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. 1995 Dec 2;131(6):1759–1773. doi: 10.1083/jcb.131.6.1759

Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function

PMCID: PMC2120671  PMID: 8557743

Abstract

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins- specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.

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Selected References

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  1. Brown C. W., McHugh K. M., Lessard J. L. A cDNA sequence encoding cytoskeletal gamma-actin from rat. Nucleic Acids Res. 1990 Sep 11;18(17):5312–5312. doi: 10.1093/nar/18.17.5312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bähler M., Moser H., Eppenberger H. M., Wallimann T. Heart C-protein is transiently expressed during skeletal muscle development in the embryo, but persists in cultured myogenic cells. Dev Biol. 1985 Dec;112(2):345–352. doi: 10.1016/0012-1606(85)90405-1. [DOI] [PubMed] [Google Scholar]
  3. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Claycomb W. C., Lanson N., Jr Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell. In Vitro. 1984 Aug;20(8):647–651. doi: 10.1007/BF02619615. [DOI] [PubMed] [Google Scholar]
  5. Claycomb W. C., Palazzo M. C. Culture of the terminally differentiated adult cardiac muscle cell: a light and scanning electron microscope study. Dev Biol. 1980 Dec;80(2):466–482. doi: 10.1016/0012-1606(80)90419-4. [DOI] [PubMed] [Google Scholar]
  6. DeNofrio D., Hoock T. C., Herman I. M. Functional sorting of actin isoforms in microvascular pericytes. J Cell Biol. 1989 Jul;109(1):191–202. doi: 10.1083/jcb.109.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eldridge J., Zehner Z., Paterson B. M. Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence. Gene. 1985;36(1-2):55–63. doi: 10.1016/0378-1119(85)90069-1. [DOI] [PubMed] [Google Scholar]
  8. Eppenberger-Eberhardt M., Flamme I., Kurer V., Eppenberger H. M. Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes. Dev Biol. 1990 Jun;139(2):269–278. doi: 10.1016/0012-1606(90)90296-u. [DOI] [PubMed] [Google Scholar]
  9. Fürst D. O., Osborn M., Weber K. Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J Cell Biol. 1989 Aug;109(2):517–527. doi: 10.1083/jcb.109.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gallione C. J., Rose J. K. A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. J Virol. 1985 May;54(2):374–382. doi: 10.1128/jvi.54.2.374-382.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  12. Gordon D. J., Boyer J. L., Korn E. D. Comparative biochemistry of non-muscle actins. J Biol Chem. 1977 Nov 25;252(22):8300–8309. [PubMed] [Google Scholar]
  13. Gregorio C. C., Fowler V. M. Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly. J Cell Biol. 1995 May;129(3):683–695. doi: 10.1083/jcb.129.3.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grove B. K., Kurer V., Lehner C., Doetschman T. C., Perriard J. C., Eppenberger H. M. A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies. J Cell Biol. 1984 Feb;98(2):518–524. doi: 10.1083/jcb.98.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gunning P., Ponte P., Okayama H., Engel J., Blau H., Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol. 1983 May;3(5):787–795. doi: 10.1128/mcb.3.5.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Handel S. E., Greaser M. L., Schultz E., Wang S. M., Bulinski J. C., Lin J. J., Lessard J. L. Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin. Cell Tissue Res. 1991 Mar;263(3):419–430. doi: 10.1007/BF00327276. [DOI] [PubMed] [Google Scholar]
  17. Hartman A. L., Sawtell N. M., Lessard J. L. Expression of actin isoforms in developing rat intestinal epithelium. J Histochem Cytochem. 1989 Aug;37(8):1225–1233. doi: 10.1177/37.8.2754253. [DOI] [PubMed] [Google Scholar]
