Skip to main content
SpringerLink
Log in

Changes in the levels of human tropomyosins IEF 52,55, and 56 do not correlate with the loss of actin cables observed in SV 40 transformed MRC-5 fibroblasts

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

A mouse monoclonal antibody (mAb 1D122G9) raised against human tropomyosin IEF 52 (HeLa protein catalogue number, Mr=35 kd) has been characterized both in terms of specificity and patterns of immunofluorescence staining in Triton extracted cultured cells. As determined by two dimensional gel immunoblotting of HeLa cell proteins the antibody recognized IEF 52 and two other acidic proteins (IEF 55, Mr=31.8 kd; IEF 56, Mr=31 kd) previously identified as putative tropomyosin-like proteins. Immunofluorescence staining of Triton extracted cultured cells revealed the striated or interrupted pattern on the actin cables characteristic of tropomyosin staining. Quantitation of the three tropomyosins in Triton cytoskeletons from normal and SV 40 transformed human MRC-5 fibroblasts showed that the latter contained significantly less of tropomyosin IEF's 52 (52%) and 56 (72%) as compared to their normal counterparts. The ratios of these two tropomyosins to actin however was very similar for both types of cytoskeletons. This was not the case for tropomyosin IEF 55, which was present in nearly twice the amount in the cytoskeletons from the SV 40 transformed cells. The ratio of actin to total tropomyosin for whole cells was found to be unchanged on transformation. This ratio however was 31% lower in the cytoskeletons from the transformed cells. These and other results presented here suggest that changes in the levels of these three tropomyosins are not enough to account for the magnitude of the loss of actin cables observed in the transformed cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

IEF:

isoelectric focusing

mAb:

monoclonal antibody

NEPHGE:

non equilibrium pH gradient electrophoresis

References

  • Bernstein BW, Bamburg JR (1982) Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin-depolymerizing factor (ADF). Cell Motility 2: 1–8

    Google Scholar 

  • Bravo R, Celis JE (1980) A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells. J Cell Biol 84: 795–802

    Google Scholar 

  • — — (1982a) Updated catalogue of HeLa cell proteins: percentages and characteristics of the major cell polypeptides labelled with a mixture of 16 (14C) amino acids. Clin Chem 28: 766–781

    Google Scholar 

  • — — (1982b) Human proteins sensitive to neoplastic transformation in cultured epithelial and fibroblast cells. Clin Chem 28: 949–954

    Google Scholar 

  • — — (1984) Catalogue of HeLa cell proteins.In:Celis JE.Bravo R (eds) Two-dimensional gel electrophoresis of proteins: Methods and applications. Academic Press, New York, pp 445–476

    Google Scholar 

  • ——,Fey SJ, Bellatin J, Mose Larsen P, Arevalo J, Celis JE (1981 a) Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation. Exp Cell Res 136: 311–319

    Google Scholar 

  • Bravo R, Small JV, Mose Larsen P, Celis JE (1981 b) Coexistence of three major isoactins in a single Sarcoma 180 cell. Cell 25: 195–202

    Google Scholar 

  • —,Small JV, Fey SJ, Mose Larsen P, Celis JE (1982) Architecture and polypeptide Composition of HeLa cell cytoskeletons. Modification of cytoarchitectural proteins during mitosis. J Mol Biol 159: 121–143

    Google Scholar 

  • Celis JE, Bravo R (1981) Cataloguing human and mouse proteins. Trends Biochem Sci 6: 197–201

    Google Scholar 

  • — —,Mose Larsen P, Fey SJ, Bellatin J, Celis A (1984 a) Expression of cellular proteins in normal and transformed human cultured cells and tumors. In:Celis JE, Bravo R (eds) Two-dimensional gel electrophoresis of proteins: Methods and applications. Academic Press, New York, pp 307–362

    Google Scholar 

  • —,Celis A (1985) Cell cycle-dependent variations in the distribution of the nuclear protein cyclin (proliferating cell nuclear antigen) in cultured cells: Subdivision of S-phase. Proc Natl Acad Sci USA 82: 3262–3266

