The mechanisms and dynamics of αvβ3 integrin clustering in living cells

Abstract

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

“…S1, which is available online). A lower mobile fraction reflects increased levels of activation (Cluzel et al, 2005), therefore, these data suggest that blocking phosphorylation at the NPxY motifs (YY>FF) increases bPS-integrin activity in this context. We generated another activating mutation in the TM domain, L796R, which mimics talininduced conformational changes by shortening the length of transmembrane domain that is buried in the plasma membrane .…”

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

“…Compared to other phosphoinositides that may also bind to talin [14,21] PIP2 is locally enriched by talin-recruited PIPKIγ kinase in vivo [22,23] and is thus a physiologically important ligand for talin to promote integrin adhesion [15,21,25]. This selectivity is supported by the recent genetic studies where specific ablation of PIPKIγ in focal adhesions significantly impaired the talin recruitment to membrane and adhesion sites [28].…”

“…Previous studies suggested that the inactive talin has an autoinhibited conformation, where a key integrin-binding site on talin-F3 is self-masked by a talin-R segment (talin-RS) [19,20]. Membrane lipid phosphotidylinositol-4,5-bisphosphate (PIP2) [19,21], which is locally enriched by talin-recruited PIPKIγ kinase [22,23], was shown to activate talin [19,21] and promote integrin-mediated cell adhesion [15,[24][25][26][27][28], but the detailed structural basis of this is not clear. The prevailing hypothesis is that PIP2 may sterically induce the conformational change of talin [2,15,16,19,21], a mechanism widely employed in the known PIP2-mediated activation of proteins, including FERM domain proteins [29,30].…”

Structural and mechanical functions of integrins

“…Protein tyrosine phosphorylation is an important event in outside-in signalling and is mediated through Src and FAK family protein tyrosine kinases (Arias-Salgado et al 2003, 2005Jiang et al 2003;Cluzel et al 2005). Src family kinases (SFKs) and their inhibitor Csk are permanently bound to integrin β-tails, but upon ligand binding, Csk dissociates to facilitate SFK activation and recruitment of tyrosine phosphatases (Arias-Salgado et al 2005).…”