  18. Hennessey E. S., Drummond D. R., Sparrow J. C. Molecular genetics of actin function. Biochem J. 1993 May 1;291(Pt 3):657–671. doi: 10.1042/bj2910657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herman I. M. Actin isoforms. Curr Opin Cell Biol. 1993 Feb;5(1):48–55. doi: 10.1016/s0955-0674(05)80007-9. [DOI] [PubMed] [Google Scholar]
  20. Herman I. M., Crisona N. J., Pollard T. D. Relation between cell activity and the distribution of cytoplasmic actin and myosin. J Cell Biol. 1981 Jul;90(1):84–91. doi: 10.1083/jcb.90.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hill M. A., Gunning P. Beta and gamma actin mRNAs are differentially located within myoblasts. J Cell Biol. 1993 Aug;122(4):825–832. doi: 10.1083/jcb.122.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoock T. C., Newcomb P. M., Herman I. M. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653–664. doi: 10.1083/jcb.112.4.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  24. Kabsch W., Vandekerckhove J. Structure and function of actin. Annu Rev Biophys Biomol Struct. 1992;21:49–76. doi: 10.1146/annurev.bb.21.060192.000405. [DOI] [PubMed] [Google Scholar]
  25. Kislauskis E. H., Li Z., Singer R. H., Taneja K. L. Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol. 1993 Oct;123(1):165–172. doi: 10.1083/jcb.123.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Labeit S., Gautel M., Lakey A., Trinick J. Towards a molecular understanding of titin. EMBO J. 1992 May;11(5):1711–1716. doi: 10.1002/j.1460-2075.1992.tb05222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lubit B. W., Schwartz J. H. An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins. J Cell Biol. 1980 Sep;86(3):891–897. doi: 10.1083/jcb.86.3.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McHugh K. M., Lessard J. L. The development expression of the rat alpha-vascular and gamma-enteric smooth muscle isoactins: isolation and characterization of a rat gamma-enteric actin cDNA. Mol Cell Biol. 1988 Dec;8(12):5224–5231. doi: 10.1128/mcb.8.12.5224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McHugh K. M., Lessard J. L. The nucleotide sequence of a rat vascular smooth muscle alpha-actin cDNA. Nucleic Acids Res. 1988 May 11;16(9):4167–4167. doi: 10.1093/nar/16.9.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Messerli J. M., Eppenberger-Eberhardt M. E., Rutishauser B. M., Schwarb P., von Arx P., Koch-Schneidemann S., Eppenberger H. M., Perriard J. C. Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy. Histochemistry. 1993 Sep;100(3):193–202. doi: 10.1007/BF00269092. [DOI] [PubMed] [Google Scholar]
  32. Messerli J. M., van der Voort H. T., Rungger-Brändle E., Perriard J. C. Three-dimensional visualization of multi-channel volume data: the amSFP algorithm. Cytometry. 1993 Oct;14(7):725–735. doi: 10.1002/cyto.990140705. [DOI] [PubMed] [Google Scholar]
  33. Ohshima S., Abe H., Obinata T. Isolation of profilin from embryonic chicken skeletal muscle and evaluation of its interaction with different actin isoforms. J Biochem. 1989 Jun;105(6):855–857. doi: 10.1093/oxfordjournals.jbchem.a122765. [DOI] [PubMed] [Google Scholar]
  34. Otey C. A., Kalnoski M. H., Bulinski J. C. Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell Motil Cytoskeleton. 1988;9(4):337–348. doi: 10.1002/cm.970090406. [DOI] [PubMed] [Google Scholar]
  35. Otey C. A., Kalnoski M. H., Lessard J. L., Bulinski J. C. Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells. J Cell Biol. 1986 May;102(5):1726–1737. doi: 10.1083/jcb.102.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pardo J. V., Pittenger M. F., Craig S. W. Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria. Cell. 1983 Apr;32(4):1093–1103. doi: 10.1016/0092-8674(83)90293-3. [DOI] [PubMed] [Google Scholar]
