Advertisement

Journal of Muscle Research & Cell Motility

, Volume 10, Issue 6, pp 446–456 | Cite as

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

  • Sandra Pierobon-Bormioli
  • Romeo Betto
  • Giovanni Salviati
Papers

Summary

Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 μm, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 μm, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.

Keywords

Reaction Site Large Protein Thin Filament Sarcomere Length Ultrastructural Level 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bonilla, E., Miranda, A., Prelle, A., Salviati, G., Betto, R., Zeviani, M., Schon, E. A., Dimauro, S. &Rowland, L. P. (1988) Immunocytochemical study of nebulin in Duchenne muscular dystrophy.Neurology 38, 1600–3.PubMedGoogle Scholar
  2. Eastwood, A. B., Wood, D. S., Bock, K. L. &Sorenson, M. M. (1979) Chemically skinned mammalian skeletal muscle. I. The nature of skinned rabbit psoas.Tissue & Cell 11, 553–66.Google Scholar
  3. Fürst, D. O., Osborn, M., Nave, R. &Weber, K. (1988) The organization of titin filaments in the halfsarcomere 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. 106, 1563–72.PubMedGoogle Scholar
  4. Garamvolgyi, N. (1965) Inter Z-bridges in the flight muscle of the bee.J. Ultrastruct. Res. 13, 435–43.PubMedGoogle Scholar
  5. Granger, B.L. &Lazarides, E. (1978) The existence of an insoluble Z-disc scaffold in chicken skeletal muscle.Cell 15, 1253–68.PubMedGoogle Scholar
  6. Higuchi, H. &Umazume, Y. (1985) Localization of elastic component in frog muscle.Biophys. J. 48, 137–47.PubMedGoogle Scholar
  7. Horowitz, R. &Podolsky, R. J. (1987) The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments.J. Cell Biol. 105, 2217–23.PubMedGoogle Scholar
  8. Itoh, Y., Suzuki, T., Kimura, S., Ohashi, K., Higuchi, H., Sawada, H., Shimizu, T., Shibata, M. &Maruyama, K. (1988) Extensible and less extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies.J. Biochem. (Tokyo) 104, 504–8.Google Scholar
  9. Leary, J. J., Brigati, D. J. &Ward, D. C. (1983) Rapid and sensitive method for visualizing biotin-labelled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose. Bio-blots.Proc. natn. Acad. Sci. U.S.A. 80, 4045–9.Google Scholar
  10. Locker, R. H. &Leet, N. G. (1976) Histology of highly-stretched beef muscle. II. Further evidence on the localization and nature of gap filaments.J. Ultrastruct. Res. 55, 157–67.PubMedGoogle Scholar
  11. Maruyama, K. (1986) Connectin, an elastic filamentous protein of striated muscle.Int. Rev. Cytol. 104, 81–114.PubMedGoogle Scholar
  12. Maruyama, K., Matsubara, S., Nonomura, Y., Kimura, S., Ohashi, K., Murakami, F., Handa, S. &Eguchi, G. (1977) Connectin, an elastic protein of muscle: characterization and function.J. Biochem.(Tokyo) 82, 317–37.Google Scholar
  13. Maruyama, K., Yoshioka, T., Higuchi, H., Ohashi, K., Kimura, S. &Natori, R. (1985) Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy.J. Cell Biol. 101, 2167–75.PubMedGoogle Scholar
  14. Mould, A. P., Holmes, D. P., Kadler, K. E. &Chapman, J. A. (1985) Mica sandwich technique for preparing macromolecules for rotary shadowing.J. Ultrastruct. Res. 91, 66–76.PubMedGoogle Scholar
  15. Natori, R. (1954) The property and contraction process of isolated myofibrils.Jikeikai Med. J. 1, 119–26.Google Scholar
  16. Page, S. &Huxley, H. E. (1963) Filament length in striated muscle.J. Cell Biol. 19, 369–91.PubMedGoogle Scholar
  17. Pierobon-Bormioli, S. (1980) Transverse sarcomere filamentous systems: ‘Z-,M’- and N′-cables.J. Musc. Res. Cell Motility 1, 445 (abstr.).Google Scholar
  18. Pierobon-Bormioli, S. (1981) Transverse sarcomere filamentous systems: ‘Z- and M’-cables.J. Musc. Res. Cell Motility 2, 401–13.Google Scholar
  19. Pierobon-Bormioli, S. (1985) Striated muscle cytohistoskeleton. In proceedings of Cell Biology and Clinical Management in Functional Electrostimulation of Neurones and Muscles (edited byCarraro, U. &Angelini, C.) pp. 57–9. Padova: Cleup.Google Scholar
  20. Pierobon-Bormioli, S. (1988) Striated muscle structure function. The transverse histo-cytoskeleton. The endoskeleton. In Associazione di Biologia Cellulare e del Differenziamento (ABCD) VII Congresso Nazionale Spoleto 16–19 ottobre, pp. B11.Eur. J. Cell Biol. (in press) (abstr.).Google Scholar
