Talin dissociates from RIAM and associates to vinculin sequentially in response to the actomyosin force.

Cells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic vinculin-binding sites, triggers this process. The binding of RIAM to talin could regulate this mechanism. However, the mechanosensitivity of the talin-RIAM complex has never been tested. It is also not known whether RIAM controls the mechanosensitivity of the talin-vinculin complex. To address these issues, we designed an in vitro microscopy assay with purified proteins in which the actomyosin force controls RIAM and vinculin-binding to talin. We demonstrate that actomyosin triggers RIAM dissociation from several talin domains. Actomyosin also provokes the sequential exchange of RIAM for vinculin on talin. The effect of RIAM on this force-dependent binding of vinculin to talin varies from one talin domain to another. This mechanism could allow talin to biochemically code a wide range of forces by selecting different combinations of partners.

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.

Raver1 interactions with vinculin and RNA suggest a feed-forward pathway in directing mRNA to focal adhesions.

The translational machinery of the cell relocalizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of vinculin's head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their colocalization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.

Preliminary crystallographic study of turkey gizzard vinculin.

Vinculin is a 117 kDa microfilament-associated protein located at the cytoplasmic aspects of focal contacts and cell-cell adherens type junctions. In both sites, vinculin participates in the formation of a submembrane 'plaque' structure which is responsible for the attachment of actin filaments to the plasma membrane. Vinculin consists of 1066 amino acids, which form a large 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two domains are separated by several stretches of proline residues where the major proteolytic cleavage sites are located. The experimental procedure for isolation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obtained. The homogeneity of the vinculin solution was analyzed prior to crystallization using dynamic light scattering. Crystals of vinculin have been obtained in buffer containing 2 mg ml(-1) protein, 0.9 M ammonium sulfate, 0.1 M MES pH 6.5 using both the hanging-drop and sitting-drop vapour-diffusion methods. The crystals have the form of rhombic plates and grow to maximal dimensions of 0.3 x 0.3 x 0.05 mm in two weeks. Preliminary X-ray data show that the crystals diffract to 3.5 A resolution at the X11 beamline of DESY and belong to the monoclinic space group P2(1). Crystal unit-cell parameters are estimated to be a = 57, b = 351, c = 70 A, alpha = 90, beta = 113, gamma = 90 degrees.

Redistribution of a 120 kDa phosphoprotein in the parietal cell associated with stimulation.

When rabbit isolated gastric glands were stimulated via the cyclic AMP pathway, a phosphorylated protein band of about 120 kDa (pp120) was markedly increased in the apical membrane-rich fraction, concomitant with an increase in the amount of H,K-ATPase and the phosphorylation of the cytoskeletal protein ezrin in the same fraction. The cytosolic fraction, but not other membrane fractions, also contained a protein with common features to the membrane-bound pp120, i.e., comigration in two-dimensional gels with a pI of approximately 4.5, anomalous mobility in SDS-PAGE, reactivity to antibodies, and phosphorylation, indicating that these two proteins were identical. The possibility that pp120 is vinculin was completely excluded. Using antibody against pp120, this protein was found to be almost exclusively in the gastric parietal cell. Biochemical and immunohistochemical analyses suggest that pp120 exists mainly in the cytosol, and that a small part of the protein binds to the apical membrane when the parietal cell is stimulated via the cyclic AMP pathway. In the presence of histone, purified pp120 produced phosphorylation on pp120 as well as histone. The inhibitor profile of this kinase activity is not consistent with any known kinase. We conclude that pp120 is closely associated with a new type of kinase, and translocates from cytosol to the apical membrane when the parietal cell is stimulated.

The growing cell plate of higher plants is a site of both actin assembly and vinculin-like antigen recruitment.

Compelling evidence supports the idea that actin filaments play an active role in the cytokinetic process of higher plant cells. However, the mechanisms that control the growth of the cell plate and its stabilization remain so far unknown. We show that a novel population of short actin filaments continuously assembles in the phragmoplast at the growing cell plate. Microinjection of rhodamine-phalloidin during these final stages of telophase revealed the dynamic assembly and organization of these actin filaments during vesicle fusion. Comparable data were obtained in endosperm syncytia during the development of the cell plate between non sister nuclei, i.e. independently of the formation of the mitotic phragmoplast. Concomitantly, plant polypeptides sharing epitopes with human vinculin are revealed within the forming cell plate, suggesting their recruitment during cytokinesis-associated actin assembly. These vinculin-like antigens may participate in membrane/F-actin anchorage protein complexes. Our data, in addition to the identification of plant integrin homologues reported by several authors, suggest the existence of a cell wall/extracellular matrix/plasma membrane/actin cytoskeleton continuum. Such an architecture may control cell-cell interactions during cell plate formation and may contribute to the establishment of polarity in higher plants.

