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Blood Feb 1993Our previous finding that normal serum immunoglobulins bind to internal platelet proteins on Western blots led us to further identify these proteins and determine the...
Our previous finding that normal serum immunoglobulins bind to internal platelet proteins on Western blots led us to further identify these proteins and determine the possible significance of autoantibodies against them. A 95-Kd protein reactive with immunoglobulins in most normal sera and easily confused with gpIIIa was shown to be a fragment of vinculin generated by calpain proteolysis. Identity was established by peptide sequencing of the protein purified from platelets stored without specific protease inhibitors. Normal immunoglobulins bound intact vinculin (117 Kd) and metavinculin (152 Kd), and their 105-, 95-, and 80- to 85-Kd proteolytic fragments. IgG in 89%, and IgA and IgM in 100% of normal sera reacted in titers of 10 to 1,000 with purified vinculin. In addition, IgG in 79%, and IgA and IgM in 93% of normal sera reacted in titers of 10 to 5,000 with talin (235 Kd), another cytoskeletal protein, and its 200-Kd proteolytic fragment. IgGs in sera from animals of several different phylogenetic classes also reacted with human vinculin and talin on Western blots. Frequency of occurrence, titers, and classes of antivinculin and antitalin autoantibodies in patients with thrombocytopenia did not differ discernibly from those of normal individuals. These antibodies had no effect on platelet aggregation or clot retraction, and no apparent pathogenic significance, but their widespread presence and the variability in extent of proteolysis of platelet preparations used for Western blots can complicate interpretation of patterns obtained with sera from patients with presumed immune-mediated thrombocytopenias.
Topics: Antibodies, Monoclonal; Autoantibodies; Blood Platelets; Blotting, Western; Calpain; Clot Retraction; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Molecular Weight; Platelet Aggregation; Talin; Vinculin
PubMed: 8427966
DOI: No ID Found -
The Journal of Cell Biology Mar 1987Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we...
Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.
Topics: Animals; Antibodies; Avian Proteins; Chickens; Gizzard, Avian; Molecular Weight; Muscle Proteins; Muscle, Smooth; Protein Conformation; Vinculin
PubMed: 3102502
DOI: 10.1083/jcb.104.3.473 -
The Journal of Cell Biology Mar 1999We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and...
We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.
Topics: Amino Acid Sequence; Animals; Base Sequence; COS Cells; Cell Membrane; DNA, Complementary; Kinesins; Mice; Microfilament Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Molecular Sequence Data; Myosins; Protein Binding; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Vinculin
PubMed: 10085297
DOI: 10.1083/jcb.144.5.1001 -
Journal of Cell Science Jan 2009
Review
Topics: Animals; Cell Adhesion; Cell Communication; Extracellular Matrix; Humans; Integrins; Paxillin; Protein Kinases; Signal Transduction; Talin; Vinculin
PubMed: 19118207
DOI: 10.1242/jcs.018093 -
Molecular Biology of the Cell Feb 2018The shaping of a multicellular body and repair of adult tissues require fine--tuning of cell adhesion, cell mechanics, and intercellular transmission of mechanical load....
The shaping of a multicellular body and repair of adult tissues require fine--tuning of cell adhesion, cell mechanics, and intercellular transmission of mechanical load. Adherens junctions (AJs) are the major intercellular junctions by which cells sense and exert mechanical force on each other. However, how AJs adapt to mechanical stress and how this adaptation contributes to cell-cell cohesion and eventually to tissue-scale dynamics and mechanics remains largely unknown. Here, by analyzing the tension-dependent recruitment of vinculin, α-catenin, and F-actin as a function of stiffness, as well as the dynamics of GFP-tagged wild-type and mutated α-catenins, altered for their binding capability to vinculin, we demonstrate that the force-dependent binding of vinculin stabilizes α-catenin and is responsible for AJ adaptation to force. Challenging cadherin complexes mechanical coupling with magnetic tweezers, and cell-cell cohesion during collective cell movements, further highlight that tension-dependent adaptation of AJs regulates cell-cell contact dynamics and coordinated collective cell migration. Altogether, these data demonstrate that the force-dependent α-catenin/vinculin interaction, manipulated here by mutagenesis and mechanical control, is a core regulator of AJ mechanics and long-range cell-cell interactions.
Topics: Actins; Adherens Junctions; Animals; Cell Adhesion; Cells, Cultured; Dogs; Fluorescent Antibody Technique; Humans; Madin Darby Canine Kidney Cells; Mechanical Phenomena; Mechanotransduction, Cellular; Protein Binding; Vinculin; alpha Catenin
PubMed: 29282282
DOI: 10.1091/mbc.E17-04-0231 -
Life Science Alliance Aug 2024Vinculin is a cytoskeletal linker strengthening cell adhesion. The IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with...
Vinculin is a cytoskeletal linker strengthening cell adhesion. The IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a and D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion.
Topics: Vinculin; Humans; Focal Adhesions; Cell Adhesion; Bacterial Proteins; Mutation; Host-Pathogen Interactions; HeLa Cells; Protein Binding; Shigella; Antigens, Bacterial; Dysentery, Bacillary
PubMed: 38834194
DOI: 10.26508/lsa.202302418 -
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 Journal of Biological Chemistry Dec 2006Sprouty-related proteins with an EVH1 domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 domain, which is related to the VASP...
Sprouty-related proteins with an EVH1 domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 domain, which is related to the VASP (vasodilator-stimulated phosphoprotein) EVH1 domain (Enabled/VASP homology 1 domain) and a C-terminal Sprouty-related domain, typical for Sprouty proteins. Spreds were, like Sproutys, initially discovered as inhibitors of the Ras/MAPK pathway, and the SPR (Sprouty-related) domains of both protein families seem to be very important for many protein interactions and cellular processes. VASP was initially characterized as a proline-rich substrate of protein kinases A and G in human platelets and later shown to be a scaffold protein, regulating both signal transduction pathways and the actin filament system. The VASP-EVH1 domain is known to bind specifically to a FP(4) binding motif, which is, for example, present in the focal adhesion proteins vinculin and zyxin. In this review we give a structural and functional overview on these three protein families and ask whether nature plays a modular protein domain puzzle with stable exchangeable elements or if these closely related domains have various functions when pasted in a different protein context.
Topics: Actins; Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Blood Platelets; Cell Adhesion Molecules; Cytoskeletal Proteins; Glycoproteins; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Membrane Proteins; Microfilament Proteins; Models, Biological; Phosphoproteins; Protein Structure, Tertiary; Signal Transduction; Vinculin; Zyxin
PubMed: 16987806
DOI: 10.1074/jbc.R600023200 -
The Journal of Cell Biology May 1998In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin...
In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and alpha-catenin. We show that alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2-4 x 10(-7) M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, were directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments were recruited to cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity.
Topics: Animals; Cadherins; Cattle; Cell Communication; Cell Line; Cross-Linking Reagents; Cytoskeletal Proteins; Intercellular Junctions; Mice; Transfection; Vinculin; alpha Catenin
PubMed: 9566974
DOI: 10.1083/jcb.141.3.755 -
Cell Reports Apr 2022Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving...
Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.
Topics: Adherens Junctions; Animals; Cadherins; Endothelial Cells; Intercellular Junctions; Mechanotransduction, Cellular; Neovascularization, Physiologic; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 35417696
DOI: 10.1016/j.celrep.2022.110658