  21. Pierobon-Bormioli, S., Salviati, G. &Betto, R. (1988) Skeletal muscle. Endosarcomeric immunoultrastructure localization of titin/connectin, nebulin and ‘Band 4’. InSarcomeric and Non-Sarcomeric Muscles: Basic and Applied Research Prospects for the 90s (edited by)Carraro, U., pp. 501–6. Padova: Unipress.Google Scholar
  22. Somerville, L. L. &Wang, K. (1988) Sarcomere matrix of striated muscle:in vivo phosphorylation of titin and nebulin in mouse diaphragm muscle.Arch. Biochem. Biophys. 262, 118–29.PubMedGoogle Scholar
  23. Towbin, H., Staehelin, T. &Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulosa sheets: procedures and some applications.Proc. natn. Acad. Sci. U.S.A. 76, 4350–4.Google Scholar
  24. Tokuyasu, K. T., Dutton, A. H. &Singer, S. J. (1983) Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle.J. Cell Biol. 96, 1727–35.PubMedGoogle Scholar
  25. Trinick, J., Knight, P. &Whiting, A. (1984) Purification and properties of native titin.J. molec. Biol. 180, 331–56.PubMedGoogle Scholar
  26. Wang, K. (1981) Nebulin, a giant protein component of N2 line of striated muscle.J. Cell Biol. 91, 355a (abstr.).Google Scholar
  27. Wang, K. (1982) Purification of titin and nebulin.Meth. Enzymol. 85b, 264–74.Google Scholar
  28. Wang, K. (1984) Cytoskeletal matrix in striated muscle: the role of titin, nebulin and intermediate filaments. InContractile Mechanisms in Muscle (edited bySugi, H. &Pollack, C. H.), pp. 285–306. New York: Plenum Publishing Corp.Google Scholar
  29. Wang, K. (1985) Sarcomere-associated cytoskeletal lattices in striated muscle. InCell and Muscle Motility (edited byShay, J. W.), pp. 315–69. New York: Plenum Press.Google Scholar
  30. Wang, K., McLure, J. &Tu, A. (1979) Titin: major myofibrillar components of striated muscle.Proc. natn. Acad. Sci. U.S.A. 76, 3698–702.Google Scholar
  31. Wang, K. &Williamson, C. L. (1980) Identification of an N2 line protein of striated muscle.Proc. natn. Acad. Sci. U.S.A. 77, 3254–8.Google Scholar
  32. Wang, K. &Wright, J. (1987a) Architecture of sarcomere matrix in skeletal muscle. Evidence that nebulin constitutes a distinct set of non-extensible filaments in parallel with titin filaments.J. Cell Biol. 105, n4 part 2 n. 138, (abstr.).Google Scholar
  33. Wang, K. &Wright, J. (1987b) Architecture of sarcomere matrix in skeletal muscle. Nebulin filament as a thin filament scaffold.Biophys. J. 5, 219a (abstr.).Google Scholar
  34. Wang, K. &Wright, J. (1988a) Sarcomere matrix of skeletal muscle: the role of thick filaments in the segmental extensibility of elastic titin filaments.Biophys. J. 53, 25a (abstr.).Google Scholar
  35. Wang, K. &Wright, J. (1988b) Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line.J. Cell Biol. 107, 2199–212.PubMedGoogle Scholar
  36. Watkins, S. C., Samuel, J. L., Marotte, F., Bertier-Savalle, B. &Rappaport, L. (1987) Microtubules and desmin filaments during onset of heart hypertrophy in rat: a double immunoelectron microscope study.Circ. Res. 60, 327–36.PubMedGoogle Scholar
  37. Wood, D. S., Zeviani, M., Prelle, A., Bonilla, E., Miranda, A. F., DiMauro, S. &Rowland, L. P. (1987) Is nebulin the defective gene product in Duchenne muscular dystrophy?N. Engl. J. Med. 316, 107–8.Google Scholar
  38. Wood, D. S., Zollman, J. &Reuben, J. P. (1978) Human skeletal muscle. Properties of the ‘chemically skinned’ fibres.Science 187, 1075–6.Google Scholar
  39. Zeviani, M., Darras, B. T., Rizzuto, R., Salviati, G., Betto, R., Miranda, E., Du, A. F., Samitt, C., Dickson, G., Walsh, F. S., Dimauro, S., Francke, U. &Schon, E. A. (1988) Cloning and expression of human nebulin cDNAs and assignment of the gene to chromosome 2q31-q32.Genomics 2, 249–56.PubMedGoogle Scholar

Copyright information

© Chapman and Hall Ltd. 1989

Authors and Affiliations

  • Sandra Pierobon-Bormioli
    • 1
  • Romeo Betto
    • 1
  • Giovanni Salviati
    • 1
  1. 1.CNR Unit for Muscle Biology and Physiopathology, Institute of General PathologyUniversity of PadovaPadovaItaly

Personalised recommendations