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. 1980 Dec 1;87(3):633–642. doi: 10.1083/jcb.87.3.633

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

JA Schloss, RD Goldman
PMCID: PMC2110773  PMID: 6893987

Abstract

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.

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Selected References

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  1. Bretscher A., Weber K. Purification of microvilli and an analysis of the protein components of the microfilament core bundle. Exp Cell Res. 1978 Oct 15;116(2):397–407. doi: 10.1016/0014-4827(78)90463-9. [DOI] [PubMed] [Google Scholar]
  2. Bretscher A., Weber K. Tropomyosin from bovine brain contains two polypeptide chains of slightly different molecular weights. FEBS Lett. 1978 Jan 1;85(1):145–148. doi: 10.1016/0014-5793(78)81267-8. [DOI] [PubMed] [Google Scholar]
  3. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  4. Clarke M., Spudich J. A. Nonmuscle contractile proteins: the role of actin and myosin in cell motility and shape determination. Annu Rev Biochem. 1977;46:797–822. doi: 10.1146/annurev.bi.46.070177.004053. [DOI] [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. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edds K. T. Isolation and characterization of two forms of a cytoskeleton. J Cell Biol. 1979 Oct;83(1):109–115. doi: 10.1083/jcb.83.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujiwara K., Porter M. E., Pollard T. D. Alpha-actinin localization in the cleavage furrow during cytokinesis. J Cell Biol. 1978 Oct;79(1):268–275. doi: 10.1083/jcb.79.1.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garrels J. I. Changes in protein synthesis during myogenesis in a clonal cell line. Dev Biol. 1979 Nov;73(1):134–152. doi: 10.1016/0012-1606(79)90143-x. [DOI] [PubMed] [Google Scholar]
  13. Goldman R. D., Lazarides E., Pollack R., Weber K. The distribution of actin in non-muscle cells. The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells. Exp Cell Res. 1975 Feb;90(2):333–344. doi: 10.1016/0014-4827(75)90323-7. [DOI] [PubMed] [Google Scholar]
  14. Goldman R. D., Milsted A., Schloss J. A., Starger J., Yerna M. J. Cytoplasmic fibers in mammalian cells: cytoskeletal and contractile elements. Annu Rev Physiol. 1979;41:703–722. doi: 10.1146/annurev.ph.41.030179.003415. [DOI] [PubMed] [Google Scholar]
  15. Goldman R. D., Yerna M. J., Schloss J. A. Localization and organization of microfilaments and related proteins in normal and virus-transformed cells. J Supramol Struct. 1976;5(2):155–183. doi: 10.1002/jss.400050206. [DOI] [PubMed] [Google Scholar]
  16. Gorovsky M. A., Carlson K., Rosenbaum J. L. Simple method for quantitive densitometry of polyacrylamide gels using fast green. Anal Biochem. 1970 Jun;35(2):359–370. doi: 10.1016/0003-2697(70)90196-x. [DOI] [PubMed] [Google Scholar]
  17. Hartshorne D. J., Mueller H. The preparation of tropomyosin and troponin from natural actomyosin. Biochim Biophys Acta. 1969 Mar;175(2):301–319. doi: 10.1016/0005-2795(69)90008-7. [DOI] [PubMed] [Google Scholar]
  18. Hayashi J. I., Hirabayashi T. Existence of common antigenic sites in tropomyosins. Biochim Biophys Acta. 1978 Apr 26;533(2):362–370. doi: 10.1016/0005-2795(78)90382-3. [DOI] [PubMed] [Google Scholar]
  19. Heggeness M. H., Wang K., Singer S. J. Intracellular distributions of mechanochemical proteins in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3883–3887. doi: 10.1073/pnas.74.9.3883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hitchcock S. E. Regulation of motility in nonmuscle cells. J Cell Biol. 1977 Jul;74(1):1–15. doi: 10.1083/jcb.74.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. ITZHAKI R. F., GILL D. M. A MICRO-BIURET METHOD FOR ESTIMATING PROTEINS. Anal Biochem. 1964 Dec;9:401–410. doi: 10.1016/0003-2697(64)90200-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kato T., Tonomura Y. Physarum tropomyosin-troponin complex. Isolation and properties. J Biochem. 1975 Sep;78(3):583–588. doi: 10.1093/oxfordjournals.jbchem.a130943. [DOI] [PubMed] [Google Scholar]
  24. Korn E. D. Biochemistry of actomyosin-dependent cell motility (a review). Proc Natl Acad Sci U S A. 1978 Feb;75(2):588–599. doi: 10.1073/pnas.75.2.588. [DOI] [PMC free article] [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. Immunofluorescence studies on the structure of actin filaments in tissue culture cells. J Histochem Cytochem. 1975 Jul;23(7):507–528. doi: 10.1177/23.7.1095651. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lehman W., Regenstein J. M., Ransom A. L. The stoichiometry of the components of arthropod thin filaments. Biochim Biophys Acta. 1976 May 20;434(1):215–222. doi: 10.1016/0005-2795(76)90053-2. [DOI] [PubMed] [Google Scholar]
