| 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.Cell 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 such as talin and vinculin, and a stress fiber interfacial zone that contains actin-associated proteins such as VASP (vasodilator-stimulated protein) and α-actinin. Although such multilaminar architecture signifies a certain degree of compartmentalization within FAs that may serve to spatially constrain protein–protein interactions and dynamics, the structural connectivity, the molecular configuration and geometry of FA proteins, and the molecular basis of their higher-order organization remain unclear.Proteomic and interactome analysis of the integrin adhesome have uncovered several direct and multitier connections between integrins and actin (17–20). This suggests that multiple highly interconnected protein–protein interactions could collectively self-organize into FA structures; such redundancy could also account for the remarkable mechanical robustness of FAs after cellular disruption or perturbation (21). Alternatively, a specific FA component may play a dominant role in regulating FA architecture. Aspects of both scenarios may also act cooperatively or function at distinct stages of FA assembly and maturation. Superresolution microscopy of cells expressing fluorescent protein (FP)-tagged FA components has revealed that talin, a large cytoskeletal adaptor protein, adopts a highly polarized orientation in FAs (16), with the N terminus residing in the membrane-proximal layer and the C terminus elevated by z ∼30 nm to the FA/stress fiber interfacial zone. This led us to hypothesize that an array of integrin–talin–actin linkages may vertically span the FA core, serving a structural role in determining FA architecture (16).To test this hypothesis, we sought to perturb FAs by substituting endogenous talin with recombinant analogs having modified lengths. These were generated by retaining both the N-terminal FERM (band 4.1/Ezrin/Radixin/Moesin) and C-terminal THATCH (Talin/HIP1R/Sla2p Actin-tethering C-terminal Homology, or R13) domains but with selective deletion of the multiple helical bundles within the central region of talin. By using a siRNA-mediated knockdown/rescue approach, we found that such talin analogs were able to support FA formation, clustering of activated integrins, and linkages to the actin cytoskeleton. By mapping the z-position of the FPs tagged at either the N or the C termini, we show that talin and its analogs are linearly extended and oriented in FAs, with their lengths regulating FA nanoscale organization. Chimeric-talin analogs with a 30-nm spacer insertion are also able to support FA assembly, facilitating the precise determination of talin geometry in FAs. Our results indicate that talin is oriented at 15° relative to the plasma membrane, measuring ∼97 nm end to end. FA nanoscale architecture in vinculin-null mouse embryonic fibroblasts (MEFs) retained its stratified organization and talin polarization similar to that in other cell types, suggesting that vinculin is dispensable for the specification of FA architecture. Our measurements demonstrate how the integrin–talin–actin module serves as the primary, and surprisingly modular, structural and tension-bearing core of FAs and geometrically define how such complexes could integrate multiple cellular forces at adhesion sites. |
