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.     

Structural determinants of integrin recognition by talin

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.     

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.     

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.     

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.     

Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.     

Distinct developmental roles for direct and indirect talin-mediated linkage to actin

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 essential to connect extracellular matrix-bound integrins to the cytoskeleton. Talin can connect to the cytoskeleton either directly, through its actin-binding motifs, or indirectly, by recruiting other actin-binding proteins. Talin's carboxy-terminal end contains a well-characterized actin-binding domain (ABD). We tested the role of the C-terminal ABD of talin in integrin function in Drosophila. We found that introduction of mutations that reduced actin binding in vitro into the isolated C-terminal Talin-ABD impaired actin binding in vivo. Moreover, when engineered into full-length talin, these mutations disrupted a subset of integrin-mediated adhesion-dependent developmental events. Specifically, morphogenetic processes that involve dynamic, short-term integrin-mediated adhesion were particularly sensitive to impaired function of the C-terminal Talin-ABD. We propose that during development talin connects integrins to the cytoskeleton in distinct ways in different types of integrin-mediated adhesion: directly in transient adhesions and indirectly in stable long-lasting adhesions. Our results provide insight into how a similar array of molecular components can contribute to diverse adhesive processes throughout development.     

Tensin stabilizes integrin adhesive contacts in Drosophila

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.     

A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin β tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin β subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in αIIbβ3 integrin activation, but not for kindlin binding to integrin β tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well β-integrin tails contribute to the ability of kindlins to activate integrins.     

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.     

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.     

The C terminus of talin links integrins to cell cycle progression

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell-matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

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.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

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.     

TA205, an anti-talin monoclonal antibody, inhibits integrin-talin interaction

Talin mediates integrin signaling by binding to integrin cytoplasmic tails through its FERM domain which consists of F1, F2 and F3 subdomains. TA205, an anti-talin monoclonal antibody, disrupts actin stress fibers and focal adhesion when microinjected into fibroblasts. Here, we showed that TA205 caused an allosteric inhibition of integrin alphaIIb beta3 binding to the talin FERM domain and mapped the TA205 epitope to residues 131-150 in talin F1. Furthermore, binding of a talin rod fragment to talin head was partially inhibited by TA205. These findings suggest that talin F1 may be important in regulation of integrin binding and talin head-rod interaction.     

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.     

Integrin adhesions: Who’s on first? What's on second?

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

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.