Talin Binding Research Articles

Ligand binding initiates single-molecule integrin conformational activation

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin β-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

FAK, vinculin, and talin control mechanosensitive YAP nuclear localization

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Talin and kindlin use integrin tail allostery and direct binding to activate integrins

Integrin affinity regulation, also termed integrin activation, is essential for metazoan life. Although talin and kindlin binding to the β-integrin cytoplasmic tail is indispensable for integrin activation, it is unknown how they achieve this function. By combining NMR, biochemistry and cell biology techniques, we found that talin and kindlin binding to the β-tail can induce a conformational change that increases talin affinity and decreases kindlin affinity toward it. We also discovered that this asymmetric affinity regulation is accompanied by a direct interaction between talin and kindlin, which promotes simultaneous binding of talin and kindlin to β-tails. Disrupting allosteric communication between the β-tail-binding sites of talin and kindlin or their direct interaction in cells severely compromised integrin functions. These data show how talin and kindlin cooperate to generate a small but critical population of ternary talin–β-integrin–kindlin complexes with high talin–integrin affinity and high dynamics.

Allosteric activation of vinculin by talin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s−1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The ‘allosteric vinculin mutant’ is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

Kindlin stabilizes the talin·integrin bond under mechanical load by generating an ideal bond

Integrin-mediated adhesion is essential for metazoan life. Integrin binding to ligand requires an activation step prior to binding ligand that depends on direct binding of talin and kindlin to the β-integrin cytoplasmic tail and the transmission of force from the actomyosin via talin to the integrin-ligand bonds. However, the affinity of talin for integrin tails is low. It is therefore still unclear how such low-affinity bonds are reinforced to transmit forces up to 10 to 40 pN. In this study, we use single-molecule force spectroscopy by optical tweezers to investigate the mechanical stability of the talin•integrin bond in the presence and absence of kindlin. While talin and integrin alone form a weak and highly dynamic slip bond, the addition of kindlin-2 induces a force-independent, ideal talin•integrin bond, which relies on the steric proximity of and the intervening amino acid sequences between the talin- and kindlin-binding sites in the β-integrin tail. Our findings show how kindlin cooperates with talin to enable transmission of high forces required to stabilize cell adhesion.

The structural basis of the talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions.

Adhesion between cells and the extracellular matrix is mediated by heterodimeric (αβ) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organizes cytosolic signalling proteins into discrete complexes on β-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we adapted a non-covalent crystallographic chaperone to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN region of KANK1 contains a novel motif where a β-hairpin stabilizes the α-helical region, explaining both its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localizes throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.

LRP12 is an endogenous transmembrane inactivator of α4 integrins

Dynamic regulation of integrin activation and inactivation is critical for precisely controlled cell adhesion and migration in physiological and pathological processes. The molecular basis for integrin activation has been intensively studied; however, the understanding of integrin inactivation is still limited. Here, we identify LRP12 as an endogenous transmembrane inhibitor for α4 integrin activation. The LRP12 cytoplasmic domain directly binds to the integrin α4 cytoplasmic tail and inhibits talin binding to the β subunit, thus keeping integrin inactive. In migrating cells, LRP12-α4 interaction induces nascent adhesion (NA) turnover at the leading-edge protrusion. Knockdown of LRP12 leads to increased NAs and enhanced cell migration. Consistently, LRP12-deficient Tcells show an enhanced homing capability in mice and lead to aggravated chronic colitis in a Tcell-transfer colitis model. Altogether, LRP12 is a transmembrane inactivator for integrins that inhibits α4 integrin activation and controls cell migration by maintaining balanced NA dynamics.

An integrin binding motif in TLN-1/talin plays a minor role in motility and ovulation.

TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cytoplasmic tail of integrin and the actin cytoskeleton. In C. elegans , TLN-1 is expressed strongly in striated muscle and the gonadal sheath cells. Here, we report that a CRISPR-generated TLN-1 allele TLN-1(W387A), predicted to affect binding of talin to integrins, results in mild phenotypes, including motility defects and ovulation defects. The arrangement of the actin cytoskeleton in the body wall muscles, spermatheca, and sheath appears identical in wild type and TLN-1(W387A) animals. This analysis suggests that W387 in TLN-1 does not have a major effect on the binding of talin to integrin in vivo .

