Actomyosin-dependent formation of the mechanosensitive talin–vinculin complex reinforces actin anchoring

Abstract

The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin–vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin–talin–vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.

Talin – the master of integrin adhesions

“…This was confirmed in vitro by using magnetic tweezers to pull open talin rod domains, demonstrating that the 13 α-helical bundles form a chain of 'spring-like' structures that progressively unfold as force increases from 5 to 25 pN, and binding of vinculin to talin requires some unfolding (del Rio et al, 2009;Yao et al, 2014;Yao et al, 2016). This provides a valuable paradigm for how force across a protein can lead to a chemical change, such as vinculin recruitment, and this can be recapitulated in a cell-free system (Ciobanasu et al, 2014). Bundle unfolding is reversible (Yao et al, 2016), and therefore the talin rod will respond to fluctuating force levels with cycles of recruitment and release of vinculin.…”

Integrin-bound talin head inhibits actin filament barbed-end elongation

“…Single molecule experiments showed that force transduction involves the mechanical stretching of talin rod domain, which exposes cryptic vinculin binding sites (VBSs) (27). Our previous work showed that vinculin binding to mechanically-stretched talin exposes its ABD, known as vinculin tail, and reinforces actin anchoring (28,29). In cells, actomyosin force acts on talin via its ABD3 to expose VBSs and ABD2, locking talin into an actin-binding configuration that stabilizes FAs (30).…”

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

“…In support of the Ballestrem model, however, an exciting new finding describes a potential mechanism for the force-dependent recruitment and residence time of vinculin within FAs. Using micropatterned islands of pure talin stretched to expose VBS via the selfassembly of F-actin and myosin II www.landesbioscience.comcontractile networks, 24 the authors demonstrated that: (1) the assembly of actomyosin network increases the stability of the talin-vinculin interaction, (2) vinculin's binding to talin's VBS sites is the rate-limiting step in talin refolding, and (3) stretched-talin-induced activation of vinculin creates a positive feedback loop that strengthens the talin-vinculin-actin association. And because the interactions between vinculin and the talin-VBS sites in this model were reversible upon the loss of connection between the talin-vinculin complex and the actomyosin machinery, this model provides a mechanism for vinculin's force-dependent recruitment and release from FAs.…”

“…The recent finding that vinculin experiences tensile forces supports this model as does the recently proposed model for actomyosin-mediated vinculin association with FA. 11,24 Indeed, recent evidence from the Ballestrem lab shows that vinculin co-localizes with subcellular areas of high tension. 33 This co-localization could be a result of tensile forces across vinculin that overcome the autoinhibitory head-tail interaction, thereby maintaining it in an active confirmation.…”

“…Importantly, Ciobanasu, et al recently demonstrated that force-mediated talin stretching can activate vinculin and reinforce its association with talin. 36 Indeed, by stretching talin, F-actin cables promoted a positive feedback loop in which an autoinhibited full-length vinculin bound to talin and limited its refolding while promoting recruitment of actin filaments. Using magnetic tweezers to measure the force required to stretch the talin rod domain, Yao, et al showed that the first three N-terminus helical bundles of the talin rod unfold is three discrete moves to expose VBS sites.…”