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Biophysical Journal Jan 2011Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and...
Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and strengthening of these focal adhesions is dependent on the interaction between talin and vinculin. This study investigates said interaction and how vinculin activation influences it. Using molecular dynamics, the interaction between talin's vinculin binding site (VBS) and vinculin's domain 1 (D1) is simulated both before and after vinculin activation. The simulations of VBS binding to vinculin before activation suggest the proximity of the vinculin tail to D1 prevents helical movement in D1 and thus prevents binding of VBS. In contrast, interaction of VBS with activated vinculin shows the possibility of complete VBS insertion into D1. In the simulations of both activated and autoinhibited vinculin where VBS fails to fully bind, VBS demonstrates significant hydrophobic interaction with surface residues in D1. These interactions link VBS to D1 even without its proper insertion into the hydrophobic core. Together these simulations suggest VBS binds to vinculin with the following mechanism: 1), VBS links to D1 via surface hydrophobic interactions; 2), vinculin undergoes activation and D1 is moved away from the vinculin tail; 3), helices in D1 undergo conformational change to allow VBS binding; and 4), VBS inserts itself into the hydrophobic core of D1.
Topics: Binding Sites; Crystallography, X-Ray; Cytoskeleton; Focal Adhesions; Hydrophobic and Hydrophilic Interactions; Mechanotransduction, Cellular; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Talin; Vinculin
PubMed: 21244829
DOI: 10.1016/j.bpj.2010.11.024 -
Journal of Immunology (Baltimore, Md. :... Sep 2013Lymphocytes use integrin-based platforms to move and adhere firmly to the surface of other cells. The molecular mechanisms governing lymphocyte adhesion dynamics are...
Lymphocytes use integrin-based platforms to move and adhere firmly to the surface of other cells. The molecular mechanisms governing lymphocyte adhesion dynamics are however poorly understood. In this study, we show that in mouse B lymphocytes, the actin binding protein vinculin localizes to the ring-shaped integrin-rich domain of the immune synapse (IS); the assembly of this platform, triggered by cognate immune interactions, is needed for chemokine-mediated B cell motility arrest and leads to firm, long-lasting B cell adhesion to the APC. Vinculin is recruited early in IS formation, in parallel to a local phosphatidylinositol (4,5)-bisphosphate wave, and requires spleen tyrosine kinase activity. Lack of vinculin at the IS impairs firm adhesion, promoting, in turn, cell migration with Ag clustered at the uropod. Vinculin localization to the B cell contact area depends on actomyosin. These results identify vinculin as a major controller of integrin-mediated adhesion dynamics in B cells.
Topics: Animals; B-Lymphocytes; Blotting, Western; Cell Adhesion; Chemotaxis, Leukocyte; Fluorescent Antibody Technique; Immunological Synapses; Integrins; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Vinculin
PubMed: 23872053
DOI: 10.4049/jimmunol.1300684 -
Experimental Cell Research Feb 1997Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two...
Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.
Topics: Actinin; Actins; Animals; Cell Adhesion; Cell Movement; Cell Size; Cytoskeletal Proteins; Cytoskeleton; Integrins; Mice; Microscopy, Confocal; Paxillin; Phosphoproteins; Pseudopodia; Stress, Mechanical; Talin; Tumor Cells, Cultured; Vinculin
PubMed: 9056408
DOI: 10.1006/excr.1996.3451 -
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 -
Nature Jan 2004Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions,...
Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.
Topics: Adherens Junctions; Animals; Chickens; Crystallography, X-Ray; Focal Adhesions; Humans; Models, Biological; Models, Molecular; Protein Conformation; Static Electricity; Talin; Vinculin
PubMed: 14702644
DOI: 10.1038/nature02281 -
Nature Cell Biology Jul 2015Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved...
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
Topics: Actins; Blotting, Western; Cell Line; Cell Line, Tumor; Fluorescence Resonance Energy Transfer; Focal Adhesions; Humans; Integrins; Luminescent Proteins; Microscopy, Fluorescence; Models, Molecular; Mutation; Nanostructures; Nanotechnology; Paxillin; Protein Binding; Protein Structure, Tertiary; RNA Interference; Talin; Vinculin
PubMed: 26053221
DOI: 10.1038/ncb3180 -
Current Biology : CB Feb 2013Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory...