New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain.

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Integrin activation is an essential component of SARS-CoV-2 infection

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's β subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

The Cryogenic Electron Microscopy Structure of the Cell Adhesion Regulator Metavinculin Reveals an Isoform-Specific Kinked Helix in Its Cytoskeleton Binding Domain.

Vinculin and its heart-specific splice variant metavinculin are key regulators of cell adhesion processes. These membrane-bound cytoskeletal proteins regulate the cell shape by binding to several other proteins at cell–cell and cell–matrix junctions. Vinculin and metavinculin link integrin adhesion molecules to the filamentous actin network. Loss of both proteins prevents cell adhesion and cell spreading and reduces the formation of stress fibers, focal adhesions, or lamellipodia extensions. The binding of talin at cell–matrix junctions or of α-catenin at cell–cell junctions activates vinculin and metavinculin by releasing their autoinhibitory head–tail interaction. Once activated, vinculin and metavinculin bind F-actin via their five-helix bundle tail domains. Unlike vinculin, metavinculin has a 68-amino-acid insertion before the second α-helix of this five-helix F-actin–binding domain. Here, we present the full-length cryogenic electron microscopy structure of metavinculin that captures the dynamics of its individual domains and unveiled a hallmark structural feature, namely a kinked isoform-specific α-helix in its F-actin-binding domain. Our identified conformational landscape of metavinculin suggests a structural priming mechanism that is consistent with the cell adhesion functions of metavinculin in response to mechanical and cellular cues. Our findings expand our understanding of metavinculin function in the heart with implications for the etiologies of cardiomyopathies.

PPM1F controls integrin activity via a conserved phospho-switch.

Control of integrin activity is vital during development and tissue homeostasis, while derailment of integrin function contributes to pathophysiological processes. Phosphorylation of a conserved threonine motif (T788/T789) in the integrin β cytoplasmic domain increases integrin activity. Here, we report that T788/T789 functions as a phospho-switch, which determines the association with either talin and kindlin-2, the major integrin activators, or filaminA, an integrin activity suppressor. A genetic screen identifies the phosphatase PPM1F as the critical enzyme, which selectively and directly dephosphorylates the T788/T789 motif. PPM1F-deficient cell lines show constitutive integrin phosphorylation, exaggerated talin binding, increased integrin activity, and enhanced cell adhesion. These gain-of-function phenotypes are reverted by reexpression of active PPM1F, but not a phosphatase-dead mutant. Disruption of the ppm1f gene in mice results in early embryonic death at day E10.5. Together, PPM1F controls the T788/T789 phospho-switch in the integrin β1 cytoplasmic tail and constitutes a novel target to modulate integrin activity.

Talin-dependent integrin activation is required for endothelial proliferation and postnatal angiogenesis.

Integrin activation contributes to key blood cell functions including adhesion, proliferation and migration. An essential step in the cell signaling pathway that activates integrin requires the binding of talin to the β-integrin cytoplasmic tail. Whereas this pathway is understood in platelets in detail, considerably less is known regarding how integrin-mediated adhesion in endothelium contributes to postnatal angiogenesis. We utilized an inducible EC-specific talin1 knock-out mouse (Tln1 EC-KO) and talin1 L325R knock-in mutant (Tln1 L325R) mouse, in which talin selectively lacks the capacity to activate integrins, to assess the role of integrin activation during angiogenesis. Deletion of talin1 during postnatal days 1-3 (P1-P3) caused lethality by P8 with extensive defects in retinal angiogenesis and widespread hemorrhaging. Tln1 EC-KO mice displayed reduced retinal vascular area, impaired EC sprouting and proliferation relative to Tln1 CTRLs. In contrast, induction of talin1 L325R in neonatal mice resulted in modest defects in retinal angiogenesis and mice survived to adulthood. Interestingly, deletion of talin1 or expression of talin1 L325R in ECs increased MAPK/ERK signaling. Strikingly, B16-F0 tumors grown in Tln1 L325R adult mice were 55% smaller and significantly less vascularized than tumors grown in littermate controls. EC talin1 is indispensable for postnatal development angiogenesis. The role of EC integrin activation appears context-dependent as its inhibition is compatible with postnatal development with mild defects in retinal angiogenesis but results in marked defects in tumor growth and angiogenesis. Inhibiting EC pan-integrin activation may be an effective approach to selectively target tumor blood vessel growth.

