-
The Journal of Biological Chemistry Jun 2006Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to...
Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.
Topics: Fluorescence; Humans; Integrin beta1; Mutation; Protein Binding; Protein Conformation; Talin; Vinculin
PubMed: 16608855
DOI: 10.1074/jbc.M600738200 -
Nature Communications Jun 2020Cells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic...
Cells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic vinculin-binding sites, triggers this process. The binding of RIAM to talin could regulate this mechanism. However, the mechanosensitivity of the talin-RIAM complex has never been tested. It is also not known whether RIAM controls the mechanosensitivity of the talin-vinculin complex. To address these issues, we designed an in vitro microscopy assay with purified proteins in which the actomyosin force controls RIAM and vinculin-binding to talin. We demonstrate that actomyosin triggers RIAM dissociation from several talin domains. Actomyosin also provokes the sequential exchange of RIAM for vinculin on talin. The effect of RIAM on this force-dependent binding of vinculin to talin varies from one talin domain to another. This mechanism could allow talin to biochemically code a wide range of forces by selecting different combinations of partners.
Topics: Actomyosin; Adaptor Proteins, Signal Transducing; Animals; Genes, Reporter; Luminescent Proteins; Membrane Proteins; Microscopy, Fluorescence; Molecular Imaging; Muscle, Skeletal; Rabbits; Recombinant Proteins; Talin; Vinculin
PubMed: 32561773
DOI: 10.1038/s41467-020-16922-1 -
Structure (London, England : 1993) Jul 2004Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large...
Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.
Topics: Animals; Binding Sites; Cell Adhesion; Chickens; Crystallography, X-Ray; Humans; Models, Molecular; Protein Structure, Secondary; Protein Structure, Tertiary; Vinculin
PubMed: 15242595
DOI: 10.1016/j.str.2004.05.009 -
European Journal of Cell Biology Jun 2024Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and...
Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs.
Topics: alpha Catenin; Humans; Protein Serine-Threonine Kinases; Vinculin; Mechanotransduction, Cellular; Adaptor Proteins, Signal Transducing; Intercellular Junctions; HEK293 Cells; rho-Associated Kinases; Transcription Factors
PubMed: 38805800
DOI: 10.1016/j.ejcb.2024.151426 -
Structure (London, England : 1993) Feb 2017Vinculin, a scaffolding protein that localizes to focal adhesions (FAs) and adherens junctions, links the actin cytoskeleton to the adhesive super-structure. While...
Vinculin, a scaffolding protein that localizes to focal adhesions (FAs) and adherens junctions, links the actin cytoskeleton to the adhesive super-structure. While vinculin binds to a number of cytoskeletal proteins, it can also associate with phosphatidylinositol 4,5-bisphosphate (PIP) to drive membrane association. To generate a structural model for PIP-dependent interaction of vinculin with the lipid bilayer, we conducted lipid-association, nuclear magnetic resonance, and computational modeling experiments. We find that two basic patches on the vinculin tail drive membrane association: the basic collar specifically recognizes PIP, while the basic ladder drives association with the lipid bilayer. Vinculin mutants with defects in PIP-dependent liposome association were then expressed in vinculin knockout murine embryonic fibroblasts. Results from these analyses indicate that PIP binding is not required for localization of vinculin to FAs or FA strengthening, but is required for vinculin activation and turnover at FAs to promote its association with the force transduction FA nanodomain.
Topics: Actin Cytoskeleton; Actins; Amino Acid Motifs; Animals; Binding Sites; Embryo, Mammalian; Fibroblasts; Focal Adhesions; Gene Expression; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Mechanotransduction, Cellular; Mice; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Recombinant Proteins; Thermodynamics; Vinculin
PubMed: 28089450
DOI: 10.1016/j.str.2016.12.002 -
Open Biology Jun 2023Adhesion between cells and the extracellular matrix is mediated by heterodimeric () integrin receptors that are intracellularly linked to the contractile actomyosin...
