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 ß-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.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

SnapShot: talin and the modular nature of the integrin adhesome.

Integrin αß transmembrane heterodimers play central roles in metazoan development and physiology by mediating adhesion and by transmitting forces and biochemical signals across the plasma membrane. In this SnapShot, we present a simplified "modular" view of the integrin adhesome, centered on the talin-integrin interaction, and provide examples of how this view can help to unravel the adhesome's remarkable functional diversity and plasticity.

Molecular dynamics simulations reveal how vinculin refolds partially unfolded talin rod helices to stabilize them against mechanical force.

Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin-talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork.

The C-terminal actin-binding domain of talin forms an asymmetric catch bond with F-actin.

SignificanceTalin is a mechanosensitive adaptor protein that links integrins to the actin cytoskeleton at cell-extracellular matrix adhesions. Although the C-terminal actin-binding domain ABS3 of talin is required for function, it binds weakly to actin in solution. We show that ABS3 binds actin strongly only when subjected to mechanical forces comparable to those generated by the cytoskeleton. Moreover, the interaction between ABS3 and actin depends strongly on the direction of force in a manner predicted to organize actin to facilitate adhesion growth and efficient cytoskeletal force generation. These characteristics can explain how force sensing by talin helps to nucleate adhesions precisely when and where they are required to transmit force between the cytoskeleton and the extracellular matrix.

Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms.

Adhesion of cells to the extracellular matrix (ECM) must be exquisitely coordinated to enable development and tissue homeostasis. Cell-ECM interactions are regulated by multiple signalling pathways that coordinate the activation state of the integrin family of ECM receptors. The protein talin is pivotal in this process, and talin's simultaneous interactions with the cytoplasmic tails of the integrins and the plasma membrane are essential to enable robust, dynamic control of integrin activation and cell-ECM adhesion. Here, we report the identification of a de novo heterozygous c.685C>T (p.Pro229Ser) variant in the TLN1 gene from a patient with a complex phenotype. The mutation is located in the talin head region at the interface between the F2 and F3 domains. The characterization of this novel p.P229S talin variant reveals the disruption of adhesion dynamics that result from disturbance of the F2-F3 domain interface in the talin head. Using biophysical, computational and cell biological techniques, we find that the variant perturbs the synergy between the integrin-binding F3 and the membrane-binding F2 domains, compromising integrin activation, adhesion and cell migration. Whilst this remains a variant of uncertain significance, it is probable that the dysregulation of adhesion dynamics we observe in cells contributes to the multifaceted clinical symptoms of the patient and may provide insight into the multitude of cellular processes dependent on talin-mediated adhesion dynamics.

Quantum Dot-Based FRET Nanosensors for Talin-Membrane Assembly and Mechanosensing.

Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter- and intra-molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin-membrane distance of 2.5 nm. We demonstrate that QD-FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub-nanometer accuracy.

Biphasic reinforcement of nascent adhesions by vinculin.

Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.

Crystal structure of the FERM-folded talin head reveals the determinants for integrin binding.

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Tension Enhances the Binding Affinity of ß1 Integrin by Clamping Talin Tightly: An Insight from Steered Molecular Dynamics Simulations.

Integrin activation is a predominant step for cell-cell and cell-ECM interactions. Talin and Kindlin are mechanosensitive adaptor proteins that bind to the integrin cytoplasmic tail and mediate integrin activation, cytoskeleton rearrangement, and focal adhesion assembly. However, knowledge about how Talin and Kindlin synergistically assist integrin activation remains unclear. Here, we performed so-called "ramp-clamp" SMD simulations, which modeled the mechanosignaling from Kindlin, to investigate the effect of tension on the interaction of the ß1 integrin cytoplasmic tail with the Talin-F3 domain. The present results showed that mild but not excessive stretching enhanced the binding of integrin with Talin. This mechanical regulation on integrin affinity to Talin referred to an event cascade, in which under stretching, the integrin cytoplasmic tail adopted allostery in response to the mechanical stimulus, remodeling of integrin in favor of Talin-association ensued, and finally, a stable, close-knit complex was formed. In the cascade, the torsion angle transition of integrin was the cue for the stable interaction of the complex under tensile force. The present work suggested a model for Talin and Kindlin to synergistically activate integrin. It should help understand integrin activation and its mechanochemical regulation mechanism, integrin-related innate cellular immune responses, cell adhesion, cell-cell interaction, and integrin-related drug development.