A distinct talin2 structure directs isoform specificity in cell adhesion

Integrin receptors regulate normal cellular processes such as signaling, cell migration, adhesion to the extracellular matrix, and leukocyte function. Talin recruitment to the membrane is necessary for its binding to and activation of integrin. Vertebrates have two highly conserved talin homologs that differ in their expression patterns. The F1-F3 FERM subdomains of cytoskeletal proteins resemble a cloverleaf, but in talin1, its F1 subdomain and additional F0 subdomain align more linearly with its F2 and F3 subdomains. Here, we present the talin2 crystal structure, revealing that its F0-F1 di-subdomain displays another unprecedented constellation, whereby the F0-F1-F2 adopts a new cloverleaf-like arrangement. Using multiangle light scattering (MALS), fluorescence lifetime imaging (FLIM), and FRET analyses, we found that substituting the corresponding residues in talin2 that abolish lipid binding in talin1 disrupt the binding of talin to the membrane, focal adhesion formation, and cell spreading. Our results provide the molecular details of the functions of specific talin isoforms in cell adhesion.

Ligand Binding to the Collagen VI Receptor Triggers a Talin-to-RhoA Switch that Regulates Receptor Endocytosis

Capillary morphogenesis gene 2 (CMG2/ANTXR2) is a cell surface receptor for both collagen VI and anthrax toxin. Biallelic loss-of-function mutations in CMG2 lead to a severe condition, hyaline fibromatosis syndrome (HFS). We have here dissected a network of dynamic interactions between CMG2 and various actin interactors and regulators, describing a different behavior from other extracellular matrix receptors. CMG2 binds talin, and thereby the actin cytoskeleton, only in its ligand-free state. Extracellular ligand binding leads to src-dependent talin release and recruitment of the actin cytoskeleton regulator RhoA and its effectors. These sequential interactions of CMG2 are necessary for the control of oriented cell division during fish development. Finally, we demonstrate that effective switching between talin and RhoA binding is required for the intracellular degradation of collagen VI in human fibroblasts, which explains why HFS mutations in the cytoskeleton-binding domain lead to dysregulation of extracellular matrix homeostasis.

Clustering of integrin \u03b2 cytoplasmic domains triggers nascent adhesion formation and reveals a protozoan origin of the integrin-talin interaction

Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130CAS, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.

Talin-Dependent Integrin Activation Regulates VE-Cadherin Localization and Endothelial Cell Barrier Function.

Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the β-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. To test the requirement of talin-dependent activation of β1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function. EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring β1-integrin activation in talin-deficient cells with a β1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins. Talin-dependent activation of EC β1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.

Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 sign pathway

Curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. inhibits platelet aggregation, suggesting its significant anticoagulant and antithrombotic effects. However, the mechanisms have not been fully elucidated. We hypothesized that curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 signaling pathway. We performed in vitro assays to evaluate the effect of curdione on thrombin-induced expression levels of the AMPK signaling molecule and integrin αIIbβ3 signaling pathway components. Platelet proteins were extracted from washed human platelets, and the effects of curdione on thrombin-induced platelet aggregation were evaluated. The expression levels of the AMPK signaling molecule and integrin αIIbβ3 signaling pathway-related proteins were examined using western blot and RT-PCR. The binding of vinculin and talin were studied using immunoprecipitation, double immunofluorescence staining and microscale thermophoresis. Platelet aggregation analysis showed that 0.02 U/ml thrombin significantly induces platelet aggregation. Western blot and RT-PCR analysis revealed that AMPK inhibits the vinculin/talin-mediated integrin αIIbβ3 signaling pathway, and curdione downregulates the thrombin-induced expression of phosphorylated AMPK (P-AMPK) and P-integrin at both the protein and mRNA levels and downregulates vinculin and talin at the protein level. Furthermore, microscale thermophoresis experiments showed that curdione inhibits the binding of vinculin and talin. The results from the immunoprecipitation and double immunofluorescence staining were consistent with the results of the microscale thermophoresis experiments. Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 signaling pathway, which suggests its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent thrombotic diseases.

Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5Å resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila.

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.