Integrin-Associated Complexes Form Hierarchically with Variable Stoichiometry in Nascent Adhesions

Abstract

Summary Background A complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from co-immunoprecipitation studies using expressed components; but few have been demonstrated or characterized functionally in living cells. Results We introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, ‘when’ and ‘where’ molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin-activation and in the integrin-actin linkage in NAs and show that these molecules form integrin containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, while also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature. Conclusions These observations suggest a working model for NA assembly, whereby transient α-actinin- integrin complexes help nucleate NAs within the lamellipodium. Subsequently integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes.

Lamellipodia are crucial for haptotactic sensing and response

“…Therefore integrins, and β1-containing integrins in particular, are necessary for haptotaxis but are not differentially localized. Kindlins and talin activate integrins, and kindlin2 has been shown to associate with β1 integrin at nascent adhesions before talin recruitment during adhesion maturation (Bachir et al, 2014). Consistent with a central role of β1-containing integrins, depletion of kindlin2 in fibroblasts (Fig.…”

Early Events in Cell Spreading as a Model for Quantitative Analysis of Biomechanical Events

“…Thus, when the pentameric fibronectin type III domain 7-10 was used as a ligand, it supported 6-to 9-fold greater force per fibronectin than the corresponding monomeric form (10). The formation of early integrin clusters typically occurs at the cell edge, when the membrane protrudes forward and encounters new matrix, and involves talin and/or kindlin binding (11)(12)(13). After the initial clusters form, some grow into mature adhesions in a myosin-II-dependent manner (14)(15)(16)(17).…”

Talin – the master of integrin adhesions

“…In this mode, integrin α5β1, kindlin2, talin-1 and vinculin enter nascent adhesions simultaneously. However, cross-variance analysis has revealed that, during adhesion assembly, α5β1 is bound to kindlin2, and talin bound to vinculin; however, these two subcomplexes are not bound to each other until the nascent adhesion becomes stabilised by myosin II activity (Bachir et al, 2014). Thus, talin can be retained at the adhesion without maintaining integrin binding, and interactions between IAPs can change following recruitment.…”

“…1, where talin dimers are recruited by binding to a ligand-bound integrin, and then can insideout activate integrins that diffuse within suitable proximity. However, examination of nascent adhesions in epithelial cells revealed a third mode of initial recruitment (Bachir et al, 2014). In this mode, integrin α5β1, kindlin2, talin-1 and vinculin enter nascent adhesions simultaneously.…”