BACKGROUND
Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear.
RESULTS
Here we show that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the actomyosin machinery. We demonstrate that the vinculin interaction with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by our observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, we establish which components of the cell-matrix adhesion network are coordinated through direct and indirect associations with vinculin. Moreover, using cyclic stretching, we demonstrate that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganization of cell polarity. Of particular importance is the actin-binding tail region of vinculin, without which the cell's ability to repolarize in response to cyclic stretching is perturbed.
CONCLUSIONS
Overall our data promote a model whereby vinculin controls the transmission of intracellular and extracellular mechanical cues that are important for the spatiotemporal assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility.
Topics: Actins; Actomyosin; Animals; Cell Adhesion; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Polarity; Cell-Matrix Junctions; Cytoskeleton; Extracellular Matrix; Focal Adhesions; Integrins; Melanoma; Mice; Mutation; Osteosarcoma; Protein Binding; Talin; Vinculin
PubMed: 23375895
DOI: 10.1016/j.cub.2013.01.009 -
Science Advances Dec 2019The vinculin-mediated mechanosensing requires establishment of stable mechanical linkages between vinculin to integrin at focal adhesions and to cadherins at adherens...
The vinculin-mediated mechanosensing requires establishment of stable mechanical linkages between vinculin to integrin at focal adhesions and to cadherins at adherens junctions through associations with the respective adaptor proteins talin and α-catenin. However, the mechanical stability of these critical vinculin linkages has yet to be determined. Here, we developed a single-molecule detector assay to provide direct quantification of the mechanical lifetime of vinculin association with the vinculin binding sites in both talin and α-catenin, which reveals a surprisingly high mechanical stability of the vinculin-talin and vinculin-α-catenin interfaces that have a lifetime of >1000 s at forces up to 10 pN and can last for seconds to tens of seconds at 15 to 25 pN. Our results suggest that these force-bearing intermolecular interfaces provide sufficient mechanical stability to support the vinculin-mediated mechanotransduction at cell-matrix and cell-cell adhesions.
Topics: Actin Cytoskeleton; Binding Sites; Cell Adhesion; Focal Adhesions; Humans; Integrins; Mechanotransduction, Cellular; Plasmids; Protein Binding; Single Molecule Imaging; Talin; Vinculin; alpha Catenin
PubMed: 31897422
DOI: 10.1126/sciadv.aav2720 -
Journal of Cardiovascular Translational... Dec 2023Predictors of myocardial recovery in heart failure (HF) are poorly understood. We explored if vinculin (VCL) variants are associated with myocardial recovery in dilated...
Predictors of myocardial recovery in heart failure (HF) are poorly understood. We explored if vinculin (VCL) variants are associated with myocardial recovery in dilated cardiomyopathy (DCM). Six infants with DCM with a VCL loss-of-function (LOF) variant were identified. Median age at diagnosis was 2 months, median LV ejection fraction was 24%, and median LV end-diastolic diameter z-score was 10.8. All patients received HF medications. Five patients (83%) showed normalization of LV function at a median age of 2.7 years. One patient progressed to end-stage HF requiring heart transplant. This case series identified a unique phenotype of HF with reduced ejection fraction at presentation that evolved to HF with recovered EF in over 80% of infant DCM cases with LOF VCL variants. These findings have prognostic implications for counseling and management of VCL-associated DCM and highlight a possible genetic basis for HF with recovered ejection fraction.
Topics: Infant; Humans; Child, Preschool; Stroke Volume; Ventricular Function, Left; Vinculin; Heart Failure; Prognosis
PubMed: 37548861
DOI: 10.1007/s12265-023-10421-6 -
Journal of Cell Science Dec 2016Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here,...
Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here, we examine how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin.
Topics: Animals; Cell Adhesion; Cytoplasm; Drosophila melanogaster; Embryo, Nonmammalian; Integrins; Models, Biological; Muscles; Protein Aggregates; Protein Binding; Protein Domains; Vinculin
PubMed: 27737911
DOI: 10.1242/jcs.189878