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Journal of Cell Science Feb 2010Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that...
Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that vinculin is important for the organization of cell-cell junctions. However, this approach removes vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of vinculin, but not of a vinculin A50I substitution mutant that is defective for beta-catenin binding. These findings suggest that an interaction between beta-catenin and vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a beta-catenin mutant that fails to bind vinculin. Thus, our study identifies vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.
Topics: Adherens Junctions; Animals; Avian Proteins; Cadherins; Cell Adhesion; Cell Line; Cell Membrane; Chickens; Female; Humans; Mutagenesis, Site-Directed; Protein Binding; RNA Interference; Recombinant Fusion Proteins; Vinculin; beta Catenin
PubMed: 20086044
DOI: 10.1242/jcs.056432 -
Scientific Reports Dec 2018Cells respond to the mechanics of their environment. Mechanical cues include extracellular matrix (ECM) stiffness and deformation, which are primarily sensed through...
Cells respond to the mechanics of their environment. Mechanical cues include extracellular matrix (ECM) stiffness and deformation, which are primarily sensed through integrin-mediated adhesions. We investigated the impact of ECM deformation on cellular forces, measuring the time-evolution of traction forces of isolated mouse fibroblasts in response to stretch and release. Stretch triggered a marked increase of traction stresses and apparent stiffness. Expression of the focal adhesion protein vinculin not only increased baseline traction forces, but also increased dissipation of mechanical energy, which was correlated with the cells' failure to recover baseline traction forces after release of stretch.
Topics: Animals; Biomarkers; Cell Adhesion; Cell Shape; Cells, Cultured; Extracellular Matrix; Fibroblasts; Fluorescent Antibody Technique; Focal Adhesions; Gene Knockout Techniques; Mechanical Phenomena; Mice; Vinculin
PubMed: 30568231
DOI: 10.1038/s41598-018-36272-9 -
FASEB Journal : Official Publication of... Nov 2015Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue...
Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.
Topics: Animals; Cell Movement; Cell Polarity; Collagen; Extracellular Matrix; Fibroblasts; Mice; Mice, Knockout; Myosin Type II; Pseudopodia; Vinculin
PubMed: 26195589
DOI: 10.1096/fj.14-268235 -
ELife Mar 2021Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated...
Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.
Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Focal Adhesions; Mice; Protein Binding; Talin; Vinculin
PubMed: 33783351
DOI: 10.7554/eLife.66151 -
Journal of Molecular Biology Apr 2019Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the...
Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the essential cytoskeletal protein vinculin. Metavinculin is co-expressed with vinculin at sub-stoichiometric ratios in cardiac tissues. CM mutations in the metavinculin tail domain (MVt) occur within the extra 68-residue insert that differentiates it from the vinculin tail domain (Vt). Vt binds actin filaments (F-actin) and promotes vinculin dimerization to bundle F-actin into thick fibers. While MVt binds to F-actin in a similar manner to Vt, MVt is incapable of F-actin bundling and inhibits Vt-mediated F-actin bundling. We performed F-actin co-sedimentation and negative-stain EM experiments to dissect the coordinated roles of metavinculin and vinculin in actin fiber assembly and the effects of three known metavinculin CM mutations. These CM mutants were found to weakly induce the formation of disordered F-actin assemblies. Notably, they fail to inhibit Vt-mediated F-actin bundling and instead promote formation of large assemblies embedded with linear bundles. Computational models of MVt bound to F-actin suggest that MVt undergoes a conformational change licensing the formation of a protruding sub-domain incorporating the insert, which sterically prevents dimerization and bundling of F-actin by Vt. Sub-domain formation is destabilized by CM mutations, disrupting this inhibitory mechanism. These findings provide new mechanistic insights into the ability of metavinculin to tune actin organization by vinculin and suggest that dysregulation of this process by CM mutants could underlie their malfunction in disease.
Topics: Actins; Animals; Cardiomyopathies; Chickens; Humans; Models, Molecular; Point Mutation; Protein Binding; Protein Domains; Protein Interaction Maps; Vinculin
PubMed: 30844403
DOI: 10.1016/j.jmb.2019.02.024 -
ELife Jul 2020Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting...
Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.
Topics: Actin Cytoskeleton; Actins; Membranes, Artificial; Phosphatidylinositols; Talin; Vinculin
PubMed: 32657269
DOI: 10.7554/eLife.56110 -
Journal of Muscle Research and Cell... Feb 1996
Review
Topics: Animals; Cell Adhesion; Cell Line; Humans; Models, Structural; Protein Structure, Secondary; Talin; Vinculin
PubMed: 8740427
DOI: 10.1007/BF00140319 -
The EMBO Journal Oct 2007Vinculin links integrin receptors to the actin cytoskeleton by binding to talin. Vinculin is held in an inactive, closed-clamp conformation through hydrophobic...
Vinculin links integrin receptors to the actin cytoskeleton by binding to talin. Vinculin is held in an inactive, closed-clamp conformation through hydrophobic interactions between its head and tail domains, and vinculin activation has long been thought to be dependent upon severing the head-tail interaction. Talin, alpha-actinin, and the invasin IpaA of Shigella flexneri sever vinculin's head-tail interaction by inserting an alpha-helix into vinculin's N-terminal four-helical bundle, provoking extensive conformational changes by a helical bundle conversion mechanism; these alterations in vinculin structure displace its tail domain, allowing vinculin to bind to its other partners. IpaA harbors two juxtaposed alpha-helical vinculin-binding sites (VBS) in its C-terminus. Here, we report that the lower affinity VBS of IpaA can also bind to the adjacent C-terminal four-helical bundle of vinculin's head domain through a helix addition mechanism. These hydrophobic interactions do not alter the conformation of this helical bundle, and the architecture of the complex suggests that IpaA can simultaneously interact with both of the four-helical bundle domains of vinculin's N-terminus to stabilize vinculin-IpaA interactions.
Topics: Actinin; Actins; Binding Sites; Cell Adhesion; Crystallography, X-Ray; Cytoskeleton; Humans; Models, Molecular; Molecular Conformation; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Vinculin
PubMed: 17932491
DOI: 10.1038/sj.emboj.7601863 -
Biochemical and Biophysical Research... Sep 2014We investigated the effect of the point mutation E29R on vinculin under cell mechanical aspects. MEFvcl KO cells were transfected with intact eGFP-vinculin (rescue) or...
We investigated the effect of the point mutation E29R on vinculin under cell mechanical aspects. MEFvcl KO cells were transfected with intact eGFP-vinculin (rescue) or mutant E29R vinculin. Cellular stiffness and adhesion strength of mutant E29R vinculin were considerably higher compared to rescue and MEFvcl KO cells. 2D traction microscopy also indicated markedly higher strain energy in E29R mutant cells compared to rescue and MEFvcl KO cells. Fluorescence recovery after photobleaching showed that the recovery time for mutant E29R cells was drastically slower than for MEFvcl rescue cells and that the mobile fraction was larger for rescue compared to E29R mutant cells. These results indicate that E29R mutation might prime the vinculin head for F-actin binding, which results in higher cell stiffness, contractile force, and strengthening of focal adhesions.
Topics: Actins; Animals; Arginine; Aspartic Acid; Cell Line; Embryo, Mammalian; Fibroblasts; Focal Adhesions; Gene Expression; Gene Knockout Techniques; Genes, Reporter; Green Fluorescent Proteins; Mechanotransduction, Cellular; Mice; Mutation; Protein Binding; Rheology; Stress, Mechanical; Transfection; Vinculin
PubMed: 25193699
DOI: 10.1016/j.bbrc.2014.08.133 -
The Journal of Biological Chemistry 2021α-Catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation....
α-Catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle (M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry showed that αT-catenin binds the β-catenin-N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates the M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.
Topics: Actin Cytoskeleton; Binding Sites; Myocardium; Protein Binding; Proteolysis; Vinculin; alpha Catenin
PubMed: 33771561
DOI: 10.1016/j.jbc.2021.100582