    Google Scholar 

  • —,Fey SJ, Mose Larsen P, Celis A (1984 b) Expression of the transformation-sensitive protein “cyclin” in normal human epidermal basal cells and simian virus 40-transformed keratinocytes. Proc Natl Acad Sci USA 81: 3128–3123

    Google Scholar 

  • Cote GP (1983) Structural and functional properties of the nonmuscle tropomyosins. Molecular and Cellular Biochemistry 57: 127–146

    Google Scholar 

  • Fattoum A, Hartwig JH, Stossel TP (1983) Isolation and some structural and functional properties of macrophage tropomyosin. Biochemistry 22: 1187–1193

    Google Scholar 

  • Forchhammer J (1982) Quantitative changes of some cellular polypeptides in C3H mouse cells following transformation by Moloney-Sarcoma virus. In:Chandra P (ed) Biochemical and biological markers of neoplastic transformation. Plenum, pp 445–455

  • Franza BR, Garrels JI (1984) Transformation sensitive proteins of REF 52 cells detected by computer-analyzed two dimensional gel electrophoresis. Cancer Cells 1: 137–146

    Google Scholar 

  • Fujime S, Ishiwata S (1971) Dynamic study of F-actin by quasielastic scattering of laser light. J Mol Biol 62: 251–265

    Google Scholar 

  • Fujiwara K, Pollard T (1976) Fluorescent antibody localization of myosin in the cytoplasm cleavage furrow and mitotic spindle of humans. J Cell Biol 71: 848–875

    Google Scholar 

  • —,Porter ME, Pollard TD (1978) Alpha-actinin localization in the cleavage furrow during cytokinesis. J Cell Biol 79: 268–275

    Google Scholar 

  • Goldman RD, Yerna MJ, Schloss JA (1976) Localization and organization of microfilaments and related proteins in normal and virus-transformed cells. J Supramol Struct 5: 155–183

    Google Scholar 

  • Heggeness MH, Wang K, Singer SJ (1977): Intracellular distributions of mechanochemical proteins in cultured fibroblasts. Proc Natl Acad Sci USA 74: 3883–3887

    Google Scholar 

  • Hendricks M, Weintraub H (1981) Tropomyosin is decreased in transformed cells. Proc Natl Acad Sci USA 78: 5633–5637

    Google Scholar 

  • — — (1984) Multiple tropomyosin polypeptides in chicken embryo fibroblasts: Differential repression of transcription by Rous sarcoma virus transformation. Mol Cell Biol 4: 1823–1833

    Google Scholar 

  • Köhler G, Milstein C (1976) Derivation of specific antibody producing tissue culture tumour lines by cell fusion. Eur J Immunol 6: 511–519

    Google Scholar 

  • Kawamura M, Maruyama K (1970) Electron microscopic particle lengths of F-actin polymerizedin vitro. J Biochem 67: 437–457

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685

    Google Scholar 

  • Lazarides E (1975) Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells. J Cell Biol 65: 459–561

    Google Scholar 

  • —,Burridge K (1965) Alpha-actinin immunofluorescent localization of a muscle structural protein in non-muscle cells. Cell 6: 289–298

    Google Scholar 

  • Lehrer SS (1981) Damage to actin filaments by glutaraldehyde: Protection by tropomyosin. J Cell Biol 90: 459–466

    Google Scholar 

  • Leonardi CL, Warren RH, Rubin RW (1982) Lack of tropomyosin correlates with the absence of stress fibres in transformed rat kidney cells. Biochim Biophys Acta 720: 154–162

    Google Scholar 

  • Lin J J-C, Helfman DM, Hughes SH, Chou C-S (1985) Tropomyosin isoforms in chicken embryo fibroblasts: Purification, characterization and changes in Rous sarcoma virus transformed cells. J Cell Biol 100: 692–703

