Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.

Relief of talin autoinhibition triggers a force-independent association with vinculin.

Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are autoinhibited and do not interact with each other. However, contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of autoinhibition is sufficient to mediate their tight interaction. We also found that paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrated that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force machinery and focal adhesion maturation.

Endothelial Progenitor Cells Enhance the Migration and Osteoclastic Differentiation of Bone Marrow-Derived Macrophages in vitro and in a Mouse Femur Fracture Model through Talin-1.

BACKGROUND/AIMS: Bone resorption mediated by osteoclasts plays an important role in bone healing. Endothelial progenitor cells (EPCs) promote bone repair by stimulating neovascularization and osteogenesis. However, the role of EPCs in osteoclast formation and function is not well defined. The aim of this study was to elucidate mechanisms of EPCs in osteoclast formation and function. METHODS: In this study, we examined the effects of EPCs on the proliferation, migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in a co-culture system in vitro. We also evaluated the effects of EPC co-transplantation on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. The technology of immunofluorescence, immunohistochemical, western blot, Rt-PCR, cell co-culture and Transwell were used in this study. RESULTS: EPCs secreted TGF-ß1 in the EPC-BMM co-culture medium and increased Talin-1 expression in the co-cultured BMMs. Treatment with a TGF-ß1 neutralizing antibody or Talin-1 silencing in BMMs completely inhibited BMM osteoclastic differentiation in the co-culture system. These results indicated that the osteoclastogenic effects of EPCs were mediated by TGF-ß1-mediated Talin-1 expression in BMMs. In the femur fracture model, BMMs co-transplanted with EPCs exhibited enhanced engraftment into the fracture site and osteoclastic differentiation compared with those transplanted alone. Mice treated with EPC-BMM co-transplantation exhibited increased neovascularization at the fracture site and accelerated fracture healing compared with those treated with BMMs alone. CONCLUSION: Taken together, the results suggest that EPCs can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Gα13 Switch Region 2 Relieves Talin Autoinhibition to Activate αIIbß3 Integrin.

Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0-F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (Gα13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of Gα13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows Gα13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo Altogether, we present a mechanistic basis for the regulation of talin through Gα13.

Airway Hyperresponsiveness in Asthma Model Occurs Independently of Secretion of ß1 Integrins in Airway Wall and Focal Adhesions Proteins Down Regulation.

The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with ß1 integrins (α1ß1 and α2ß1) in guinea pig. The putative role of ß1 integrin subunit alterations due to shedding in airway smooth muscle (ASM) in an allergic asthma model was evaluated. Guinea pigs were sensitized and challenged with antigen. Antigenic challenges induced bronchoobstruction and hyperresponsiveness at the third antigenic challenge. Immunohistochemistry and immunoelectronmicroscopy studies showed that the cytosolic and extracellular domains of the ß1 integrin subunit shared the same distribution in airway structures in both groups. Various polypeptides with similar molecular weights were detected with both the cytosolic and extracellular ß1 integrin subunit antibodies in isolated airway myocytes and the connective tissue that surrounds the ASM bundle. Flow cytometry and Western blot studies showed that the expression of cytosolic and extracellular ß1 integrin subunit domains in ASM was similar between groups. An increment of ITGB1 mRNA in ASM was observed in the asthma model group. RACE-PCR of ITGB1 in ASM did not show splicing variants. The expression levels of integrin-linked kinase (ILK) and paxillin diminished in the asthma model, but not talin. The levels of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr(696) increased in asthma model. Our work suggests that ß1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, ß1 integrin cytodomain assembly proteins in focal cell adhesions in which ILK and paxillin are involved are altered in asthma model. J. Cell. Biochem. 117: 2385-2396, 2016. © 2016 Wiley Periodicals, Inc.

Talin1 targeting potentiates anti-angiogenic therapy by attenuating invasion and stem-like features of glioblastoma multiforme.

Glioblastoma multiforme (GBM) possesses florid angiogenesis. However, the anti-angiogenic agent, Bevacizumab, did not improve overall survival of GBM patients. For more durable anti-angiogenic treatment, we interrogated resistant mechanisms of GBM against Bevacizumab. Serial orthotopic transplantation of in vivo Bevacizumab-treated GBM cells provoked complete refractoriness to the anti-angiogenic treatment. These tumors were also highly enriched with malignant phenotypes such as invasiveness, epithelial to mesenchymal transition, and stem-like features. Through transcriptome analysis, we identified that Talin1 (TLN1) significantly increased in the refractory GBMs. Inhibition of TLN1 not only attenuated malignant characteristics of GBM cells but also reversed the resistance to the Bevacizumab treatment. These data implicate TLN1 as a novel therapeutic target for GBM to overcome resistance to anti-angiogenic therapies.

