Talin and kindlin cooperate to control the density of integrin clusters

Abstract

Focal adhesions are important mechanosensitive structures, composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Cellular adhesion to the extracellular matrix depends on the activation of integrins by intracellular mechanisms. Talin and kindlin are major activators of integrins that are recruited to the inner membrane and bind to Beta-integrin cytoplasmic tails. Many studies showed the importance of integrin activation and clustering and how the organization of extracellular ligands guides the nanoscale organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we performed experiments using a biomimetic in vitro system, made of Giant Unilamellar Vesicles, containing transmembrane integrins, on which purified talin, kindlin, and actomyosin assemble. Here we first show that talin and kindlin individually have the ability to cluster integrins. When added together, talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density in the talin-integrin, kindlin-integrin, and talin-kindlin-integrin clusters reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin significantly enhances the segregation of talin-integrin clusters induced by actomyosin contractility, suggesting that it increases the coupling of these clusters to the actin cytoskeleton. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

QD-based biosensors for protein assembly and mechanosensing

“…Talin variants appended with a C-terminal hexahistidine (His 6 ) were purified as described elsewhere [6]. CdSe/CdS/CdZnS/ZnS core/multishell QDs were prepared and coated with CL4 capping ligands as described previously [45].…”

“…Talin is largely responsible for the mechanosensing of FA [4]. This protein directly binds FA receptors (integrins) and phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on the membrane [5], [6]. Filamentous actin binds to the distal end of Talin and with myosin exerts a pulling force [7], triggering the maturation of focal adhesions [4].…”

Quantum Dot-based biosensors for protein assembly and mechanosensing

“…Talin is largely responsible for the mechanosensing of FA [4]. This protein directly binds FA receptors (integrins) and phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on the membrane [5], [6]. Filamentous Actin binds to the distal end of Talin and with Myosin exert a pulling force [7], triggering the maturation of focal adhesions [4].…”

Quantum dot-based FRET nanosensors for Talin-membrane assembly and mechanosensing

“…Its head domain binds to the cell plasma membrane. On the one hand, it interacts with inositol phosphate groups [5], [6] of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) lipids at the plasma membrane, releasing Talin autoinhibition [7], [8]. Interestingly, it was demonstrated that F 3 interacts with R 9 and F 2 with R 12 , resulting in an inactive snake like biting its own tail conformation [6], [9].…”