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The Journal of Biological Chemistry Feb 2014Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus...
Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr(1065) on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr(1065) in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr(1065) (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr(1065) phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr(1065) phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.
Topics: Acetylcholine; Actins; Animals; Cell Membrane; Cholinergic Agonists; Dogs; Muscle Contraction; Muscle, Smooth; Phosphorylation; Protein Structure, Tertiary; Talin; Trachea; Vinculin
PubMed: 24338477
DOI: 10.1074/jbc.M113.508077 -
Frontiers in Immunology 2022Activation of the integrin phagocytic receptors CR3 (αβ, CD11b/CD18) and CR4 (αβ, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin...
Activation of the integrin phagocytic receptors CR3 (αβ, CD11b/CD18) and CR4 (αβ, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (α), ITGAX (α) and ITGB2 (β) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored α expression. In general, the expression of α was less responsive to jasplakinolide treatment than α, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αβ expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored α expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αβ and αβ during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.
Topics: Actins; Adaptor Proteins, Signal Transducing; Cell Adhesion Molecules; Co-Repressor Proteins; HL-60 Cells; Humans; Integrin alphaXbeta2; Integrins; Macrophage-1 Antigen; Membrane Proteins; Microfilament Proteins; Neutrophils; Phosphoproteins; RNA, Messenger; Serum Response Factor; Talin; Vinculin
PubMed: 36238292
DOI: 10.3389/fimmu.2022.951280 -
Journal of Cell Science Jan 2009Cells govern tissue shape by exerting highly regulated forces at sites of matrix adhesion. As the major force-bearing adhesion-receptor protein, integrins have a central... (Review)
Review
Cells govern tissue shape by exerting highly regulated forces at sites of matrix adhesion. As the major force-bearing adhesion-receptor protein, integrins have a central role in how cells sense and respond to the mechanics of their surroundings. Recent studies have shown that a key aspect of mechanotransduction is the cycle by which integrins bind to the matrix at the leading cell edge, attach to the cytoskeleton, transduce mechanical force, aggregate in the plasma membrane as part of increasingly strengthened adhesion complexes, unbind and, ultimately, are recycled. This mechanical cycle enables the transition from early complexes to larger, more stable adhesions that can then rapidly release. Within this mechanical cycle, integrins themselves exhibit intramolecular conformational change that regulates their binding affinity and may also be dependent upon force. How the cell integrates these dynamic elements into a rigidity response is not clear. Here, we focus on the steps in the integrin mechanical cycle that are sensitive to force and closely linked to integrin function, such as the lateral alignment of integrin aggregates and related adhesion components.
Topics: Animals; Cell Adhesion; Cell Movement; Cytoskeleton; Humans; Integrins; Mechanotransduction, Cellular; Phosphorylation; Stress, Mechanical; Syndecans; Talin; Vinculin
PubMed: 19118210
DOI: 10.1242/jcs.042127 -
α-catenin, vinculin, and F-actin in strengthening E-cadherin cell-cell adhesions and mechanosensing.Cell Adhesion & Migration 2013Classical cadherins play a crucial role in establishing intercellular adhesion, regulating cortical tension, and maintaining mechanical coupling between cells. The...
Classical cadherins play a crucial role in establishing intercellular adhesion, regulating cortical tension, and maintaining mechanical coupling between cells. The mechanosensitive regulation of intercellular adhesion strengthening depends on the recruitment of adhesion complexes at adhesion sites and their anchoring to the actin cytoskeleton. Thus, the molecular mechanisms coupling cadherin-associated complexes to the actin cytoskeleton are actively being studied, with a particular focus on α-catenin and vinculin. We have recently addressed the role of these proteins by analyzing the consequences of their depletion and the expression of α-catenin mutants in the formation and strengthening of cadherin-mediated adhesions. We have used the dual pipette assay to measure the forces required to separate cell doublets formed in suspension. In this commentary, we briefly summarize the current knowledge on the role of α-catenin and vinculin in cadherin-actin cytoskeletal interactions. These data shed light on the tension-dependent contribution of α-catenin and vinculin in a mechanoresponsive complex that promotes the connection between cadherin and the actin cytoskeleton and their requirement in the development of adhesion strengthening.
Topics: Animals; Cadherins; Gene Expression Regulation; Vinculin; alpha Catenin
PubMed: 23739176
DOI: 10.4161/cam.25139 -
ELife Nov 2020Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling...
Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Actins; Animals; Cell Adhesion; Cryoelectron Microscopy; Electron Microscope Tomography; Extracellular Matrix; Humans; Integrins; Microscopy, Confocal; Sf9 Cells; Talin; Vinculin
PubMed: 33185186
DOI: 10.7554/eLife.53990 -
Proceedings of the National Academy of... Jul 2021Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap...
Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell-cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II-dependent changes in cytoskeletal tension. We also show that in epithelial cells, ∼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.
Topics: Actins; Animals; Cadherins; Cell Adhesion; Cytoskeleton; Dogs; Madin Darby Canine Kidney Cells; Myosin Type II; Protein Binding; Vinculin
PubMed: 34301871
DOI: 10.1073/pnas.2104090118 -
Journal of Molecular Biology Mar 2023Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via...
Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.
Topics: Actin Cytoskeleton; Actins; alpha Catenin; Cadherins; Cell Adhesion; Protein Binding; Vinculin; Allosteric Regulation
PubMed: 36682678
DOI: 10.1016/j.jmb.2023.167969 -
Aging Feb 2021In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness,...
High expression of vinculin predicts poor prognosis and distant metastasis and associates with influencing tumor-associated NK cell infiltration and epithelial-mesenchymal transition in gastric cancer.
In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness, resulting in the spread of tumor cells. Immune cells and inflammation in the tumor microenvironment are the driving factors of EMT, but few studies have explored the core targets of the interaction between EMT and tumor immune cells. We analyzed thousands of cases of gastric cancer and gastric tissue specimens of TCGA, CPTAC, GTEx and analyzing QPCR and IHC data of 56 gastric cancer patients in SYSU Gastric Cancer Research Center. It was known that EMT has an important connection with the infiltration of NK cells, and that the expression of vinculin may be the target of the phenomenon. The increased expression of vinculin is closely related to the aggressiveness and distant metastasis of cancer, which affects the survival prognosis of the patient. Moreover, through experiments under 3D conditions, we found that vinculin, cell invasion and metastasis are clearly linked. VCL can affect EMT and tumor immunity by regulating EPCAM gene expression. The role and mechanism of action of vinculin have been controversial, but this molecule may downregulate EpCAM (epithelial cellular adhesion molecule) and its own role in gastric cancer through DNA methylation, causing NK cells to enrich into tumor cells and kill tumor cells. At the same time, it promotes the occurrence of EMT, which in turn causes tumor metastasis and thus poorer prognosis.
Topics: Adenocarcinoma; Aged; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Humans; Killer Cells, Natural; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Stomach Neoplasms; Vinculin
PubMed: 33535187
DOI: 10.18632/aging.202440 -
PloS One 2017The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of...
The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of adhesion to the extracellular matrix (ECM) and cell neighbors, not only transmit these mechanical forces, but also allow cells to respond to changes in force by inducing biochemical feedback pathways. Such force-induced signaling processes are termed mechanotransduction. Vinculin is a central protein in mechanotransduction that in both integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesions mediates force-induced cytoskeletal remodeling and adhesion strengthening. Vinculin was found to be important for the integrity and remodeling of epithelial tissues in cell culture models and could therefore be expected to be of broad importance in epithelial morphogenesis in vivo. Besides a function in mouse heart development, however, the importance of vinculin in morphogenesis of other vertebrate tissues has remained unclear. To investigate this further, we knocked out vinculin functioning in zebrafish, which contain two fully functional isoforms designated as vinculin A and vinculin B that both show high sequence conservation with higher vertebrates. Using TALEN and CRISPR-Cas gene editing technology we generated vinculin-deficient zebrafish. While single vinculin A mutants are viable and able to reproduce, additional loss of zygotic vinculin B was lethal after embryonic stages. Remarkably, vinculin-deficient embryos do not show major developmental defects, apart from mild pericardial edemas. These results lead to the conclusion that vinculin is not of broad importance for the development and morphogenesis of zebrafish tissues.
Topics: Animals; Cell Adhesion; Dogs; Embryonic Development; Extracellular Matrix; Gene Expression Regulation, Developmental; Gene Knockout Techniques; HEK293 Cells; Humans; Madin Darby Canine Kidney Cells; Mechanotransduction, Cellular; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 28767718
DOI: 10.1371/journal.pone.0182278 -
The Journal of Cell Biology Oct 2022The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM...
The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5β1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
Topics: Cell Adhesion; Extracellular Matrix; Fibronectins; Focal Adhesions; Integrins; Talin; Tensins; Vinculin
PubMed: 36074065
DOI: 10.1083/jcb.202107022