Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Aug 1;107(2):563–572. doi: 10.1083/jcb.107.2.563

Differential localization of tropomyosin isoforms in cultured nonmuscle cells

PMCID: PMC2115218  PMID: 3047141

Abstract

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two- dimensional gel analysis to have specificities to human EJ TM-3 and TM- 5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity- purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernstein B. W., Bamburg J. R. Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin-depolymerizing factor (ADF). Cell Motil. 1982;2(1):1–8. doi: 10.1002/cm.970020102. [DOI] [PubMed] [Google Scholar]
  2. Bonder E. M., Mooseker M. S. Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process. J Cell Biol. 1983 Apr;96(4):1097–1107. doi: 10.1083/jcb.96.4.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Burgess D. R., Broschat K. O., Hayden J. M. Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins. J Cell Biol. 1987 Jan;104(1):29–40. doi: 10.1083/jcb.104.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen I., Cohen C. A tropomyosin-like protein from human platelets. J Mol Biol. 1972 Jul 21;68(2):383–387. doi: 10.1016/0022-2836(72)90220-3. [DOI] [PubMed] [Google Scholar]
  6. Cooper H. L., Feuerstein N., Noda M., Bassin R. H. Suppression of tropomyosin synthesis, a common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes. Mol Cell Biol. 1985 May;5(5):972–983. doi: 10.1128/mcb.5.5.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Côté A., Doucet J. P., Trifaró J. M. Adrenal medullary tropomyosins: purification and biochemical characterization. J Neurochem. 1986 Jun;46(6):1771–1782. doi: 10.1111/j.1471-4159.1986.tb08495.x. [DOI] [PubMed] [Google Scholar]
  8. Côté G. P., Smillie L. B. Preparation and some properties of equine platelet tropomyosin. J Biol Chem. 1981 Nov 10;256(21):11004–11010. [PubMed] [Google Scholar]
  9. Côté G. P. Structural and functional properties of the non-muscle tropomyosins. Mol Cell Biochem. 1983;57(2):127–146. doi: 10.1007/BF00849190. [DOI] [PubMed] [Google Scholar]
  10. Endo T., Masaki T. Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins. J Biochem. 1982 Nov;92(5):1457–1468. doi: 10.1093/oxfordjournals.jbchem.a134070. [DOI] [PubMed] [Google Scholar]
  11. Fattoum A., Hartwig J. H., Stossel T. P. Isolation and some structural and functional properties of macrophage tropomyosin. Biochemistry. 1983 Mar 1;22(5):1187–1193. doi: 10.1021/bi00274a031. [DOI] [PubMed] [Google Scholar]
  12. Fine R. E., Blitz A. L. A chemical comparison of tropomyosins from muscle and non-muscle tissues. J Mol Biol. 1975 Jul 5;95(3):447–454. doi: 10.1016/0022-2836(75)90202-8. [DOI] [PubMed] [Google Scholar]
  13. Fine R. E., Blitz A. L., Hitchcock S. E., Kaminer B. Tropomyosin in brain and growing neurones. Nat New Biol. 1973 Oct 10;245(145):182–186. doi: 10.1038/newbio245182a0. [DOI] [PubMed] [Google Scholar]
  14. Fowler V. M., Bennett V. Erythrocyte membrane tropomyosin. Purification and properties. J Biol Chem. 1984 May 10;259(9):5978–5989. [PubMed] [Google Scholar]
  15. Giometti C. S., Anderson N. L. A variant of human nonmuscle tropomyosin found in fibroblasts by using two-dimensional electrophoresis. J Biol Chem. 1981 Nov 25;256(22):11840–11846. [PubMed] [Google Scholar]