  29. Lehman W., Szent-Györgyi A. G. Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom. J Gen Physiol. 1975 Jul;66(1):1–30. doi: 10.1085/jgp.66.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mahendran C., Berl S. Isolation of troponin-like complex from bovine brain cortex. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2273–2277. doi: 10.1073/pnas.74.6.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Masaki T. Tropomyosin-like protein in chick embryo fibroblasts. J Biochem. 1975 Apr;77(4):901–904. doi: 10.1093/oxfordjournals.jbchem.a130798. [DOI] [PubMed] [Google Scholar]
  32. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McLachlan A. D., Stewart M. The 14-fold periodicity in alpha-tropomyosin and the interaction with actin. J Mol Biol. 1976 May 15;103(2):271–298. doi: 10.1016/0022-2836(76)90313-2. [DOI] [PubMed] [Google Scholar]
  34. Mooseker M. S. Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium. J Cell Biol. 1976 Nov;71(2):417–433. doi: 10.1083/jcb.71.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nachmias V. T. Properties of Physarum myosin purified by a potassium iodide procedure. J Cell Biol. 1974 Jul;62(1):54–65. doi: 10.1083/jcb.62.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Osborn M., Weber K. The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res. 1977 May;106(2):339–349. doi: 10.1016/0014-4827(77)90179-3. [DOI] [PubMed] [Google Scholar]
  37. Pollard T. D., Thomas S. M., Niederman R. Human platelet myosin. I. Purification by a rapid method applicable to other nonmuscle cells. Anal Biochem. 1974 Jul;60(1):258–266. doi: 10.1016/0003-2697(74)90152-3. [DOI] [PubMed] [Google Scholar]
  38. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  39. Potter J. D. The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils. Arch Biochem Biophys. 1974 Jun;162(2):436–441. doi: 10.1016/0003-9861(74)90202-1. [DOI] [PubMed] [Google Scholar]
  40. Schloss J. A., Goldman R. D. Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4484–4488. doi: 10.1073/pnas.76.9.4484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schloss J. A., Milsted A., Goldman R. D. Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study. J Cell Biol. 1977 Sep;74(3):794–815. doi: 10.1083/jcb.74.3.794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sender P. M. Muscle fibrils: Solubilization and gel electrophoresis. FEBS Lett. 1971 Sep 15;17(1):106–110. doi: 10.1016/0014-5793(71)80575-6. [DOI] [PubMed] [Google Scholar]
  43. Stossel T. P., Hartwig J. H. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. J Cell Biol. 1976 Mar;68(3):602–619. doi: 10.1083/jcb.68.3.602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Szent-Györgyi A. G., Cohen C., Kendrick-Jones J. Paramyosin and the filaments of molluscan "catch" muscles. II. Native filaments: isolation and characterization. J Mol Biol. 1971 Mar 14;56(2):239–258. doi: 10.1016/0022-2836(71)90462-1. [DOI] [PubMed] [Google Scholar]
  45. Taylor D. L., Rhodes J. A., Hammond S. A. The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts. J Cell Biol. 1976 Jul;70(1):123–143. doi: 10.1083/jcb.70.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Trotter J. A., Foerder B. A., Keller J. M. Intracellular fibres in cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis. J Cell Sci. 1978 Jun;31:369–392. doi: 10.1242/jcs.31.1.369. [DOI] [PubMed] [Google Scholar]
  47. Wang K., Ash J. F., Singer S. J. Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4483–4486. doi: 10.1073/pnas.72.11.4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weber A., Murray J. M. Molecular control mechanisms in muscle contraction. Physiol Rev. 1973 Jul;53(3):612–673. doi: 10.1152/physrev.1973.53.3.612. [DOI] [PubMed] [Google Scholar]
  49. Weber K., Groeschel-Stewart U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4561–4564. doi: 10.1073/pnas.71.11.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yerna M. J., Aksoy M. O., Hartshorne D. J., Goldman R. D. BHK21 myosin: isolation, biochemical characterization and intracellular localization. J Cell Sci. 1978 Jun;31:411–429. doi: 10.1242/jcs.31.1.411. [DOI] [PubMed] [Google Scholar]

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