    Google Scholar 

  • —,Yamashiro-Matsumara S, Matsumara F (1984) Microfilaments in normal and transformed cells: changes in the multiple forms of tropomyosin. Cancer Cells 1: 57–65

    Google Scholar 

  • Linder S, Krondahl U, Sennerstam R, Ringertz N (1981) Retinoic acid-induced differentiation of F 9 embryonal carcinoma cells. Exp Cell Res 132: 453–460

    Google Scholar 

  • Matsumara F, Lin J-C, Yamashiro-Matsumara S, Thomas GP, Topp WC (1983 a) Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem 258: 13954–13964

    Google Scholar 

  • —,Yamashiro-Matsumara S (1985) Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells. J Biol Chem 260: 13851–13859

    Google Scholar 

  • — —,Lin J J-C (1983 b) Isolation and characterization of tropomyosin-containing microfilaments from cultured cells. J Biol Chem 258: 6636–6644

    Google Scholar 

  • Maupin-Szamier P, Pollard TD (1978) Actin filament destruction by osmium tetroxide. J Cell Biol 77: 837–852

    Google Scholar 

  • McNutt NS, Culp LA, Black PH (1973) Contact-inhibited revertant cell lines isolated from SV40-transformed cells. J Cell Biol 56: 412–428

    Google Scholar 

  • Miyachi K, Fritzler MJ, Tan E (1978) Autoantibody to a nuclear antigen in proliferating cells. J Immunol 121: 2228–2234

    Google Scholar 

  • Nakamura T, Yamaguchi M, Yanagisawa T (1979). Comparative studies of actins from various sources. J Biochem (Tokyo) 85: 627–631

    Google Scholar 

  • O'Farrell PH (1975) High-resolution two dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021

    Google Scholar 

  • O'Farrell PZ, Goodmann HM, O'Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12: 1131–1142

    Google Scholar 

  • Paulin D, Perreu J, Jakob F, Yaniv M (1979) Tropomyosin synthesis accompanies formation of actin filaments in embryonal carcinoma cells induced to differentiate by hexamethylene bisacetamide. Proc Natl Acad Sci USA 76: 1891–1895

    Google Scholar 

  • Pollack R, Osborn M, Weber K (1975) Patterns of organization of actin and myosin in normal and transformed non-muscle cells. Proc Natl Acad Sci USA 72: 994–998

    Google Scholar 

  • —,Rifkin D (1975) Actin-containing cables within anchoragedependent rat embryo cells are dissociated by plasmin and trypsin. Cell 6: 495–506

    Google Scholar 

  • Small JV, Celis JE (1978) Filament arrangement in negatively stained cultured cells: The organization of actin. Cytobiology 16: 308–325

    Google Scholar 

  • Takebayashi T, Morita Y, Oosawa F (1977) Electron microscopic investigation of the flexibility of F-actin. Biochim Biophys Acta 492: 357–363

    Google Scholar 

  • Towbin H, Staehelin T, Gordon M (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc Natl Acad Sci USA 76: 4350–4354

    Google Scholar 

  • Wang K, Ash JF, Singer SJ (1975) Filamin, a new high-molecular-weight protein found in smooth muscle and non muscle cells. Proc Natl Acad Sci USA 72: 4483–4486

    Google Scholar 

  • Weber K, Groeschel-Stewart U (1974) Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci USA 71: 4561–4564

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Biochemistry, Aarhus University, Ole Worms Alle, Building 170, University Park, DK-8000, Aarhus C, Denmark

    J. E. Celis, Borbala Gesser, S. Nielsen & Ariana Celis

  2. Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg

    J. V. Small

Authors
  1. J. E. Celis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Borbala Gesser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. V. Small
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ariana Celis
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Celis, J.E., Gesser, B., Small, J.V. et al. Changes in the levels of human tropomyosins IEF 52,55, and 56 do not correlate with the loss of actin cables observed in SV 40 transformed MRC-5 fibroblasts. Protoplasma 135, 38–49 (1986). https://doi.org/10.1007/BF01277051

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01277051

Keywords

Search

Navigation