Talin determines the nanoscale architecture of focal adhesions

Abstract

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15°relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.superresolution microscopy | focal adhesions | talin | mechanobiology | nanoscale architecture C ell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2-5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7-10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11-15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins s...

Distinct focal adhesion protein modules control different aspects of mechanotransduction

“…Interestingly, classifying FA proteins according to their dynamic behavior correlates well with the compartments identified by superresolution microscopy (Kanchanawong et al, 2010;Liu et al, 2015): (1) the 'integrin signaling layer' (containing FAK and paxillin), (2) the 'force transduction layer' (talin and vinculin) and (3) the 'actin regulatory layer' (zyxin, VASP and α-actinin). According to our data, proteins residing within these individual layers have very similar rates of turnover.…”

“…At these sites, the integrin transmembrane receptors connect ECM proteins to the cytoplasmic FA plaque complex, providing a link to the contractile actomyosin machinery (Hynes, 2002). While the overall molecular architecture of FAs has been established (Kanchanawong et al, 2010;Liu et al, 2015), much less is known about the dynamic processes that occur within FAs, and their functional relevance to mechanotransduction, the conversion of a mechanical signal into a cellular response.…”

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

“…Tilting of integrins on the cell surface was predicted using molecular-dynamics simulation to model integrin conformations in the presence of lateral forces, and may be allowed by the flexible residues connecting the extracellular domains and transmembrane domains (11). The tilt of integrins described here is similar to the tilts of both F-actin and talin in FAs (31,32), suggesting force coaligns the integrin-talin-F-actin link (Fig. 4I).…”

Talin – the master of integrin adhesions

“…This orientation was also observed in muscle attachments in Drosophila, but not in its epithelial cells (Klapholz et al, 2015). Vinculin is positioned between the talin head and C-terminus and oriented in the same direction as talin, fitting with it binding to the talin rod with its head domain and to the actin layer with its C-terminal actinbinding domain (Kanchanawong et al, 2010;Case et al, 2015a;Liu et al, 2015). Importantly, talin specifies the separation between the layers, as deletions within its rod domain bring the actin regulatory layer closer to the membrane .…”