Advertisement

Journal of Muscle Research & Cell Motility

, Volume 12, Issue 3, pp 225–234 | Cite as

Visualization of myosin exchange between synthetic thick filaments

  • A. D. Saad
  • J. E. Dennis
  • I. P. Tan
  • D. A. Fischman
Papers

Summary

Exchange of myosin molecules between synthetic thick filaments was examined by fluorescence energy transfer and visualized by electron microscopy using streptavidin-gold to detect exchanged biotinylated myosin molecules. N-hydroxysuccinimidobiotin (NHS-biotin) was covalently linked to purified adult chicken pectoralis myosin to obtain assembly-competent biotinylated myosin molecules. Two distinct classes of synthetic filaments, distinguishable by length, were prepared. Biotinylated filaments (575±100 nm) were assembled by a quick dilution (QD) method and unlabelled filaments (1025±250 nm) were obtained by a sequential dilution (SD). The two filament population maintained their distinct length distributions even when mixed. To measure exchange, biotinylated short (QD) filaments were combined with unlabelled long (SD) filaments at a 1∶5 ratio, sampled at varying times and the entry of biotinylated myosin into the previously unlabelled long filaments visualized by the addition of streptavidin-gold. The number of gold particles per micron was examined for fully biotinylated short filaments (<700 nm), unlabelled long filaments (>900 nm), and exchanged filaments. Equivalent binding of streptavidin-gold to the two filament types was detected by 60 min suggesting randomization of biotinylated monomers by this time. The precise location of streptavidin-gold sites on the long filaments was also measured. Although labeling was detected along the full length of the filaments, at the earliest time points (5 min) filament ends contained twice the number of gold particles as the filament centers. Approximately equivalent labeling along the entire length of the filaments was observed by 60 min. These results provide additional support for our earlier report of extensive myosin exchange between synthetic thick filaments and show that extensive exchange takes place rapidly along the full length of synthetic thick filaments.

Keywords

Gold Particle Length Distribution Early Time Point Extensive Exchange Filament Type 
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. Bader, D., Masaki, T. &Fischman, D. A. (1982) Immunochemical analysis of myosin heavy chain during avian myogenesisin vivo andin vitro.J. Cell Biol. 95, 763–70.PubMedGoogle Scholar
  2. Bahler, M., Eppenberger, H. M. &Wallimann, T. (1985a) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. I. Purification and characterization.J. Mol. Biol. 186, 381–91.PubMedGoogle Scholar
  3. Bahler, M., Eppenberger, H. M. &Wallimann, T. (1985b) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. II. Distribution and localization.J. Mol. Biol. 186, 393–401.PubMedGoogle Scholar
  4. Gauthier, G. F. (1990) Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers.J. Cell. Biol. 110, 693–701.PubMedGoogle Scholar
  5. Godfrey, J. E. &Harrington, W. F. (1970) Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment.Biochemistry 9, 886–93.PubMedGoogle Scholar
  6. Green, N. M. (1975). Avidin.Advances in Protein Chemistry 29, 85–133.PubMedGoogle Scholar
  7. Grove, B. K., Kurer, V., Lehner, C., Doetschman, T. C., Perriard, J.-C. &Eppenberger, H. M. (1984) A new 185.000-dalton skeletal muscle protein detected by monoclonal antibodies.J. Cell Biol. 98, 518–24.PubMedGoogle Scholar
  8. Horisberger, M. &Rosett, J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy.J. Histochem. Cytochem. 25, 295–305.PubMedGoogle Scholar
  9. Huxley, H. E. (1963) Electron microscope studies on structure of natural and synthetic protein filaments from striated muscle.J. Molec. Biol. 7, 281–308.Google Scholar
  10. Johnson, C. S., Mckenna, N. M. &Wang, Y. (1988) Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.J. Cell Biol. 107, 2213–21.PubMedGoogle Scholar
  11. Josephs, R. &Harrington, W. F. (1968) On the stability of myosin filaments.Biochemistry 7, 2834–47.PubMedGoogle Scholar
  12. Katsura, I. &Noda, H. (1971) Studies on the formation and physical chemical properties of synthetic myosin filaments.J. Biochem. 69, 219–29.PubMedGoogle Scholar
  13. Kreis, T. E., Geiger, B. &Schlessinger, J. (1982) Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.Cell 29, 835–45.PubMedGoogle Scholar
  14. Kristofferson, D., Mitchison, T. &Kirschner, M. (1986) Direct observation of steady-state microtubule dynamics.J. Cell Biol. 102, 1007–19.PubMedGoogle Scholar
  15. Masaki, T. &Takaiti, O. (1974) M-protein.J. Biochem. (Tokyo) 75, 367–80.Google Scholar
  16. Maw, M. C. &Rowe, A. J. (1980) Fraying of A-filaments into three sub-filaments.Nature 286, 412–14.PubMedGoogle Scholar
  17. Maruyama, K., Kimura, S., Ohashi, K. &Kuwano, Y. (1981) Connectin, and elastic protein of muscle. Identification of titin with connectin.J. Biochem. (Tokyo)89, 701–9.Google Scholar
  18. Mckenna, N. M., Meigs, J. B. &Wang, Y. (1985) Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells.J. Cell Biol. 101, 2223–32.PubMedGoogle Scholar
  19. Mittal, B., Sanger, J. M. &Sanger, J. W. (1987) Visualization of myosin in living cells.J. Cell Biol. 105, 1753–60.PubMedGoogle Scholar
  20. Offer, G., Moos, C. S. &Starr, R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. Extraction, purification and characterization.J. Mol. Biol. 74, 653–76.PubMedGoogle Scholar
  21. Pardee, J. D., Simpson, P. A., Stryer, L. &Spudich, J. A. (1982) Actin filaments undergo limited subunit exchange in physiological salt conditions.J. Cell Biol. 94, 316–24.PubMedGoogle Scholar
  22. Pepe, F. A. (1983) Macromolecular assembly of myosin. In:Muscle and Nonmuscle Motility. (edited byStracher, A.) pp. 105–149. New York: Academic Press.Google Scholar
  23. Pepe, F. A., Drucker, B. &Chowrashi, P. K. (1986) The myosin filament, XI: filament assembly.Prep. Biochem. 16, 99–132.PubMedGoogle Scholar
  24. Price, M. (1987) Skelemins: cytoskeletal proteins located at the periphery of M-discs in mammalian striated muscle.J. Cell Biol. 104, 1325–36.PubMedGoogle Scholar
  25. Saad, A. D., Fischman, D. A. &Pardee, J. D. (1986a) Fluorescence energy transfer studies of myosin thick filament assembly.Biophys. J. 49, 140–2.Google Scholar
  26. Saad, A. D., Mathews, A. P., Tan, I. P. &Sorrentino, A. M. (1990) Myosin thick filament stability in the absence and presence of C-protein.Anatomical Record (in Press).Google Scholar
  27. Saad, A. D., Pardee, J. D. &Fischman, D. A. (1986b) Dymanic exchange of myosin molecules between thick filaments.Proc. Natl. Acad. Sci. (USA)83, 9483–7.Google Scholar
  28. Salmon, E. D., Leslie, R. J., Saxton, W. M., Krow, M. L. &Mcintosh, J. R. (1984) Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.J. Cell Biol. 99, 2165–74.PubMedGoogle Scholar
  29. Shimuzu, T., Dennis, J. E., Masaki, T. &Fischman, D. A. (1985) Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.J. Cell Biol. 101, 1115–23.PubMedGoogle Scholar
  30. Sutoh, K., Yamamoto, K. &Wakabayashi, T. (1984) Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system.J. Molec. Bio. 178, 323–39.Google Scholar
  31. Trybus, K. M. &Lowey, S. (1987) Subunit exchange between smooth muscle myosin filaments.J. Cell Biol. 105, 3021–30.PubMedGoogle Scholar
  32. Tyler, J. M. &Branton, D. (1980) Rotary shadowing of extended molecules dried from glycerol.J. Ultrastruct. Res. 71, 95–102.PubMedGoogle Scholar
  33. Wallimann, T., Turner, D. C. &Eppenberger, H. M. (1977) Localization of creatine kinase isozymes in myofibrils.J. Cell Biol. 75, 297–317.PubMedGoogle Scholar
  34. Wang, D., McClure, J. &Tu, A. (1979) Titin: major myofibrillar components of striated muscle.Proc. Natl. Acad. Sci. (USA)76, 3698–702.Google Scholar
  35. Wenderoth, M. P. &Eisenberg, B. R. (1987) Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid treated rabbits.J. Cell Biol. 105, 2771–80.PubMedGoogle Scholar
  36. Wrigley, N. G. (1968) The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy.J. Ultrastruct. Res. 24, 454–64.PubMedGoogle Scholar
  37. Zak, R., Martin, A. F., Prior, G. &Rabinowitz, M. (1977) Comparison of turnover of several myofibrillar proteins and critical evaluation of double isotope method.J. Cell Biol. 252, 3430–5.Google Scholar
  38. Zak, R., Martin, A. F. &Blough, R. (1979) Assessment of protein turnover by use of radioisotopic tracers.Physiological Rev. 59, 407–47.Google Scholar

Copyright information

© Chapman and Hall Ltd. 1991

Authors and Affiliations

  • A. D. Saad
    • 1
  • J. E. Dennis
    • 1
  • I. P. Tan
    • 1
  • D. A. Fischman
    • 1
  1. 1.Department of Cell Biology and AnatomyCornell University Medical CollegeNew YorkUSA

Personalised recommendations