The conformational states of talin autoinhibition complex and its activation under forces.

Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.

Non-monotonic cellular responses to heterogeneity in talin protein expression-level.

Talin is a key cell-matrix adhesion component with a central role in regulating adhesion complex maturation, and thereby various cellular properties including adhesion and migration. However, knockdown studies have produced inconsistent findings regarding the functional influence of talin in these processes. Such discrepancies may reflect non-monotonic responses to talin expression-level variation that are not detectable via canonical "binary" comparisons of aggregated control versus knockdown cell populations. Here, we deployed an "analogue" approach to map talin influence across a continuous expression-level spectrum, which we extended with sub-maximal RNAi-mediated talin depletion. Applying correlative imaging to link live cell and fixed immunofluorescence data on a single cell basis, we related per cell talin levels to per cell measures quantitatively defining an array of cellular properties. This revealed both linear and non-linear correspondences between talin expression and cellular properties, including non-monotonic influences over cell shape, adhesion complex-F-actin association and adhesion localization. Furthermore, we demonstrate talin level-dependent changes in networks of correlations among adhesion/migration properties, particularly in relation to cell migration speed. Importantly, these correlation networks were strongly affected by talin expression heterogeneity within the natural range, implying that this endogenous variation has a broad, quantitatively detectable influence. Overall, we present an accessible analogue method that reveals complex dependencies on talin expression-level, thereby establishing a framework for considering non-linear and non-monotonic effects of protein expression-level heterogeneity in cellular systems.

Signalling complexes at the cell-matrix interface.

The extracellular matrix critically controls cell behaviour. Many cell-matrix interactions are mediated by transmembrane receptors of the integrin family. In the last two years, the structural changes resulting from ligand binding to integrins α5ß1, αvß3 and αIIbß3 have been mapped in unprecedented detail. The structure of integrin αXß2 has revealed how ligand binding to the α I domain is transmitted to the rest of the ectodomain. The structural characterisation of the cytosolic regulator talin has been continued, revealing how the integrin binding site is blocked in auto-inhibited talin. Finally, structures of the discoidin domain receptors DDR1 and DDR2 have begun to reveal how these atypical receptor tyrosine kinases become activated by the major matrix component collagen.

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation.

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1-deficient neutrophils, whereas only arrest is defective in kindlin-3-deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Structural basis for the autoinhibition of talin in regulating integrin activation.

Activation of heterodimeric (alpha/beta) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin beta membrane-proximal cytoplasmic tails (beta-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin beta-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.

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.

The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway.

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and possesses apoptotic and tumor-suppressive functions. However, it is unclear whether DAPK elicits apoptosis-independent activity to suppress tumor progression. We show that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These effects are mainly mediated by an inhibitory role of DAPK in talin head domain association with integrin, thereby suppressing the integrin-Cdc42 polarity pathway. We present evidence indicating that the antimigratory effect of DAPK represents a mechanism through which DAPK suppresses tumors. First, DAPK can block migration and invasion in certain tumor cells that are resistant to DAPK-induced apoptosis. Second, using an adenocarcinoma cell line and its highly invasive derivative, we demonstrate DAPK level as a determining factor in tumor invasiveness. Collectively, our study identifies a novel function of DAPK in regulating cell polarity during migration, which may act together with its apoptotic function to suppress tumor progression.

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.

Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans.

Integrin receptors for extracellular matrix transmit mechanical and biochemical information through molecular connections to the actin cytoskeleton and to several intracellular signaling pathways. In Caenorhabditis elegans, integrins are essential for embryonic development, muscle cell adhesion and contraction, and migration of nerve cell axons and gonadal distal tip cells. To identify key components involved in distal tip cell migration, we are using an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. We have found that talin, a cytoskeletal-associated protein and focal adhesion component, is expressed in the distal tip cell and plays a central role in regulating its migration. Reduction of talin expression caused severe defects in gonad formation because of aberrant distal tip cell migration and also disrupted oocyte maturation and gonad sheath cell structure. Contractile muscle cells showed disorganization of the actin cytoskeleton leading to complete paralysis, a phenotype that was also observed with depletion of pat-2 and pat-3 integrins. These in vivo analyses show that talin is required not only for strong adhesion and cytoskeletal organization by contractile cells, but also for dynamic regulation of integrin signals during cell migration. In addition, induction of distal tip cell migration defects by bacterial RNAi in C. elegans provides an effective screen to identify genes involved in integrin signaling and function.