Differential actin organization by vinculin isoforms: implications for cell type-specific microfilament anchorage.

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 1-258 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VT-based form, suggesting that metavinculin has a specific role in muscle.

The use of two-dimensional gel electrophoresis in studies on the role of cytoskeletal plaque proteins as tumor suppressors.

The morphology and functions of cells and tissues are determined, in a large part, by mechanical forces generated at cell-cell and cell-extracellular matrix (ECM) contacts. At these sites, transmembrane adhesion receptors of the integrin and cadherin families are linked, via their cytoplasmic domain, to the cytoskeleton by submembranal plaque proteins such as vinculin, alpha-actinin and the cell-cell junctional plaque proteins alpha- and beta-catenin and plakoglobin (or gamma-catenin). Recent studies have implicated this link of structural molecules between the outside and inside of the cell in signal transduction. We have shown that the expression of junctional plaque proteins is modulated during growth stimulation and differentiation, and is dramatically reduced in certain tumor cells. To study the functional significance of these changes in expression, we have used recombinant DNA technologies to overexpress or suppress the levels of junctional plaque proteins. In addition, we eliminated the expression of vinculin in embryonal stem (ES) cells and in the embryonal carcinoma F9 line by gene disruption employing homologous recombination. The results have indicated that moderate overexpression of cell-ECM plaque proteins results in reduced cell motility. In contrast, suppression of their expression, by antisense transfection, led to enhanced motility and conferred anchorage independent growth and tumorigenicity, upon injection into nude mice. These findings suggest that submembranal plaque proteins can act as effective tumor suppressors. In agreement with this notion, we found in several tumor cell lines diminished levels of junctional plaque proteins. Restoration of their level to that found in normal cells resulted in tumor suppression after their injection into experimental animals. Here we demonstrate the usefulness of the application of two dimensional (2-D) gel electrophoresis in these studies.

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton.

The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.

Rapid induction and isolation of focal adhesion complexes.

Focal adhesion complexes (FACs) containing integrin beta 1, talin, vinculin, talin, alpha-actinin, and paxillin formed within 15 min when round cells bound magnetic microbeads coated with integrin ligands, such as fibronectin or RGD-containing peptide, but not when coated with acetylated-low density lipoprotein. Newly formed FACs were isolated and collected for biochemical analysis using a combination of detergent extraction, sonication, dounce homogenization, and magnetic pelleting. Isolated bead complexes were greatly enriched for all FAC proteins when compared with either the whole cytoskeleton or basal cell membranes whereas actin (a general cytoskeletal marker) was relatively depleted. This method which permits isolation of intact FACs within minutes following integrin ligation should facilitate analysis of both FAC assembly and the molecular basis of integrin signaling.

Immunoglobulins from normal sera bind platelet vinculin and talin and their proteolytic fragments.

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.

Restricted tissue distribution of a 37-kD possible adherens junction protein.

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.

Vinculin binding site mapped on talin with an anti-idiotypic antibody.

Vinculin and talin are major adhesion plaque components which interact in vitro and presumably in vivo. The amino acid sequence of talin is now known so details of its domain structure can be mapped. We localized vinculin binding sites in the talin sequence by overlaying peptide maps of talin with an anti-idiotypic vinculin antibody that recognizes talin and with 125I-vinculin. A rabbit injected only twice with vinculin and producing anti-vinculin antibodies spontaneously generated a second antibody that recognizes talin. Vinculin and anti-vinculin antibodies specifically compete with this second antibody for binding to talin as determined by solid-phase binding and overlay assays. The antibody is thus most likely an anti-idiotypic antibody which mimics a region of vinculin that interacts with talin. The binding site of the anti-idiotypic antibody on talin was mapped to the 196 amino acids spanning residues 1653 to 1848. A second vinculin binding site identified with an 125I-vinculin blot overlay technique was located between residues 483 and 1652. The observation that talin has two immunologically distinct vinculin binding sites suggests that vinculin may have two different talin binding sites or one "complex" site with two interacting regions.

Alpha-actinin and vinculin in human neutrophils: reorganization during adhesion and relation to the actin network.

We have studied the reorganization of vinculin and alpha-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent F-actin-rich large pseudopods. alpha-Actinin became enriched during cell adhesion in retraction fibers and, in 40-50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of alpha-actinin at a certain stage. Disruption of the actin network with cytochalasin D revealed a differential interaction of alpha-actinin and vinculin with the actin network. alpha-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10(-5) M of the drug. Vinculin organization in contrast was not affected by up to 10(-6) M cytochalasin. At 10(-5) M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas alpha-actinin may structure the actin network in retraction fibers and in less adherent pseudopods.

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.