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The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Jan 2;120(2):411–420. doi: 10.1083/jcb.120.2.411

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

PMCID: PMC2119515  PMID: 8421055

Abstract

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.

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Selected References

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  1. Bennett V. The spectrin-actin junction of erythrocyte membrane skeletons. Biochim Biophys Acta. 1989 Jan 18;988(1):107–121. doi: 10.1016/0304-4157(89)90006-3. [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. Broschat K. O. Tropomyosin prevents depolymerization of actin filaments from the pointed end. J Biol Chem. 1990 Dec 5;265(34):21323–21329. [PubMed] [Google Scholar]
  4. Broschat K. O., Weber A., Burgess D. R. Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization. Biochemistry. 1989 Oct 17;28(21):8501–8506. doi: 10.1021/bi00447a035. [DOI] [PubMed] [Google Scholar]
  5. Byers T. J., Branton D. Visualization of the protein associations in the erythrocyte membrane skeleton. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6153–6157. doi: 10.1073/pnas.82.18.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casella J. F., Craig S. W., Maack D. J., Brown A. E. Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle. J Cell Biol. 1987 Jul;105(1):371–379. doi: 10.1083/jcb.105.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chowrashi P. K., Pepe F. A. The Z-band: 85,000-dalton amorphin and alpha-actinin and their relation to structure. J Cell Biol. 1982 Sep;94(3):565–573. doi: 10.1083/jcb.94.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Dabiri G. A., Sanger J. M., Portnoy D. A., Southwick F. S. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6068–6072. doi: 10.1073/pnas.87.16.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daniels M. P. Localization of actin, beta-spectrin, 43 x 10(3) Mr and 58 x 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes. J Cell Sci. 1990 Dec;97(Pt 4):615–626. doi: 10.1242/jcs.97.4.615. [DOI] [PubMed] [Google Scholar]
  11. Drenckhahn D., Engel K., Höfer D., Merte C., Tilney L., Tilney M. Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein. J Cell Biol. 1991 Feb;112(4):641–651. doi: 10.1083/jcb.112.4.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flicker P. F., Phillips G. N., Jr, Cohen C. Troponin and its interactions with tropomyosin. An electron microscope study. J Mol Biol. 1982 Dec 5;162(2):495–501. doi: 10.1016/0022-2836(82)90540-x. [DOI] [PubMed] [Google Scholar]
  13. Fowler V. M., Bennett V. Erythrocyte membrane tropomyosin. Purification and properties. J Biol Chem. 1984 May 10;259(9):5978–5989. [PubMed] [Google Scholar]
  14. Fowler V. M. Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes. J Biol Chem. 1987 Sep 15;262(26):12792–12800. [PubMed] [Google Scholar]
  15. Fowler V. M. Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin. J Cell Biol. 1990 Aug;111(2):471–481. doi: 10.1083/jcb.111.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fürst D. O., Osborn M., Nave R., Weber K. The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J Cell Biol. 1988 May;106(5):1563–1572. doi: 10.1083/jcb.106.5.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. HUXLEY H., HANSON J. Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature. 1954 May 22;173(4412):973–976. doi: 10.1038/173973a0. [DOI] [PubMed] [Google Scholar]
  18. Hitchcock-DeGregori S. E., Sampath P., Pollard T. D. Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments. Biochemistry. 1988 Dec 27;27(26):9182–9185. doi: 10.1021/bi00426a016. [DOI] [PubMed] [Google Scholar]
  19. Ishikawa R., Yamashiro S., Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989 May 5;264(13):7490–7497. [PubMed] [Google Scholar]
  20. Ishiwata S., Funatsu T. Does actin bind to the ends of thin filaments in skeletal muscle? J Cell Biol. 1985 Jan;100(1):282–291. doi: 10.1083/jcb.100.1.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jin J. P., Wang K. Cloning, expression, and protein interaction of human nebulin fragments composed of varying numbers of sequence modules. J Biol Chem. 1991 Nov 5;266(31):21215–21223. [PubMed] [Google Scholar]
  22. Knight P. J., Trinick J. A. Preparation of myofibrils. Methods Enzymol. 1982;85(Pt B):9–12. doi: 10.1016/0076-6879(82)85004-0. [DOI] [PubMed] [Google Scholar]
  23. Kruger M., Wright J., Wang K. Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile. J Cell Biol. 1991 Oct;115(1):97–107. doi: 10.1083/jcb.115.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Labeit S., Gibson T., Lakey A., Leonard K., Zeviani M., Knight P., Wardale J., Trinick J. Evidence that nebulin is a protein-ruler in muscle thin filaments. FEBS Lett. 1991 May 6;282(2):313–316. doi: 10.1016/0014-5793(91)80503-u. [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. Lal A. A., Korn E. D. Effect of muscle tropomyosin on the kinetics of polymerization of muscle actin. Biochemistry. 1986 Mar 11;25(5):1154–1158. doi: 10.1021/bi00353a031. [DOI] [PubMed] [Google Scholar]
  27. Lees-Miller J. P., Helfman D. M. The molecular basis for tropomyosin isoform diversity. Bioessays. 1991 Sep;13(9):429–437. doi: 10.1002/bies.950130902. [DOI] [PubMed] [Google Scholar]