  37. Paterson B. M., Eldridge J. D. alpha-Cardiac actin is the major sarcomeric isoform expressed in embryonic avian skeletal muscle. Science. 1984 Jun 29;224(4656):1436–1438. doi: 10.1126/science.6729461. [DOI] [PubMed] [Google Scholar]
  38. Ponte P., Ng S. Y., Engel J., Gunning P., Kedes L. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res. 1984 Feb 10;12(3):1687–1696. doi: 10.1093/nar/12.3.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rozycki M. D., Myslik J. C., Schutt C. E., Lindberg U. Structural aspects of actin-binding proteins. Curr Opin Cell Biol. 1994 Feb;6(1):87–95. doi: 10.1016/0955-0674(94)90121-x. [DOI] [PubMed] [Google Scholar]
  40. Rubenstein P. A. The functional importance of multiple actin isoforms. Bioessays. 1990 Jul;12(7):309–315. doi: 10.1002/bies.950120702. [DOI] [PubMed] [Google Scholar]
  41. Ruzicka D. L., Schwartz R. J. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation. J Cell Biol. 1988 Dec;107(6 Pt 2):2575–2586. doi: 10.1083/jcb.107.6.2575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sawtell N. M., Lessard J. L. Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma-enteric isoform in differentiating striated myocytes. J Cell Biol. 1989 Dec;109(6 Pt 1):2929–2937. doi: 10.1083/jcb.109.6.2929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schevzov G., Lloyd C., Gunning P. High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture. J Cell Biol. 1992 May;117(4):775–785. doi: 10.1083/jcb.117.4.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schäfer B. W., Perriard J. C. Intracellular targeting of isoproteins in muscle cytoarchitecture. J Cell Biol. 1988 Apr;106(4):1161–1170. doi: 10.1083/jcb.106.4.1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sen A., Dunnmon P., Henderson S. A., Gerard R. D., Chien K. R. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem. 1988 Dec 15;263(35):19132–19136. [PubMed] [Google Scholar]
  46. Shuster C. B., Herman I. M. Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity. J Cell Biol. 1995 Mar;128(5):837–848. doi: 10.1083/jcb.128.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Skalli O., Ropraz P., Trzeciak A., Benzonana G., Gillessen D., Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986 Dec;103(6 Pt 2):2787–2796. doi: 10.1083/jcb.103.6.2787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Soldati T., Perriard J. C. Intracompartmental sorting of essential myosin light chains: molecular dissection and in vivo monitoring by epitope tagging. Cell. 1991 Jul 26;66(2):277–289. doi: 10.1016/0092-8674(91)90618-9. [DOI] [PubMed] [Google Scholar]
  49. Solomon L. R., Rubenstein P. A. Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis. J Biol Chem. 1987 Aug 15;262(23):11382–11388. [PubMed] [Google Scholar]
  50. Sparrow J. C., Drummond D. R., Hennessey E. S., Clayton J. D., Lindegaard F. B. Drosophila actin mutants and the study of myofibrillar assembly and function. Symp Soc Exp Biol. 1992;46:111–129. [PubMed] [Google Scholar]
  51. Vandekerckhove J., Weber K. Actin typing on total cellular extracts: a highly sensitive protein-chemical procedure able to distinguish different actins. Eur J Biochem. 1981 Jan;113(3):595–603. doi: 10.1111/j.1432-1033.1981.tb05104.x. [DOI] [PubMed] [Google Scholar]
  52. Vandekerckhove J., Weber K. At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol. 1978 Dec 25;126(4):783–802. doi: 10.1016/0022-2836(78)90020-7. [DOI] [PubMed] [Google Scholar]
  53. Vandekerckhove J., Weber K. The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation. Differentiation. 1979;14(3):123–133. doi: 10.1111/j.1432-0436.1979.tb01021.x. [DOI] [PubMed] [Google Scholar]
  54. Wang S. M., Greaser M. L., Schultz E., Bulinski J. C., Lin J. J., Lessard J. L. Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin. J Cell Biol. 1988 Sep;107(3):1075–1083. doi: 10.1083/jcb.107.3.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]

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