Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.     

A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.     

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

Localization of the actin-binding protein fesselin in chicken smooth muscle

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.     

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.     

Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle

The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.     

Localization of filamin in smooth muscle

The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.     

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.     

Molecular heterogeneity of adherens junctions

We describe here the subcellular distributions of three junctional proteins in different adherens-type contacts. The proteins examined include vinculin, talin, and a recently described 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 10:2249-2260). Immunofluorescent localization of the three proteins indicated that while vinculin was ubiquitously present in all adherens junctions, the other two showed selective and mutually exclusive association with either cell-substrate or cell-cell adhesions. Talin was abundant in focal contacts and in dense plaques of smooth muscle, but was essentially absent from intercellular junctions such as intercalated disks or adherens junctions of lens fibers. The 135-kD protein, on the other hand, was present in the latter two loci and was apparently absent from membrane-bound plaques of gizzard or from focal contacts. Radioimmunoassay of tissue extracts and immunolabeling of cultured chick lens cells indicated that the selective presence of talin and of the 135-kD protein in different cell contacts is spatially regulated within individual cells. On the basis of these findings it was concluded that adherens junctions are molecularly heterogeneous and consist of at least two major subgroups. Contacts with noncellular substrates contain talin and vinculin but not the 135-kD protein, whereas their intercellular counterparts contain the latter two proteins and are devoid of talin. The significance of these results and their possible relationships to contact-induced regulation of cell behavior are discussed.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Geometry of actin-membrane attachments in the smooth muscle cell: the localisations of vinculin and alpha-actinin.

Antibodies to vinculin, a component of actin-membrane attachment sites, revealed by immunofluorescence microscopy a parallel co-axial array of continuous rib-like bands on the surface of isolated vertebrate smooth muscle cells. Images of extended and shortened cells showed that these ribs remain co-axially organised on contraction. Reference to earlier studies and labelling of thin sections indicates that the ribs correspond in position to the adhesion plaques previously described in electron microscope studies. Alpha-actinin showed a punctate distribution consistent with its presence in the cytoplasmic dense bodies, but did not show a constant association with the vinculin-containing ribs. It is suggested that alpha-actinin is an intracellular actin linker and not membrane associated, as earlier supposed, and that vinculin is, as deduced by others, a mediator of actin membrane attachment. The apparent co-association of these two proteins, noted previously, is concluded to arise from the inevitable geometrical apposition of peripheral and pre-terminal parts of the contractile machinery with the cell membrane.     

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.     

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.     

Smooth muscle cells of the chicken aortic arch differ from those in the gizzard and the femoral artery in the distribution of F-actin, alpha-actinin and filamin

The distribution of F-actin, alpha-actinin and filamin in smooth muscle cells of the chicken was examined by immunofluorescent and immunoelectron microscopy. Those from the gizzard, the femoral artery and the aortic arch were compared. F-Actin labeled by NBD-phallacidin was seen diffusely distributed in the sarcoplasm in the gizzard and the femoral artery, but in the aorta it was observed as streaks and spots, with unstained areas in between. Epon sections of the aortic arch showed that bundles of thin myofilaments run in various directions interspersed with areas mostly occupied by intermediate filaments. alpha-Actinin labelling occurred in dense plaques along the sarcolemma in all the muscles examined. While dense bodies in the sarcoplasm were common and labelled for alpha-actinin in the gizzard and the femoral artery, hardly any were seen in the aortic arch and little labelling for alpha-actinin was observed in the sarcoplasm. Filamin was concentrated along the periphery of dense bodies and plaques in the gizzard and the femoral artery, but it was seen diffusely in the sarcoplasm of the aortic muscle. After chemical skinning of the latter, filamin labelling persisted only in the F-actin bundles, and other areas became negative. The present results show that smooth muscle cells of the aortic arch contrast with those of the gizzard and even with those of the femoral artery in the distribution of F-actin, alpha-actinin and filamin. The mechanisms of contraction and/or stress maintenance in the aortic smooth muscle may be different from those in other smooth muscles.     

Immunolocalization of meta-vinculin in human smooth and cardiac muscles

Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.     

Distribution of active protein kinase C in smooth muscle.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.     

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.     

LPP,a LIM protein highly expressed in smooth muscle

An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118–129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (0.5–1 ng/µg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker -actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or -actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase. LIM protein; dense plaque; Rho-kinase; nuclear transport; cell migration     

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.     

Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodriguez Fernandez, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zoller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.