  28. Maher P. A., Cox G. F., Singer S. J. Zeugmatin: a new high molecular weight protein associated with Z lines in adult and early embryonic striated muscle. J Cell Biol. 1985 Nov;101(5 Pt 1):1871–1883. doi: 10.1083/jcb.101.5.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mak A. S., Roseborough G., Baker H. Tropomyosin from human erythrocyte membrane polymerizes poorly but binds F-actin effectively in the presence and absence of spectrin. Biochim Biophys Acta. 1987 Apr 8;912(2):157–166. doi: 10.1016/0167-4838(87)90084-7. [DOI] [PubMed] [Google Scholar]
  30. Maruyama K., Kurokawa H., Oosawa M., Shimaoka S., Yamamoto H., Ito M., Maruyama K. Beta-actinin is equivalent to Cap Z protein. J Biol Chem. 1990 May 25;265(15):8712–8715. [PubMed] [Google Scholar]
  31. Matsuzaki F., Sutoh K., Ikai A. Structural unit of the erythrocyte cytoskeleton. Isolation and electron microscopic examination. Eur J Cell Biol. 1985 Nov;39(1):153–160. [PubMed] [Google Scholar]
  32. 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]
  33. Ohtsuki I. Molecular arrangement of troponin-T in the thin filament. J Biochem. 1979 Aug;86(2):491–497. doi: 10.1093/oxfordjournals.jbchem.a132549. [DOI] [PubMed] [Google Scholar]
  34. PAGE S. G., HUXLEY H. E. FILAMENT LENGTHS IN STRIATED MUSCLE. J Cell Biol. 1963 Nov;19:369–390. doi: 10.1083/jcb.19.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peng I., Fischman D. A. Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity. Cell Motil Cytoskeleton. 1991;20(2):158–168. doi: 10.1002/cm.970200208. [DOI] [PubMed] [Google Scholar]
  36. Pinder J. C., Weeds A. G., Gratzer W. B. Study of actin filament ends in the human red cell membrane. J Mol Biol. 1986 Oct 5;191(3):461–468. doi: 10.1016/0022-2836(86)90141-5. [DOI] [PubMed] [Google Scholar]
  37. Prulière G., d'Albis A., der Terrossian E. Effect of tropomyosin on the interactions of actin with actin-binding proteins isolated from pig platelets. Eur J Biochem. 1986 Sep 15;159(3):535–547. doi: 10.1111/j.1432-1033.1986.tb09920.x. [DOI] [PubMed] [Google Scholar]
  38. Rabilloud T., Carpentier G., Tarroux P. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis. 1988 Jun;9(6):288–291. doi: 10.1002/elps.1150090608. [DOI] [PubMed] [Google Scholar]
  39. Saide J. D., Chin-Bow S., Hogan-Sheldon J., Busquets-Turner L., Vigoreaux J. O., Valgeirsdottir K., Pardue M. L. Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J Cell Biol. 1989 Nov;109(5):2157–2167. doi: 10.1083/jcb.109.5.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sanger J. W., Mittal B., Sanger J. M. Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins. J Cell Biol. 1984 Mar;98(3):825–833. doi: 10.1083/jcb.98.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shen B. W., Josephs R., Steck T. L. Ultrastructure of the intact skeleton of the human erythrocyte membrane. J Cell Biol. 1986 Mar;102(3):997–1006. doi: 10.1083/jcb.102.3.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Southwick F. S., Hartwig J. H. Acumentin, a protein in macrophages which caps the "pointed" end of action filaments. Nature. 1982 May 27;297(5864):303–307. doi: 10.1038/297303a0. [DOI] [PubMed] [Google Scholar]
  43. Sung L. A., Fowler V. M., Lambert K., Sussman M. A., Karr D., Chien S. Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein. J Biol Chem. 1992 Feb 5;267(4):2616–2621. [PubMed] [Google Scholar]
  44. Sussman M. A., Battenberg E., Bloom F. E., Fowler V. M. Identification of two nerve growth factor-induced polypeptides in PC12 cells. J Mol Neurosci. 1990;2(3):163–174. doi: 10.1007/BF02896841. [DOI] [PubMed] [Google Scholar]
  45. Sussman M. A., Fowler V. M. Tropomodulin binding to tropomyosins. Isoform-specific differences in affinity and stoichiometry. Eur J Biochem. 1992 Apr 1;205(1):355–362. doi: 10.1111/j.1432-1033.1992.tb16787.x. [DOI] [PubMed] [Google Scholar]
  46. Tilney L. G., DeRosier D. J., Weber A., Tilney M. S. How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper. J Cell Biol. 1992 Jul;118(1):83–93. doi: 10.1083/jcb.118.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Traeger L., Goldstein M. A. Thin filaments are not of uniform length in rat skeletal muscle. J Cell Biol. 1983 Jan;96(1):100–103. doi: 10.1083/jcb.96.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wegner A. Equilibrium of the actin-tropomyosin interaction. J Mol Biol. 1979 Jul 15;131(4):839–853. doi: 10.1016/0022-2836(79)90204-3. [DOI] [PubMed] [Google Scholar]
  49. Wegner A. Kinetic analysis of actin assembly suggests that tropomyosin inhibits spontaneous fragmentation of actin filaments. J Mol Biol. 1982 Oct 25;161(2):217–227. doi: 10.1016/0022-2836(82)90149-8. [DOI] [PubMed] [Google Scholar]
  50. Weigt C., Schoepper B., Wegner A. Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization. FEBS Lett. 1990 Jan 29;260(2):266–268. doi: 10.1016/0014-5793(90)80119-4. [DOI] [PubMed] [Google Scholar]
  51. Zot A. S., Potter J. D. Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annu Rev Biophys Biophys Chem. 1987;16:535–559. doi: 10.1146/annurev.bb.16.060187.002535. [DOI] [PubMed] [Google Scholar]
  52. de Arruda M. V., Watson S., Lin C. S., Leavitt J., Matsudaira P. Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins. J Cell Biol. 1990 Sep;111(3):1069–1079. doi: 10.1083/jcb.111.3.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]

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