Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell

La Jolla Cancer Research Foundation, California 92037.
The Journal of Cell Biology (Impact Factor: 9.83). 06/1993; 121(4):909-21.
Source: PubMed


A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

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Available from: Jean-Luc Coll
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    • "The reasons for choosing these molecules are: (1) αactinin and vinculin are located at the attachment points of actin filaments to the plasma membrane and both molecules are involved in the organization of actin filament bundles (Craig and Pardo, 1979; Geiger, 1982; Geiger et al., 1980; Lazarides and Burridge, 1975); (2) at the adherens junctions, α-actinin is a linkage between the cadherin-catenin complex and the cytoskeleton, since it binds α-catenin (Knudsen et al., 1995; Nieset et al., 1997), vinculin (Kroemker et al., 1994; McGregor et al., 1994; Wachsstock et al., 1987), and actin (Burridge and Feramisco, 1981; Duhaiman and Bamburg, 1984; Kuhlmann et al., 1992); (3) α-actinin and vinculin are substrates of PKC phosphorylation in vitro and in vivo (Kawamoto and Hidaka, 1984; Schwienbacher et al., 1996; Werth et al., 1983; Werth and Pastan, 1984). Furthermore, since phosphorylated vinculin is enriched in the insoluble fraction of detergent treated cells (Geiger, 1982), phosphorylation might be important in vinculin function; and (4) vinculin is a crucial molecule in the maintenance of the epithelial phenotype (Coll et al., 1995; Goldman et al., 1995; Rodriguez-Fernandez et al., 1992; Samuels et al., 1993). "
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    ABSTRACT: The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.
    Full-text · Article · Jan 1999 · Journal of Cell Science
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    • "A differentially spliced version of vinculin called metavinculin (125 kDa) is found most abundantly in smooth, cardiac and skeletal muscle, and in blood platelets (Gimona et al., 1988; Turner and Burridge, 1989). A role for vinculin in cell-matrix adhesion is indicated by the 'rescue' of a non-adhesive mutant clone of F9 cells called 5.51, which expresses no vinculin (Calogero et al., 1991; Grover et al., 1987) but is induced to adhere to the matrix after the introduction of a vinculin expression vector (Samuels et al., 1993). The 5.51 cell line has multiple adhesion gene mutations and may give misleading information about the specific role of vinculin, therefore, we undertook the selective inactivation of the vinculin gene in F9 cells in order to determine its specific effects on cell-matrix and cell-cell adhesion and other cell behaviors. "
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    ABSTRACT: Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.
    Full-text · Article · Jul 1998 · Journal of Cell Science
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    • "Vinculin is an abundant cytoskeletal protein present at cell-matrix and cell-cell contacts that interacts with a-actinin, talin, F-actin, tensin, and paxillin (for review see Hemmings et al., 1995). An important role for vinculin in adhesion has been inferred by a number of studies, including the capacity of exogenous expression of vinculin to complement nonadherence of a vinculin-deficient embryonal carcinoma cell line (Samuels et al., 1993). Additionally, a role for vinculin in cell morphology, motility, and anchorage-dependent growth has been demonstrated using antisense suppression of vinculin expression in NIH3T3 cells (Fern~indez et al., 1993). "
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    ABSTRACT: Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.
    Preview · Article · Nov 1996 · The Journal of Cell Biology
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