Adhesion between cells and the extracellular matrix is mediated by heterodimeric () 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.
Topics: Actins; Focal Adhesions; Talin; Actomyosin; Cell Adhesion; Cytoskeleton; Vinculin; Integrins; Microtubules
PubMed: 37339751
DOI: 10.1098/rsob.230058 -
Proceedings of the National Academy of... Aug 2016The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due...
The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2 Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling.
Topics: Amino Acid Sequence; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Gene Expression; Genetic Vectors; Humans; Models, Molecular; Mutation; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Isoforms; Protein Multimerization; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Thermodynamics; Vinculin
PubMed: 27503891
DOI: 10.1073/pnas.1600702113 -
Scientific Reports Jan 2021Focal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were...
Focal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.
Topics: Animals; Cell Movement; Cytoskeleton; Focal Adhesions; Immunity, Cellular; Paxillin; Vinculin; Zyxin
PubMed: 33504939
DOI: 10.1038/s41598-021-81898-x -
Biophysical Journal Oct 2017Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The...
Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The lifecycle of a focal adhesion is a highly coordinated process involving spatial and temporal variations of protein composition, interaction, and cellular tension. The assembly of focal adhesions requires the recruitment and activation of vinculin. Vinculin is present in the cytoplasm in an autoinhibited conformation in which its tail is held pincerlike by its head domains, further stabilized by two high-affinity head-tail interfaces. Vinculin has binding sites for talin and F-actin, but effective binding requires vinculin activation to release its head-tail associations. In migrating cells, it has been shown that the locations of vinculin activation coincide with areas of high cellular tension, and that the highest recorded tensions across vinculin are associated with adhesion assembly. Here, we use a structure-based model to investigate vinculin activation by talin modulated by tensile force generated by transient associations with F-actin. We show that vinculin activation may proceed from an intermediate state stabilized by partial talin-vinculin association. There is a low-force regime and a high-force regime where vinculin activation is dominated by two different pathways with distinct responses to force. Specifically, at zero or low forces, vinculin activation requires substantial destabilization of the main head-tail interface, which is rigid and undergoes very limited fluctuations, despite the other being relatively flexible. This pathway is not significantly affected by force; instead, higher forces favor an alternative pathway, which seeks to release the vinculin tail from its pincerlike head domains before destabilizing the head-tail interfaces. This pathway has a force-sensitive activation barrier and is significantly accelerated by force. Experimental data of vinculin during various stages of the focal adhesion lifecycle are consistent with the proposed force-regulated activation pathway.
Topics: Actins; Biomechanical Phenomena; Computer Simulation; Cytoplasm; Focal Adhesions; Humans; Kinetics; Models, Molecular; Protein Domains; Protein Stability; Protein Structure, Secondary; Talin; Vinculin
PubMed: 29045864
DOI: 10.1016/j.bpj.2017.08.037 -
Proceedings of the National Academy of... Dec 2020Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the...
Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the "focal adhesion clutch," in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we measured force on talin together with actin flow speed. While force on talin in small lamellipodial adhesions correlated with actin flow, talin tension in large adhesions further from the cell edge was mainly flow-independent. Stiff substrates shifted force transfer toward the flow-independent mechanism. Flow-dependent force transfer required talin's C-terminal actin binding site, ABS3, but not vinculin. Flow-independent force transfer initially required vinculin and at later times the central actin binding site, ABS2. Force transfer through integrins thus occurs not through a continuous clutch but through a series of discrete states mediated by distinct protein interactions, with their ratio modulated by substrate stiffness.
Topics: Actins; Animals; Binding Sites; Fluorescence Resonance Energy Transfer; Focal Adhesions; Integrins; Mice; Mutation; NIH 3T3 Cells; Talin; Time-Lapse Imaging; Vinculin
PubMed: 33262280
DOI: 10.1073/pnas.2010292117