  16. Helfman D. M., Cheley S., Kuismanen E., Finn L. A., Yamawaki-Kataoka Y. Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol. 1986 Nov;6(11):3582–3595. doi: 10.1128/mcb.6.11.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hendricks M., Weintraub H. Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation. Mol Cell Biol. 1984 Sep;4(9):1823–1833. doi: 10.1128/mcb.4.9.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hendricks M., Weintraub H. Tropomyosin is decreased in transformed cells. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5633–5637. doi: 10.1073/pnas.78.9.5633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hinssen H. An actin-modulating protein from Physarum polycephalum. II. Ca++-dependence and other properties. Eur J Cell Biol. 1981 Feb;23(2):234–240. [PubMed] [Google Scholar]
  20. Hitchcock S. E., Carisson L., Lindberg U. Depolymerization of F-actin by deoxyribonuclease I. Cell. 1976 Apr;7(4):531–542. doi: 10.1016/0092-8674(76)90203-8. [DOI] [PubMed] [Google Scholar]
  21. Ishimoda-Takagi T. Immunological purification of sea urchin egg tropomyosin. J Biochem. 1978 Jun;83(6):1757–1762. doi: 10.1093/oxfordjournals.jbchem.a132090. [DOI] [PubMed] [Google Scholar]
  22. Keiser T., Wegner A. Isolation from bovine brain of tropomyosins that bind to actin filaments with different affinities. FEBS Lett. 1985 Jul 22;187(1):76–80. doi: 10.1016/0014-5793(85)81218-7. [DOI] [PubMed] [Google Scholar]
  23. Kobayashi R., Tawata M., Mace M. L., Jr, Bradley W. A., Field J. B. Purification and characterization of tropomyosin from bovine thyroid. Biochim Biophys Acta. 1982 Apr 3;702(2):220–232. doi: 10.1016/0167-4838(82)90506-4. [DOI] [PubMed] [Google Scholar]
  24. Koteliansky V. E., Shirinsky V. P., Glukhova M. A., Nowak E., Dabrowska R. The effect of non-muscle tropomyosin on the interaction of filamin with F-actin. FEBS Lett. 1983 May 2;155(1):85–87. doi: 10.1016/0014-5793(83)80214-2. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lazarides E. Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells. J Cell Biol. 1975 Jun;65(3):549–561. doi: 10.1083/jcb.65.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lazarides E. Two general classes of cytoplasmic actin filaments in tissue culture cells: the role of tropomyosin. J Supramol Struct. 1976;5(4):531(383)–563(415). doi: 10.1002/jss.400050410. [DOI] [PubMed] [Google Scholar]
  28. Leavitt J., Latter G., Lutomski L., Goldstein D., Burbeck S. Tropomyosin isoform switching in tumorigenic human fibroblasts. Mol Cell Biol. 1986 Jul;6(7):2721–2726. doi: 10.1128/mcb.6.7.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leonardi C. L., Warren R. H., Rubin R. W. Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells. Biochim Biophys Acta. 1982 Apr 29;720(2):154–162. doi: 10.1016/0167-4889(82)90007-6. [DOI] [PubMed] [Google Scholar]
  30. Lin J. J., Chou C. S., Lin J. L. Monoclonal antibodies against chicken tropomyosin isoforms: production, characterization, and application. Hybridoma. 1985 Fall;4(3):223–242. doi: 10.1089/hyb.1985.4.223. [DOI] [PubMed] [Google Scholar]
  31. Lin J. J., Feramisco J. R. Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody. Cell. 1981 Apr;24(1):185–193. doi: 10.1016/0092-8674(81)90514-6. [DOI] [PubMed] [Google Scholar]
  32. Lin J. J., Helfman D. M., Hughes S. H., Chou C. S. Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells. J Cell Biol. 1985 Mar;100(3):692–703. doi: 10.1083/jcb.100.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lin J. J., Matsumura F., Yamashiro-Matsumura S. Tropomyosin-enriched and alpha-actinin-enriched microfilaments isolated from chicken embryo fibroblasts by monoclonal antibodies. J Cell Biol. 1984 Jan;98(1):116–127. doi: 10.1083/jcb.98.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. MacLeod A. R., Houlker C., Reinach F. C., Smillie L. B., Talbot K., Modi G., Walsh F. S. A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7835–7839. doi: 10.1073/pnas.82.23.7835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maruyama K., Ohashi K. Tropomyosin inhibits the interaction of F-actin and filamin. J Biochem. 1978 Oct;84(4):1017–1019. doi: 10.1093/oxfordjournals.jbchem.a132184. [DOI] [PubMed] [Google Scholar]
