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Biochemical and Biophysical Research... Sep 2010Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this...
Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cricetinae; Cricetulus; Gene Silencing; Humans; Integrin alpha Chains; Integrin beta3; Mice; Talin; Vinculin
PubMed: 20728432
DOI: 10.1016/j.bbrc.2010.08.056 -
The Journal of Biological Chemistry Dec 1997Cadherins mediate calcium-dependent cell-cell adhesion, and this activity is regulated by cytoplasmic interactions between cadherins, catenins, and the actin-based...
Cadherins mediate calcium-dependent cell-cell adhesion, and this activity is regulated by cytoplasmic interactions between cadherins, catenins, and the actin-based cytoskeleton. alpha-Catenin plays a critical role in the transmembrane anchorage of cadherins, and deletion of alpha-catenin has been shown to inactivate cadherin-mediated adhesion, resulting in a nonadhesive phenotype. Here we show that serum starvation increases E-cadherin expression and induces E-cadherin-dependent adhesion in the MDA-MB-468 breast cancer cell line. This adhesion occurred despite a lack of alpha-catenin expression, which was caused by mutations in the alpha-catenin gene. Coprecipitation analysis suggests that this adhesion may be mediated by cytoplasmic connections from cadherins to the cytoskeleton involving vinculin. A high level of vinculin associated with E-cadherin immunoprecipitates was observed in MDA-MB-468 cells. In contrast, vinculin was not detected in E-cadherin complexes in the A431 and MCF-7 epithelial carcinoma cell lines, which express alpha-catenin. However, in reciprocal immunoprecipitations using anti-vinculin antibodies, E-cadherin associated strongly with vinculin in MDA-MB-468 cells and, to a lesser extent, in A431 and MCF-7 cells. These results suggest that both alpha-catenin and vinculin may be present in the adhesion complex. To test the hypothesis that vinculin associates with E-cadherin complexes via beta-catenin, excess recombinant beta-catenin or alpha-catenin fusion protein was added to MDA-MB-468 cell lysates. Both specifically inhibited the coprecipitation of E-cadherin with vinculin, suggesting competition for the same binding site. These results suggest that vinculin plays a role in the establishment or regulation of the cadherin-based cell adhesion complex by direct interaction with beta-catenin.
Topics: Amino Acid Sequence; Cadherins; Cell Adhesion; Culture Media, Serum-Free; Cytoskeletal Proteins; Humans; Molecular Sequence Data; Protein Binding; Tumor Cells, Cultured; Vinculin; alpha Catenin
PubMed: 9405455
DOI: 10.1074/jbc.272.51.32448 -
Experimental Cell Research Oct 2019Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, playing an important role in linkage of integrin adhesion molecules to the actin...
Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, playing an important role in linkage of integrin adhesion molecules to the actin cytoskeleton. The planarian nervous system is a fascinating system for studying the organogenesis during regeneration. In this paper, a homolog gene of Vinculin, DjVinculin, was identified and characterized in Dugesia japonica. The DjVinculin sequence analysis revealed that it contains an opening reading frame encoding a putative protein of 975 amino acids with functionally domains that are highly conserved, including eight anti-parallel α-helical bundles organized into five distinct domains. Whole mount in situ hybridization showed that DjVinculin was predominantly expressed in the brain of intact and regenerating planarians. RNA interference of DjVinculin caused distinct defects in brain morphogenesis and influences the regeneration of planarian GABAergic neurons. The expression level of DjGAD protein was decreased in the DjVinculin-knockdown planarians. These findings suggest that DjVinculin is required for GABAergic neurons regeneration.
Topics: Amino Acid Sequence; Animals; GABAergic Neurons; Gene Expression Regulation, Developmental; Helminth Proteins; Planarians; Regeneration; Sequence Homology; Vinculin
PubMed: 31369753
DOI: 10.1016/j.yexcr.2019.111540 -
The Journal of Cell Biology Sep 1997To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By...
To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.
Topics: Actins; Animals; Antibody Specificity; Bacterial Proteins; Blood Platelets; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Cross Reactions; Dysentery, Bacillary; Fluorescent Antibody Technique; Humans; Kidney; Macropodidae; Membrane Proteins; Microfilament Proteins; Microinjections; Peptide Fragments; Phosphoproteins; Proline; Shigella flexneri; Vinculin
PubMed: 9298981
DOI: 10.1083/jcb.138.6.1255 -
Laboratory Investigation; a Journal of... Aug 2020Talin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter...
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.
Topics: Actin Cytoskeleton; Animals; Cell Membrane; Cells, Cultured; Guanine Nucleotide Exchange Factors; Hemorrhagic Fever, Ebola; Humans; Liver; Mice, Knockout; Protein Binding; Rickettsia; Spotted Fever Group Rickettsiosis; Talin; Vinculin
PubMed: 32238906
DOI: 10.1038/s41374-020-0420-9 -
Trends in Biochemical Sciences Nov 2004The ability of cells to tightly adhere to one another and to the extracellular matrix is fundamentally important in numerous biological processes, including...
The ability of cells to tightly adhere to one another and to the extracellular matrix is fundamentally important in numerous biological processes, including embryogenesis, wound healing and maintenance of tissue integrity. Vinculin, a protein localized at the cytoplasmic face of cell-matrix and cell-cell adhesions, is required for strong cell adhesion. Two new crystal structures reveal that vinculin exhibits a high degree of structural plasticity upon ligand binding that might promote rapid changes in cell adhesion.
Topics: Animals; Binding Sites; Cell Adhesion; Cytoskeletal Proteins; Humans; Models, Molecular; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Protein Structure, Tertiary; Vinculin
PubMed: 15501673
DOI: 10.1016/j.tibs.2004.09.001 -
The Journal of Cell Biology May 2004Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to...
Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.
Topics: Animals; Apoptosis; Binding Sites; Cell Adhesion; Cell Movement; Cell Survival; Cytoskeletal Proteins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Mice; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Neoplastic Stem Cells; Paxillin; Peptide Fragments; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Transfection; Up-Regulation; Vinculin
PubMed: 15138291
DOI: 10.1083/jcb.200308011 -
Biochemistry Nov 2019Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order.... (Review)
Review
Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order. Measurement and quantitation of these biological interactions are of value to scientists and are major goals of biochemistry, as affinities provide insight into biological processes. In an organism, these interactions occur in the context of forces and the need for a consideration of binding affinities in the context of a changing mechanical landscape necessitates a new way to consider the biochemistry of protein-protein interactions. In the past few decades, the field of mechanobiology has exploded, as both the appreciation of, and the technical advances required to facilitate the study of, how forces impact biological processes have become evident. The aim of this review is to introduce the concept of force dependence of biomolecular interactions and the requirement to be able to measure force-dependent binding constants. The focus of this discussion will be on the mechanotransduction that occurs at the integrin-mediated adhesions with the extracellular matrix and the major mechanosensors talin and vinculin. However, the approaches that the cell uses to sense and respond to forces can be applied to other systems, and this therefore provides a general discussion of the force dependence of biomolecule interactions.
Topics: Animals; Biophysical Phenomena; Cell Adhesion; Extracellular Matrix; Focal Adhesions; Humans; Integrins; Mechanotransduction, Cellular; Protein Binding; Talin; Vinculin
PubMed: 31315399
DOI: 10.1021/acs.biochem.9b00453 -
The Journal of Cell Biology Jul 2013In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and...
In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium-lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin-F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.
Topics: Actins; Amino Acid Substitution; Animals; Cell Movement; Cloning, Molecular; Extracellular Matrix; Fibroblasts; Focal Adhesions; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Point Mutation; Protein Binding; Protein Interaction Mapping; Protein Transport; Proteolysis; Pseudopodia; Vinculin
PubMed: 23836933
DOI: 10.1083/jcb.201303129 -
Journal of Cell Science Apr 2005The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic... (Comparative Study)
Comparative Study
The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP(2)-dependent disassembly of adhesions was suppressed. Thus, PIP(2) binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP(2) levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor.
Topics: Animals; Binding Sites; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cloning, Molecular; Collagen; Extracellular Matrix; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; Ligands; Mice; Mutagenesis, Site-Directed; Mutation; Phosphatidylinositol 4,5-Diphosphate; Phosphotransferases (Alcohol Group Acceptor); Protein Conformation; Vinculin
PubMed: 15769850
DOI: 10.1242/jcs.01734