Abstract
We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alpha- actinin 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 alpha-actinin 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.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amato P. A., Unanue E. R., Taylor D. L. Distribution of actin in spreading macrophages: a comparative study on living and fixed cells. J Cell Biol. 1983 Mar;96(3):750–761. doi: 10.1083/jcb.96.3.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloom G. S., Lockwood A. H. Redistribution of myosin during morphological reversion of Chinese hamster ovary cells induced by db-cAMP. Exp Cell Res. 1980 Sep;129(1):31–45. doi: 10.1016/0014-4827(80)90328-6. [DOI] [PubMed] [Google Scholar]
- Boreiko C., Mondal S., Narayan K. S., Heidelberger C. Effect of 12-O-tetradecanoylphorbol-13-acetate on the morphology and growth of C3H/10T1/2 mouse embryo cells. Cancer Res. 1980 Dec;40(12):4709–4716. [PubMed] [Google Scholar]
- Boschek C. B., Jockusch B. M., Friis R. R., Back R., Grundmann E., Bauer H. Early changes in the distribution and organization of microfilament proteins during cell transformation. Cell. 1981 Apr;24(1):175–184. doi: 10.1016/0092-8674(81)90513-4. [DOI] [PubMed] [Google Scholar]
- Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
- Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
- Chen W. T., Singer S. J. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol. 1982 Oct;95(1):205–222. doi: 10.1083/jcb.95.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Collett M. S., Purchio A. F., Erikson R. L. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature. 1980 May 15;285(5761):167–169. doi: 10.1038/285167a0. [DOI] [PubMed] [Google Scholar]
- Cooper J. A., Bowen-Pope D. F., Raines E., Ross R., Hunter T. Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins. Cell. 1982 Nov;31(1):263–273. doi: 10.1016/0092-8674(82)90426-3. [DOI] [PubMed] [Google Scholar]
- Croop J., Dubyak G., Toyama Y., Dlugosz A., Scarpa A., Holtzer H. Effects of 12-O-tetradecanoyl-phorbol-13-acetate on Myofibril integrity and Ca2+ content in developing myotubes. Dev Biol. 1982 Feb;89(2):460–474. doi: 10.1016/0012-1606(82)90334-7. [DOI] [PubMed] [Google Scholar]
- Driedger P. E., Blumberg P. M. The effect of phorbol diesters on chicken embryo fibroblasts. Cancer Res. 1977 Sep;37(9):3257–3265. [PubMed] [Google Scholar]
- Ek B., Westermark B., Wasteson A., Heldin C. H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature. 1982 Feb 4;295(5848):419–420. doi: 10.1038/295419a0. [DOI] [PubMed] [Google Scholar]
- Evans R. R., Robson R. M., Stromer M. H. Properties of smooth muscle vinculin. J Biol Chem. 1984 Mar 25;259(6):3916–3924. [PubMed] [Google Scholar]
- Feramisco J. R., Burridge K. A rapid purification of alpha-actinin, filamin, and a 130,000-dalton protein from smooth muscle. J Biol Chem. 1980 Feb 10;255(3):1194–1199. [PubMed] [Google Scholar]
- Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
- Gilmore T., Martin G. S. Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine. Nature. 1983 Dec 1;306(5942):487–490. doi: 10.1038/306487a0. [DOI] [PubMed] [Google Scholar]
- Herman B., Pledger W. J. Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells. J Cell Biol. 1985 Apr;100(4):1031–1040. doi: 10.1083/jcb.100.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hynes R. Phosphorylation of vinculin by pp60src: what might it mean? Cell. 1982 Mar;28(3):437–438. doi: 10.1016/0092-8674(82)90196-9. [DOI] [PubMed] [Google Scholar]
- Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
- MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McNeil P. L., McKenna M. P., Taylor D. L. A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate. J Cell Biol. 1985 Aug;101(2):372–379. doi: 10.1083/jcb.101.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mellstroöm K., Höglund A. S., Nistér M., Heldin C. H., Westermark B., Lindberg U. The effect of platelet-derived growth factor on morphology and motility of human glial cells. J Muscle Res Cell Motil. 1983 Oct;4(5):589–609. doi: 10.1007/BF00712117. [DOI] [PubMed] [Google Scholar]
- Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rifkin D. B., Crowe R. M., Pollack R. Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton. Cell. 1979 Oct;18(2):361–368. doi: 10.1016/0092-8674(79)90055-2. [DOI] [PubMed] [Google Scholar]
- Rohrschneider L. R. Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3514–3518. doi: 10.1073/pnas.77.6.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rohrschneider L., Rosok M. J. Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus. Mol Cell Biol. 1983 Apr;3(4):731–746. doi: 10.1128/mcb.3.4.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlessinger J., Geiger B. Epidermal growth factor induces redistribution of actin and alpha-actinin in human epidermal carcinoma cells. Exp Cell Res. 1981 Aug;134(2):273–279. doi: 10.1016/0014-4827(81)90426-2. [DOI] [PubMed] [Google Scholar]
- Schliwa M., Nakamura T., Porter K. R., Euteneuer U. A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells. J Cell Biol. 1984 Sep;99(3):1045–1059. doi: 10.1083/jcb.99.3.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
- Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vasiliev J. M. Spreading of non-transformed and transformed cells. Biochim Biophys Acta. 1985;780(1):21–65. doi: 10.1016/0304-419x(84)90006-4. [DOI] [PubMed] [Google Scholar]
- Wang E., Goldberg A. R. Changes in microfilament organization and surface topogrophy upon transformation of chick embryo fibroblasts with Rous sarcoma virus. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4065–4069. doi: 10.1073/pnas.73.11.4065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Y. L. Reorganization of actin filament bundles in living fibroblasts. J Cell Biol. 1984 Oct;99(4 Pt 1):1478–1485. doi: 10.1083/jcb.99.4.1478. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Y. L., Taylor D. L. Distribution of fluorescently labeled actin in living sea urchin eggs during early development. J Cell Biol. 1979 Jun;81(3):672–679. doi: 10.1083/jcb.81.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Werth D. K., Niedel J. E., Pastan I. Vinculin, a cytoskeletal substrate of protein kinase C. J Biol Chem. 1983 Oct 10;258(19):11423–11426. [PubMed] [Google Scholar]