  36. Matsumura F., Lin J. J., Yamashiro-Matsumura S., Thomas G. P., Topp W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983 Nov 25;258(22):13954–13964. [PubMed] [Google Scholar]
  37. Matsumura F., Yamashiro-Matsumura S., Lin J. J. Isolation and characterization of tropomyosin-containing microfilaments from cultured cells. J Biol Chem. 1983 May 25;258(10):6636–6644. [PubMed] [Google Scholar]
  38. Matsumura F., Yamashiro-Matsumura S. Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin. J Biol Chem. 1986 Apr 5;261(10):4655–4659. [PubMed] [Google Scholar]
  39. Matsumura F., Yamashiro-Matsumura S. Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells. J Biol Chem. 1985 Nov 5;260(25):13851–13859. [PubMed] [Google Scholar]
  40. Nishida E., Muneyuki E., Maekawa S., Ohta Y., Sakai H. An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin. Biochemistry. 1985 Nov 5;24(23):6624–6630. doi: 10.1021/bi00344a049. [DOI] [PubMed] [Google Scholar]
  41. Nomura M., Yoshikawa K., Tanaka T., Sobue K., Maruyama K. The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin). Eur J Biochem. 1987 Mar 16;163(3):467–471. doi: 10.1111/j.1432-1033.1987.tb10892.x. [DOI] [PubMed] [Google Scholar]
  42. Paulin D., Perreau J., Jakob H., Jacob F., Yaniv M. Tropomyosin synthesis accompanies formation of actin filaments in embryonal carcinoma cells induced to differentiate by hexamethylene bisacetamide. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1891–1895. doi: 10.1073/pnas.76.4.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pinder J. C., Gratzer W. B. Structural and dynamic states of actin in the erythrocyte. J Cell Biol. 1983 Mar;96(3):768–775. doi: 10.1083/jcb.96.3.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Puszkin S., Maimon J., Puszkin E. Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins. Biochim Biophys Acta. 1978 Nov 2;513(2):205–220. doi: 10.1016/0005-2736(78)90174-8. [DOI] [PubMed] [Google Scholar]
  45. Schloss J. A., Goldman R. D. Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization. J Cell Biol. 1980 Dec;87(3 Pt 1):633–642. doi: 10.1083/jcb.87.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Small J. V. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J Cell Biol. 1981 Dec;91(3 Pt 1):695–705. doi: 10.1083/jcb.91.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Talbot K., MacLeod A. R. Novel form of non-muscle tropomyosin in human fibroblasts. J Mol Biol. 1983 Feb 15;164(1):159–174. doi: 10.1016/0022-2836(83)90091-8. [DOI] [PubMed] [Google Scholar]
  49. Warren R. H., Gordon E., Azarnia R. Tropomyosin in peripheral ruffles of cultured rat kidney cells. Eur J Cell Biol. 1985 Sep;38(2):245–253. [PubMed] [Google Scholar]
  50. Zeece M. G., Robson R. M., Bechtel P. J. Interaction of alpha-actinin, filamin and tropomyosin with F-actin. Biochim Biophys Acta. 1979 Dec 14;581(2):365–370. doi: 10.1016/0005-2795(79)90258-7. [DOI] [PubMed] [Google Scholar]
  51. der Terrossian E., Fuller S. D., Stewart M., Weeds A. G. Porcine platelet tropomyosin. Purification, characterization and paracrystal formation. J Mol Biol. 1981 Nov 25;153(1):147–167. doi: 10.1016/0022-2836(81)90531-3. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES