”

“…Tyrosine phosphorylation at adhesion sites occurs after the initial recruitment of talin and paxillin apparently followed by FAK and vinculin to focal sites [74][75][76][77]. Tensin and zyxin are generally absent from nascent adhesions [74]. This ordered assembly promotes sequential tyrosine phosphorylation at nascent complexes and regulates the maturation and life-time of adhesions [74,77,78].…”

“…This ordered assembly promotes sequential tyrosine phosphorylation at nascent complexes and regulates the maturation and life-time of adhesions [74,77,78]. However, it should be noted that focal adhesions do not mature solely by accumulating signaling molecules; there is also an ordered exit of molecules during maturation, as evidenced by the decreased content of FAK and zyxin in mature adhesions [74,79]. RhoA might play a role in focal adhesion disassembly by recruiting c-Src to focal adhesions through activation of mDia [80].…”

Structural and mechanical functions of integrins

“…This PTB domain is important for binding multiple integrin adaptor proteins, in particular kindlin and talin. In this regard, the β-tails function as a hub for the 'adhesome' interactions that help mediate 'outside-in' signalling (Zaidel-Bar et al 2003). …”