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The Journal of Cell Biology Apr 2014Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at...
Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell-cell and cell-matrix adhesions remains unanswered. Here, we show vinculin is phosphorylated at Y822 in cell-cell, but not cell-matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for vinculin functions in cell-matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate vinculin; Abl inhibition mimicked the loss of vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions.
Topics: Cadherins; Cell Adhesion; Cell Communication; Cell Line, Tumor; Extracellular Matrix; Humans; Mechanotransduction, Cellular; Phosphorylation; Proto-Oncogene Proteins c-abl; Vinculin
PubMed: 24751539
DOI: 10.1083/jcb.201309092 -
Proceedings of the National Academy of... Jun 2013Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct...
Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V(H)) and tail domains. V(H) enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V(H) has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.
Topics: Actins; Animals; Blotting, Western; Cells, Cultured; Cytoskeleton; Embryo, Mammalian; Extracellular Matrix; Fibroblasts; Fluorescence Recovery After Photobleaching; Focal Adhesions; Green Fluorescent Proteins; Integrins; Mice; Mice, Knockout; Microscopy, Fluorescence; Models, Biological; Protein Binding; Stress, Mechanical; Talin; Vinculin
PubMed: 23716647
DOI: 10.1073/pnas.1216209110 -
Journal of Thrombosis and Haemostasis :... Oct 2010Summary.
UNLABELLED
Summary.
BACKGROUND
Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown.
OBJECTIVE
To determine whether vinculin is required for the functions of platelets and their major integrin, α(IIb) β(3) .
METHODS
The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo.
RESULTS
Vinculin was undetectable in platelets from Vcl fl/fl Cre(+) mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α(IIb) β(3) with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to α(IIb) β(3) , aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl(3) .
CONCLUSION
Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α(IIb) β(3) , vinculin is not required for the traditional functions of α(IIb) β(3) or the platelet actin cytoskeleton.
Topics: Actins; Animals; Blood Platelets; Cell Lineage; Collagen; Cytoskeleton; Fibrinogen; Gene Deletion; Megakaryocytes; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Vinculin
PubMed: 20670372
DOI: 10.1111/j.1538-7836.2010.03998.x -
European Journal of Clinical... Jan 2022
Topics: Antibodies; Bacterial Toxins; Gastrointestinal Diseases; Gastrointestinal Microbiome; Humans; Irritable Bowel Syndrome; Vinculin
PubMed: 34738648
DOI: 10.1111/eci.13702 -
Cell Biology International Aug 2018The focal adhesion protein vinculin has been implicated in associating with soluble and membranous phospholipids. Detailed investigations over the past ten years...
The focal adhesion protein vinculin has been implicated in associating with soluble and membranous phospholipids. Detailed investigations over the past ten years describe the intermolecular interactions of the vinculin tail domain with soluble and membrane phospholipids. Previous studies have implied that the tail's unstructured C-terminal region affects the mechanical behavior of cells and that the same region, at the molecular level, has bi-stable behavior sensitive to different protonation states. The aim of this short communication is to discuss whether the C-terminal vinculin tail (Vt) domain interacts favorably with membrane-embedded phospholipids such as PIP and that the region is also an anchor for lipid membranes.
Topics: Animals; Circular Dichroism; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylinositol 4,5-Diphosphate; Phospholipids; Protein Binding; Protein Domains; Vinculin
PubMed: 29696730
DOI: 10.1002/cbin.10978 -
Nature Communications Dec 2015The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding...
The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.
Topics: Actin Cytoskeleton; Actins; Actomyosin; Animals; Cell Polarity; Focal Adhesions; Mice; NIH 3T3 Cells; Protein Binding; Protein Structure, Tertiary; Talin; Vinculin
PubMed: 26634421
DOI: 10.1038/ncomms10038 -
The Biochemical Journal Feb 2015Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been...
Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited. In the present study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further, mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we discuss the viability of models detailing the F-actin-binding surface on vinculin in the context of our mutational analysis.
Topics: Actin Cytoskeleton; Actins; Animals; Cell Movement; Mechanotransduction, Cellular; Mice; Mice, Knockout; Protein Binding; Protein Structure, Secondary; Vinculin
PubMed: 25358683
DOI: 10.1042/BJ20140872 -
Structure (London, England : 1993) Oct 2019Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin...
Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences, we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However, the metavinculin tail (MVt) domain contains a 68 amino acid insert, with helix 1 (H1) sequestered into a globular subdomain, which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model, deletion and selective point mutations within the MVt H1 disrupt this protruding structure, and facilitate actin bundling similar to vinculin tail (Vt) domain.
Topics: Actins; Alternative Splicing; Animals; Binding Sites; Cryoelectron Microscopy; Models, Molecular; Mutation; Protein Binding; Protein Domains; Protein Structure, Secondary; Vinculin
PubMed: 31422909
DOI: 10.1016/j.str.2019.07.013 -
Biophysical Journal May 2012Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are...
Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.
Topics: Binding Sites; Computer Simulation; Models, Chemical; Models, Molecular; Phosphorylation; Protein Binding; Protein Conformation; Protein Kinases; Protein Structure, Tertiary; Vinculin
PubMed: 22824265
DOI: 10.1016/j.bpj.2012.01.062 -
Integrative Biology : Quantitative... Jun 2016Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor...
Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head-tail interaction that regulates the recruitment of, and interactions with its partners in a force-dependent manner. It is unknown, however, whether this mechanism is involved in substrate rigidity sensing. Here, we use a range of quantitative fluorescence microscopies on live human Mesenchymal Stem Cells to address this question. We identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head-tail interaction, and targets cell morphology. Vinculin and talin are recruited independently in a rigidity-dependent manner to FAs where they directly interact in a rigidity-independent stoichiometry at a site proximal to talin head. Vinculin head-tail interaction is required on soft substrates to destabilize vinculin and talin in FAs, and to allow hMSCs branching. Another module involves paxillin and FAK, which soft substrates also destabilize, but independently of vinculin head-tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues.
Topics: Cell Adhesion; Cell Size; Cells, Cultured; Cytoskeleton; Elastic Modulus; Focal Adhesions; Humans; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Stress, Mechanical; Talin; Vinculin
PubMed: 27169142
DOI: 10.1039/c5ib00307e