Search

Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding its activation, regulation and function. Here we discuss the current understanding of the roles of vinculin in cell-cell and cell-matrix adhesions. Emphasis is placed on the how vinculin is recruited, activated and regulated. We also highlight the recent understanding of how vinculin responds to and transmits force at integrin- and cadherin-containing adhesion complexes to the cytoskeleton. Furthermore, we discuss roles of vinculin in binding to and rearranging the actin cytoskeleton.

Using immunoblotting, immunprecipitation with subsequent fragment mass spectrometry, and immunolocalization techniques, we have detected the actin-binding ca. 120-kDa protein drebrin, originally identified in - and thought to be specific for - neuronal cells, in diverse kinds of human and bovine non-neuronal cells. Drebrin has been found in numerous cell culture lines and in many tissues of epithelial, endothelial, smooth muscle and neural origin but not in, for example, cardiac, skeletal and certain types of smooth muscle cells, in hepatocytes and in the human epithelium-derived cell culture line A-431. By double-label fluorescence microscopy we have found drebrin enriched in actin microfilament bundles associated with plaques of cell-cell contact sites representing adhering junctions. These drebrin-positive, adhering junction-associated bundles, however, are not identical with the vinculin-containing, junction-attached bundles, and in the same cell both subtypes of microfilament-anchoring plaques are readily distinguished by immunolocalization comparing drebrin and vinculin. The intracellular distribution of the drebrin- and the vinculin-based microfilament systems has been studied in detail by confocal fluorescence laser scanning microscopy in monolayers of the polar epithelial cell lines, MCF-7 and PLC, and drebrin has been found to be totally and selectively absent in the notoriously vinculin-rich focal adhesions. The occurrence and the possible functions of drebrin in non-neuronal cells, notably epithelial cells, and the significance of the existence of two different actin-anchoring junctional plaques is discussed.

Orientation of collagen fibrils is a key event in the development of many tissues. In the intervertebral disc, the outer annulus fibrosus comprises lamellae of parallel collagen fibres, the direction of orientation of the long axis of which alternates in angle between lamellae. In development, this organisation is preceded by the formation of sheets of oriented fibroblasts, which then deposit the oriented lamellae. Here, using fluorescent labelling, confocal and electron microscopic techniques on developmental series, we show that the orientation of cells in lamellae is associated with the formation of adherens junctions intercellularly, involving cadherins and vinculin, and longitudinal stress fibres (label for filamentous actin and tropomyosin) intracellularly. The stress fibres direct the initial elongation of cells and control the deposition of oriented extracellular matrix via junctional complexes with the matrix involving vinculin and alpha 5 beta 1 integrins, which in turn promote the formation of oriented fibronectin at the cell surface; oriented collagen is deposited between cells at the same stages. Shortly after birth, the stress fibres disappear, probably because cells now gain orientational cues from the matrix, and are undergoing differentiation-related changes to form fibrocartilage cells. Dev Dyn 1999;215:179-189., (Copyright 1999 Wiley-Liss, Inc.)

Cell adhesion to laminin 1 or to fibronectin is mediated by distinct sets of integrins and is differentially regulated by protein kinase C (PKC). It suggests that upon integrin ligation to laminin 1 or to fibronectin different intracellular signaling pathways could be activated. we have therefore investigated the formation of signaling complexes induced during cell adhesion to laminin 1 or to fibronectin. Following cell adhesion to laminin 1 the re-arrangement of the cytoskeleton was slower than that observed on fibronectin and it was activated by treating the cells with H-7, an inhibitor of PKC. Conversely, treatment of laminin-adhering cells with a PKC activator resulted in a rapid disorganization of the actin cyto skeleton while a similar treatment had no effect on fibronectin-adhering cells. These results suggested that the structural organization of the adhesion complexes might be substrate-specific and might correspond to a different arrangement of cytoskeletal and/or cytoplasmic proteins. Reflection interference contrast microscopy (RICM) images revealed that cell-substratum contacts formed on laminin 1 were not well differentiated in contrast to those developed on fibronectin. However, immunofluorescence staining revealed a similar organisation of actin microfilaments, talin and phosphotyrosyl-containing proteins on both substrates. In contrast, differences were observed for vinculin distribution within cells spread on fibronectin or on laminin 1. Following cell adhesion to fibronectin most of the vinculin appeared as thick patches at the tips of the actin stress fibers while in laminin-adhering cells vinculin was recruited into thin streaks localized at the end of only some actin stress fibers.

