Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells.

Abstract

Abstract. We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alphaactinin and vinculin in living cultured African green monkey kidney (BSC-1) cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Before treatment, microinjected alpha-actinin shows characteristic distribution along stress fibers and at adhesion plaques; vinculin is localized predominantly at adhesion plaques. Soon after the addition of TPA, highly dynamic membrane ruffles begin to form. These incorporate a large amount of alpha-actinin but little vinculin. Alpha-actinin is subsequently depleted, more or less uniformly, from stress fibers. Disrupted stress fibers often fragment into aggregates and move into the perinuclear region. Careful analyses of fluorescence intensity distribution indicate that alphaactinin is depleted more rapidly from adhesion plaques than from stress fibers. Furthermore, the depletion of alpha-actinin from adhesion plaques is also faster than either the depletion of vinculin or the disappearance of focal contacts. These observations indicate that TPA may initiate disruption of stress fibers by interfering with a link between alpha-actinin and vinculin, causing alpha-actinin to be preferentially depleted from adhesion plaques. MANY cultured nonmuscle cells contain a well-organized set of stress fibers, which are large bundles of actin filaments associated with various accessory proteins such as alpha-actinin, myosin, and tropomyosin (for a review see reference 22). One of the most consistent characteristics of cells infected with transforming viruses, cells treated with tumor-promoting phorbol esters, and cells stimulated with growth factors, is the disorganization of stress fibers (for a review see reference 33). Since all of these agents stimulate various kinds of protein kinases (6,8,9,12,17,25), it is possible that a common pathway is followed during the disruption process.The reorganization of actin and vinculin induced by the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA),~ has recently been studied using fluorescent phalloidin staining, immunofluorescence, and electron microscopy. Shortly after treatment, large actin-containing rufties appear and stress fibers start to disappear from the cytoplasm (26, 30). Vinculin can be detected at the ends of stress fibers, where adhesion plaques are located, during the early stage of disruption. At steady state, the cell contains prominent ruffles and numerous aggregates of actin and vinculin. Although these studies have shown the dramatic effects of Abbreviations used in this paper." DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; IATR, iodoacetamidotetramethylrhodamine; IRM, interference reflection microscopy; 4-alpha-PDD, 4-alpha-phorbol 12,13-didecanoate: TPA, 12-O-tetradecanoylphorbol-13-acetate.TPA, the actual process of structural reorganization can only be speculated upon since most observations were performed on different, ...

Cell locomotion and focal adhesions are regulated by substrate flexibility

“…Rhodamine and fluorescein-conjugated secondary antibodies were obtained from Sigma. Staining of cells with fluorescein-phalloidin (Molecular Probes), microinjection of fluorescently labeled vinculin, and fluorescence microscopy were performed as described (21)(22)(23) …”

Morphological Changes and Reorganization of Actinfilaments in Human Myeloid Leukemia Cells Induced by a Novel Protein Phosphatase Inhibitor, Tautomycin.

“…Actinfilaments bind to plasma membrane through various actin binding proteins (20) including vinculin and annexin which are phosphorylated by several kinases (21 , 22). Phorbol esters reorganize actin and its binding proteins (23,24). Blebbing was also observed in melanomacells deficient in the actinfilament cross-linking protein, filamin (25).…”

Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

“…These residual focal adhesions continued to stain for vinculin, talin, and integrin but not a-actinin . Evidence consistent with the loss of focal adhesions being secondary to the loss of stress fibers comes from the studies of Meigs and Wang (1986) who noted that loss of a-actinin from stress fibers and focal adhesions preceded the loss of vinculin from focal adhesions in BSC-1 cells treated with phorbol esters . Stress fibers can also be disrupted by microinjection into cells of a number of agents which appear to decrease or inhibit the interaction of myosin with actin, such as the catalytic subunit of the cAMP-dependent protein kinase, or antibodies to the myosin light chain kinase (Lamb et al, 1988) or type 1 protein phosphatase (Fernandez et al, 1990) .…”