The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.     

Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.     

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.     

The N-terminal domains of talin cooperate with the phosphotyrosine binding-like domain to activate beta1 and beta3 integrins

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.     

Structural basis of integrin activation by talin

Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.     

The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.     

An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is thought to have two roles: mediating inside-out activation of integrins, and connecting extracellular matrix (ECM)-bound integrins to the cytoskeleton. Talin's amino-terminal head, which consists of a FERM domain, binds an NPxY motif within the cytoplasmic tail of most integrin beta subunits. This is consistent with the role of FERM domains in recruiting other proteins to the plasma membrane. We tested the role of the talin-head-NPxY interaction in integrin function in Drosophila. We found that introduction of a mutation that perturbs this binding in vitro into the isolated talin head disrupts its recruitment by integrins in vivo. Surprisingly, when engineered into the full-length talin, this mutation did not disrupt talin recruitment by integrins nor its ability to connect integrins to the cytoskeleton. However, it reduced the ability of talin to strengthen integrin adhesion to the ECM, indicating that the function of the talin-head-NPxY interaction is solely to regulate integrin adhesion.     

A Spatial Model for Integrin Clustering as a Result of Feedback between Integrin Activation and Integrin Binding

Integrins are transmembrane adhesion receptors that bind extracellular matrix (ECM) proteins and signal bidirectionally to regulate cell adhesion and migration. In many cell types, integrins cluster at cell-ECM contacts to create the foundation for adhesion complexes that transfer force between the cell and the ECM. Even though the temporal and spatial regulation of these integrin clusters is essential for cell migration, how cells regulate their formation is currently unknown. It has been shown that integrin cluster formation is independent of actin stress fiber formation, but requires active (high-affinity) integrins, phosphoinositol-4,5-bisphosphate (PIP2), talin, and immobile ECM ligand. Based on these observations, we propose a minimal model for initial formation of integrin clusters, facilitated by localized activation and binding of integrins to ECM ligands as a result of biochemical feedback between integrin binding and integrin activation. By employing a diffusion-reaction framework for modeling these reactions, we show how spatial organization of bound integrins into clusters may be achieved by a local source of active integrins, namely protein complexes formed on the cytoplasmic tails of bound integrins. Further, we show how such a mechanism can turn small local increases in the concentration of active talin or active integrin into integrin clusters via positive feedback. Our results suggest that the formation of integrin clusters by the proposed mechanism depends on the relationships between production and diffusion of integrin-activating species, and that changes to the relative rates of these processes may affect the resulting properties of integrin clusters.     

The Structure of the talin head reveals a novel extended conformation of the FERM domain

FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.     

Competition for talin results in trans-dominant inhibition of integrin activation

The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin beta subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i). beta tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii). trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii). expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type Igamma-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.     

Differences in regulation of Drosophila and vertebrate integrin affinity by talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2betaPS with those of human alphaIIbbeta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2betaPS with those of alphaIIbbeta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIbbeta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2betaPS affinity because of structural features inherent in the alphaPS2betaPS extracellular and/or transmembrane domains.     

Structure of a double ubiquitin‐like domain in the talin head: a role in integrin activation

Talin is a 270‐kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N‐terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β‐integrin tails, F0 and F1 are also required for activation of β1‐integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin‐like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin‐mediated activation of β1‐integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.     

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.     

Recreation of the terminal events in physiological integrin activation

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin αIIbβ3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.     

Differential Binding of Active and Inactive Integrin to Talin

Bi-directional signaling of integrins plays an important role in platelet and leukocyte function. Talin plays a key role in integrin bi-directional signaling and its binding to integrin is highly regulated. The precise regulation of the recruitment and binding of talin to integrin is still being elucidated. In particular, the recruitment of talin to integrin is controlled by the RAP-1 and RIAM/lamellipodin signaling axis and the affinity between talin and integrin is regulated by the conformation or protease cleavage of talin. However, whether the binding between integrin and talin is also regulated by integrin conformation has not been thoroughly explored before. In this work, we used biochemical binding assays to study the potential role of integrin conformational changes in integrin–talin interactions. Constitutively active integrin αIIbb3 binds markedly stronger to talin than inactive αIIbb3. Inactive αIIbb3 markedly increases its binding to talin once activated, regardless of how αIIbb3 is activated. Further, the increased binding to talin is b3 tail dependent. Our results suggest that integrin conformation is another regulatory mechanism for integrin–talin interaction.     

Structural diversity in integrin/talin interactions

The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.     

Mechanisms of talin-dependent integrin signaling and crosstalk

Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins. Extracellular or intracellular signals emanating from different signaling cascades cause inside-out signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton. Various integrin subfamilies communicate with each other and growth factor receptors under diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an essential mediator of integrin activation, much of the integrin crosstalk would therefore be influenced by talin. However, despite the existence of an extensive body of knowledge on the role of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in regulating inter-integrin communication is limited. This review will focus on the structure of talin, its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

The structure of an integrin/talin complex reveals the basis of inside‐out signal transduction

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside‐out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the β integrin subunit. Here, we report the first structure of talin bound to an authentic full‐length β integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane–proximal helix of the β tail disrupts an integrin α/β salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside‐out TM signalling.     

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.