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Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin‐specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin‐specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0‐7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin‐specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin‐rich focal contacts, while cells grown on poorly adhesive substrate poly(2‐hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5‐fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5‐fold decrease in the levels of vinculin‐specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adherens‐type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105–120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine‐specific phosphatases (PTP), namely H2O2 and vanadate, leads to a dramatic increase in AJ‐associated phosphotyrosine which was apparent already within 2–5 min of treatment and progressed upon further incubation. Examination of H2O2 vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding‐up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO‐I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carried out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell‐cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5‐1 h) in a conspicuous belt‐like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA‐treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle‐containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane‐detached vinculin‐rich material from the zonula adhaerens . The experiments show that, upon release from their junction‐mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt‐like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half‐desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial cells contain a class of intermediate‐sized filaments formed by proteins related to epidermal alpha‐keratins (‘cytokeratins’). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000‐68 000) and isoelectric pH (5.0‐8.5). We have separated, by two‐dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one‐ or two‐dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the ‘basic cytokeratin subfamily’ defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis‐specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad‐range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

We report here on a new 135‐kd membrane protein which is specifically associated with intercellular adherens‐type junctions. This surface component was identified by a monoclonal antibody, ID‐7.2.3, raised against detergent‐extracted components of membranes of chicken cardiac muscle rich in intercalated discs. The antibodies stain extensively adherens junctions in intact cardiac muscle and in lens, as well as in cultured cells derived from these tissues. In living cultured cells only very little immunolabelling was obtained with ID‐7.2.3 antibodies, probably due to the limited accessibility of the antibodies to the intercellular gap. However, upon the removal of extracellular Ca2+ ions a dissociation of the junction occurred, leading to the rapid exposure of the 135‐kd protein. Immunoelectron microscopic labelling of EGTA‐treated, or detergent‐permeabilized cells indicated that the antigen is found along the plasma membrane and highly enriched in contact areas. Double immunolabelling for both the 135‐kd protein and vinculin pointed to the close association of the two in intercellular junctions and to the apparent absence of the former protein from the vinculin‐rich focal contacts of cultured cells and from dense plaque of smooth muscle. Immunoblotting indicated that the 135‐kd protein is present in many tissues but is particularly enriched in heart, lens and brain.