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.

Peptide-PAINT Enables Investigation of Endogenous Talin with Molecular Scale Resolution in Cells and Tissues.

Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10 nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions.

Cyanidin-3-glucoside binds to talin and modulates colon cancer cell adhesions and 3D growth.

Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with ß1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-ß-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.

Mechanotransduction in talin through the interaction of the R8 domain with DLC1.

The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes.

MAP4K4 regulates integrin-FERM binding to control endothelial cell motility.

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

The interaction of talin with the cell membrane is essential for integrin activation and focal adhesion formation.

Multicellular organisms have well-defined, tightly regulated mechanisms for cell adhesion. Heterodimeric αß integrin receptors play central roles in this function and regulate processes for normal cell functions, including signaling, cell migration, and development, binding to the extracellular matrix, and senescence. They are involved in hemostasis and the immune response, participate in leukocyte function, and have biological implications in angiogenesis and cancer. Proper control of integrin activation for cellular communication with the external environment requires several physiological processes. Perturbation of these equilibria may lead to constitutive integrin activation that results in bleeding disorders. Furthermore, integrins play key roles in cancer progression and metastasis in which certain tumor types exhibit higher levels of various integrins. Thus, the integrin-associated signaling complex is important for cancer therapy development. During inside-out signaling, the cytoskeletal protein talin plays a key role in regulating integrin affinity whereby the talin head domain activates integrin by binding to the cytoplasmic tail of ß-integrin and acidic membrane phospholipids. To understand the mechanism of integrin activation by talin, we determined the crystal structure of the talin head domain bound to the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), allowing us to design a lipid-binding-deficient talin mutant. Our confocal microscopy with talin knockout cells suggests that the talin-cell membrane interaction seems essential for focal adhesion formation and stabilization. Basal integrin activation in Chinese hamster ovary cells suggests that the lipid-binding-deficient talin mutant inhibits integrin activation. Thus, membrane attachment of talin seems necessary for integrin activation and focal adhesion formation.

Increased talin-vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections.

Talin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter protein to mediate cytoskeleton-driven dynamics of the plasma membrane. However, little is known regarding the potential role of the talin-vinculin complex during spotted fever group rickettsial and Ebola virus infections, two dreadful infectious diseases in humans. Many functional properties of proteins are determined by their participation in protein-protein complexes, in a temporal and/or spatial manner. To resolve the limitation of application in using mouse primary antibodies on archival, multiple formalin-fixed mouse tissue samples, which were collected from experiments requiring high biocontainment, we developed a practical strategic proximity ligation assay (PLA) capable of employing one primary antibody raised in mouse to probe talin-vinculin spatial proximal complex in mouse tissue. We observed an increase of talin-vinculin spatial proximities in the livers of spotted fever Rickettsia australis or Ebola virus-infected mice when compared with mock mice. Furthermore, using EPAC1-knockout mice, we found that deletion of EPAC1 could suppress the formation of spatial proximal complex of talin-vinculin in rickettsial infections. In addition, we observed increased colocalization between spatial proximity of talin-vinculin and filamentous actin-specific phalloidin staining in single survival mouse from an ordinarily lethal dose of rickettsial or Ebola virus infection. These findings may help to delineate a fresh insight into the mechanisms underlying liver specific pathogenesis during infection with spotted fever rickettsia or Ebola virus in the mouse model.

Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1.

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3-5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.