The endothelial cell is unique because it must undergo a transition from a resting cell with a cytoskeleton organized for barrier function to one which promotes cell translocation following denuding endothelial injury. Since actin microfilaments are critical for both maintaining the integrity of the resting monolayer and for optimum reendothelialization, we carried out a detailed study of the organization of microfilaments as the cell undergoes the transition from a resting to a translocating cell. We used an in vitro model in which a linear wound was made in a confluent monolayer of porcine aortic endothelial cells. The complex reorganization of actin microfilament bundles following injury and their relationship to microtubules and vinculin was studied in cells at the wound edge using immunofluorescent scanning laser confocal microscopy and time-lapse videomicroscopy. In the resting confluent monolayer, microfilaments were present as a dense peripheral band (DPB) located toward the upper part of the cell and as central microfilament bundles at the substratum. Three distinct stages of microfilament reorganization occurred sequentially during early repair. Stage 1 followed wounding and involved the reduction of the DPBs of microfilaments and associated peripheral cell-cell vinculin plaques. This was associated with rapid forward actin-based lamellipodia extrusions and cell elongation. Low-dose cytochalasin, which did not disrupt the morphology of microfilament bundles, reduced elongation. Stage 2 was characterized by central microfilaments behind the lamellipodia distributed parallel to the wound edge with vinculin plaques at their tips. This was associated with prominent spreading at the front of the cell which enhanced the extent of coverage of the denuded wound area. Stage 3 was characterized by the orientation of central microfilaments perpendicular to the wound edge with vinculin plaques at their tips and was associated with the initiation of cell translocation. There was no specific structural association between central microfilaments and microtubules as the former were toward the substratum while the latter were toward the center and upper part of the cell. Thus, the sequential appearance of three patterns of microfilament distribution define the cytoskeletal events that regulate the reestablishment of endothelial integrity following denuding endothelial injury.

We used quick-freeze, deep-etch, rotary-replication transmission electron microscopy to determine at molecular resolution the organization of microfilaments at the cytoplasmic surface of the sarcolemma of Xenopus myocytes. We demonstrate that actin microfilaments interact with the sarcolemma in two distinct ways. In one, which resembled focal contacts in Xenopus fibroblasts [Samuelsson et al., 1993: J. Cell Biol. 122:485-496], bundles of microfilaments approached the sarcolemma at sites containing aggregates of membrane-associated particles. Immunogold cytochemistry showed that these particle aggregates contained vinculin, talin and beta 1-integrin. In the second, which covered most of the cytoplasmic surface of the sarcolemma, individual actin microfilaments formed an extensive, lattice-like array. Particle aggregates associated with this array of actin microfilaments also labeled with antibodies to vinculin, talin and beta 1-integrin. The unique, lattice-like association of actin microfilaments with the membrane in Xenopus myocytes suggests that the organization of actin filaments over most of the sarcolemma is distinct from focal contacts, mediating widespread associations of the actin cytoskeleton with the cytoplasmic membrane face.

Adhesion to proteins of the extracellular matrix exerts a profound influence upon cell function and behavior. Similar adhesive interactions mediate the spreading of cultured cells upon artificial substrata. Recently we observed that thyrotropin (TSH) and intercellular contact regulated thyroid cell-substrate adhesion to inhibit cell spreading, but not initial attachment. This is a mechanism which preserves thyroid follicular differentiation in culture. In the present study we have investigated the role of cytoplasmic components in mediating thyroid cell adhesion to collagen. The earliest change associated with cell spreading was the accumulation of vinculin and phosphotyrosine in developing focal adhesions, which was followed by stress fiber and microtubule assembly. Genistein, an inhibitor of tyrosine kinases, and cytochalasin B inhibited cell spreading and focal adhesion formation without affecting initial attachment to substrate. In contrast microtubule disorganization by colchicine did not alter any parameter of thyroid cell-substrate adhesion. These observations indicate that protein tyrosine phosphorylation and dynamic microfilament integrity are essential for attached thyroid cells to spread upon substrate. They are therefore potential intracellular loci at which TSH and intercellular contact may regulate cell adhesion to extracellular matrix and influence thyroid cell behavior.

The beta-thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin beta 4 (T beta 4) and thymosin beta 10 (T beta 10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of beta-thymosins, we have used isoform-specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of T beta 4 and T beta 10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both beta-thymosins are uniformly distributed within the cytoplasm. cDNA-mediated overexpression of beta-thymosins in CV1 fibroblasts induced extensive loss of phalloidin-stained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing beta-thymosin were observed to divide. In these cells, beta-thymosin was excluded from the midbody which contains an actin filament-rich contractile ring. Our results indicate that T beta 4 and T beta 10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells.

Addition of protein kinase inhibitor H-7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169-178] it was demonstrated that intercellular junctions in H-7-treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H-7-treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact-attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N-cadherin associated with cell-cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H-7 suppresses cellular contractility. Thus, short pretreatment with H-7 leads to strong inhibition of the ATP-induced contraction of saponin permeabilized cells. Comparison of H-7 effects with those of other kinase inhibitors revealed that H-7-induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31-8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H-7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell-cell junctions in low-calcium medium.

Contact guidance refers to the reactions of cells with the topography of their substratum. Current hypotheses on the mechanism of contact guidance focus on the dynamic behaviour of the cytoskeletal components, but most observations have been made on cells that have already become oriented with topographic features of the substratum. The purpose of this study was to examine the sequence in which microtubules, focal contacts and microfilament bundles become aligned to the substratum topography as fibroblasts spread on grooved substrata. Human gingival fibroblasts were trypsinized and seeded onto grooved titanium surfaces produced by micromachining, as well as onto control smooth surfaces. After observation and photography of the spreading cells at times up to 6 hours, the cells were fixed and exposed to one or more of the following antibodies or fluorescent stains: phallacidin to stain actin filaments, monoclonal anti-tubulin, monoclonal anti-vinculin, anti-mouse IgG labelled with Texas-Red or FITC, and/or an aldehyde-reactive stain to identify the cell outline. The cells were photographed and cell area, shape and orientation were calculated. Cells were also examined with confocal microscopy to obtain optical sections so that cell height as well as the precise locations of the cytoskeletal components with respect to the vertical dimension of the grooved substrata could be determined. Microtubules were the first element to become oriented parallel to the direction of the grooves and were first aligned at the bottom of the grooves. This alignment of microtubules was evident as early as 20 minutes after plating and preceded the orientation of the cell as a whole. Aligned actin microfilament bundles were not observed until 40-60 minutes and were observed first at the wall-ridge edges. At early times, focal contacts were distributed radially, but only after 3 hours did the majority of cells demonstrate aligned focal contacts. If the first cytoskeletal component to become aligned is the prime determinant of cell orientation, then these data suggest that microtubules in human gingival fibroblasts may determine cell orientation on grooved titanium surfaces. By analogy with microtubule behaviour in other systems, we suggest that microtubule orientation on grooved substrata may occur as a result of the substratum establishing shear-free planes.

We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.

Thrombin is present at sites of vascular injury. The objective of this study was to determine if thrombin may regulate endothelial repair. To address this our study was designed to determine the effect of thrombin on cell shape and microfilament distribution of porcine aortic endothelial cells in low-density cultures. Since these cells grow as islands of cells, low-density cultures serve as a model for conditions in which reendothelialization occurs from several foci following prominent patchy endothelial denudation. Thrombin incubation for 1, 2, and 4 hr at 0.5, 4, and 8 U/ml caused a significant increase in both the surface area of endothelial cells and the amount of microfilament bundles occupying the cell surface area. The increase in microfilament bundles was more than would be expected from the increased surface area. However, the pattern of distribution of microfilaments and of vinculin adhesion plaques within the cells was not altered by thrombin. Attempts at understanding the molecular mechanisms involved were undertaken. The role of protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) caused cell spreading as well but no increase in microfilaments within the first 2 hr of incubation. Some cells showed mild disruption of microfilaments. Following 24 hr incubation with PMA, which reduces protein kinase C, the cells showed retraction and arborization along with thinning and disruption of microfilaments. Removal of PMA and addition of thrombin reversed these changes to normal within 2 hr while washout of PMA on its own did not alter cell shape or microfilaments. Thrombin also partially reversed the effects of H-7, a protein kinase C inhibitor, which on its own also caused cell retraction and disruption of microfilaments. Neomycin sulfate did not alter the thrombin effect suggesting that the participation of the phosphotidylinositol system is not directly required. In conclusion, thrombin promotes endothelial activities which are associated with long-term endothelial repair; however, these effects do not appear to be directly due to the involvement of conventional isoforms of protein kinase C or the phosphotidylinositol system.

The primary function of the osteoclast is that of the major cell mediating bone resorption. They are actively migrating cells but during resorption they polarize to form a specialized tight attachment structure, the sealing zone, adjacent to the mineralized bone matrix. The processes of adhesion to, and migration on, bone involves cell adhesion molecules, integrins, interacting with their ligands in bone. We have used confocal microscopy to analyse, in rat osteoclasts cultured on bone and glass substrata, the distribution of vitronectin receptor, the major integrin of osteoclasts, and cytoskeletal proteins that it may be linked to. Double staining for F-actin and vinculin, and for vinculin with talin, revealed that cytoskeletal organization differs at various activation states of osteoclasts. Microfilament structures were flat, of 1.5 microns size, and concentrated near the bone surface. The vitronectin receptor was localized both in the basolateral membrane (away from the bone surface) and in the ruffled border (adjacent to bone) in osteoclasts cultured on bone, but was detected mainly in the basolateral membrane when cultured on glass. The vitronectin receptor appeared to be condensed on small microvilli-like projections on the basolateral membrane of osteoclasts on either bone or glass and may provide a route for alternative signalling pathways to modify osteoclast behaviour, other than by influencing cell adhesion directly. The leading edges of migrating osteoclasts, and the attachment structure, a broad vinculin band, which forms before bone resorption, also expressed vitronectin receptor, particularly when the antibody against the alpha v subunit was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.