Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Sep 1;101(3):1115–1123. doi: 10.1083/jcb.101.3.1115

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin

PMCID: PMC2113698  PMID: 3897243

Abstract

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A- bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross- bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15- nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ashby B., Frieden C., Bischoff R. Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle. J Cell Biol. 1979 May;81(2):361–373. doi: 10.1083/jcb.81.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bader D., Masaki T., Fischman D. A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982 Dec;95(3):763–770. doi: 10.1083/jcb.95.3.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CASPAR D. L. ASSEMBLY AND STABILITY OF THE TOBACCO MOSAIC VIRUS PARTICLE. Adv Protein Chem. 1963;18:37–121. doi: 10.1016/s0065-3233(08)60268-5. [DOI] [PubMed] [Google Scholar]
  4. Cohen C., Lowey S., Harrison R. G., Kendrick-Jones J., Szent-Gyorgyi A. G. Segments from myosin rods. J Mol Biol. 1970 Feb 14;47(3):605–609. doi: 10.1016/0022-2836(70)90329-3. [DOI] [PubMed] [Google Scholar]
  5. Craig R., Offer G. Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle. J Mol Biol. 1976 Apr 5;102(2):325–332. doi: 10.1016/s0022-2836(76)80057-5. [DOI] [PubMed] [Google Scholar]
  6. Craig R., Offer G. The location of C-protein in rabbit skeletal muscle. Proc R Soc Lond B Biol Sci. 1976 Mar 16;192(1109):451–461. doi: 10.1098/rspb.1976.0023. [DOI] [PubMed] [Google Scholar]
  7. Craig R. Structure of A-segments from frog and rabbit skeletal muscle. J Mol Biol. 1977 Jan 5;109(1):69–81. doi: 10.1016/s0022-2836(77)80046-6. [DOI] [PubMed] [Google Scholar]
  8. Dennis J. E., Shimizu T., Reinach F. C., Fischman D. A. Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies. J Cell Biol. 1984 Apr;98(4):1514–1522. doi: 10.1083/jcb.98.4.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elliott A., Offer G., Burridge K. Electron microscopy of myosin molecules from muscle and non-muscle sources. Proc R Soc Lond B Biol Sci. 1976 Mar 30;193(1110):45–53. doi: 10.1098/rspb.1976.0030. [DOI] [PubMed] [Google Scholar]
  10. Elliott A., Offer G. Shape and flexibility of the myosin molecule. J Mol Biol. 1978 Aug 25;123(4):505–519. doi: 10.1016/0022-2836(78)90204-8. [DOI] [PubMed] [Google Scholar]
  11. Elliott G. F., Lowy J., Millman B. M. Low-angle x-ray diffraction studies of living striated muscle during contraction. J Mol Biol. 1967 Apr 14;25(1):31–45. doi: 10.1016/0022-2836(67)90277-x. [DOI] [PubMed] [Google Scholar]
  12. Etlinger J. D., Zak R., Fischman D. A. Compositional studies of myofibrils from rabbit striated muscle. J Cell Biol. 1976 Jan;68(1):123–141. doi: 10.1083/jcb.68.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. HALL C. E. Method for the observation of macromolecules with the electron microscope illustrated with micrographs of DNA. J Biophys Biochem Cytol. 1956 Sep 25;2(5):625–628. doi: 10.1083/jcb.2.5.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HUXLEY H. E. The double array of filaments in cross-striated muscle. J Biophys Biochem Cytol. 1957 Sep 25;3(5):631–648. doi: 10.1083/jcb.3.5.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hanson J., O'Brien E. J., Bennett P. M. Structure of the myosin-containing filament assembly (A-segment) separated from frog skeletal muscle. J Mol Biol. 1971 Jun 28;58(3):865–871. doi: 10.1016/0022-2836(71)90045-3. [DOI] [PubMed] [Google Scholar]
  17. Harrison R. G., Lowey S., Cohen C. Assembly of myosin. J Mol Biol. 1971 Aug 14;59(3):531–535. doi: 10.1016/0022-2836(71)90317-2. [DOI] [PubMed] [Google Scholar]
  18. Huxley H. E., Brown W. The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J Mol Biol. 1967 Dec 14;30(2):383–434. doi: 10.1016/s0022-2836(67)80046-9. [DOI] [PubMed] [Google Scholar]
  19. Huxley H. E. The fine structure of striated muscle and its functional significance. Harvey Lect. 1966;60:85–118. [PubMed] [Google Scholar]
  20. Ip W., Heuser J. Direct visualization of the myosin crossbridge helices on relaxed rabbit psoas thick filaments. J Mol Biol. 1983 Nov 25;171(1):105–109. doi: 10.1016/s0022-2836(83)80317-9. [DOI] [PubMed] [Google Scholar]
  21. Kensler R. W., Levine R. J. Determination of the handedness of the crossbridge helix of Limulus thick filaments. J Muscle Res Cell Motil. 1982 Sep;3(3):349–361. doi: 10.1007/BF00713042. [DOI] [PubMed] [Google Scholar]
  22. Knappeis G. G., Carlsen F. The ultrastructure of the M line in skeletal muscle. J Cell Biol. 1968 Jul;38(1):202–211. doi: 10.1083/jcb.38.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Levine R. J., Kensler R. W., Reedy M. C., Hofmann W., King H. A. Structure and paramyosin content of tarantula thick filaments. J Cell Biol. 1983 Jul;97(1):186–195. doi: 10.1083/jcb.97.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
  25. Lowey S., Steiner L. A. An immunochemical approach to the structure of myosin and the thick filament. J Mol Biol. 1972 Mar 14;65(1):111–126. doi: 10.1016/0022-2836(72)90495-0. [DOI] [PubMed] [Google Scholar]
  26. Maruyama K., Kimura S., Ohashi K., Kuwano Y. Connectin, an elastic protein of muscle. Identification of "titin" with connectin. J Biochem. 1981 Mar;89(3):701–709. doi: 10.1093/oxfordjournals.jbchem.a133249. [DOI] [PubMed] [Google Scholar]
  27. Masaki T., Takaiti O. M-protein. J Biochem. 1974 Feb;75(2):367–380. doi: 10.1093/oxfordjournals.jbchem.a130403. [DOI] [PubMed] [Google Scholar]
  28. O'Brien E. J., Bennett P. M., Hanson J. Optical diffraction studies of myofibrillar structure. Philos Trans R Soc Lond B Biol Sci. 1971 May 27;261(837):201–208. doi: 10.1098/rstb.1971.0051. [DOI] [PubMed] [Google Scholar]
  29. OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1–78. [PubMed] [Google Scholar]
  30. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Pepe F. A., Drucker B. The myosin filament. III. C-protein. J Mol Biol. 1975 Dec 25;99(4):609–617. doi: 10.1016/s0022-2836(75)80175-6. [DOI] [PubMed] [Google Scholar]
  33. Pepe F. A. Structure of muscle filaments from immunohistochemical and ultrastructural studies. J Histochem Cytochem. 1975 Jul;23(7):543–562. doi: 10.1177/23.7.1095653. [DOI] [PubMed] [Google Scholar]
  34. Pepe F. A. The myosin filament. II. Interaction between myosin and actin filaments observed using antibody staining in fluorescent and electron microscopy. J Mol Biol. 1967 Jul 28;27(2):227–236. doi: 10.1016/0022-2836(67)90017-4. [DOI] [PubMed] [Google Scholar]
  35. Reinach F. C., Masaki T., Shafiq S., Obinata T., Fischman D. A. Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies. J Cell Biol. 1982 Oct;95(1):78–84. doi: 10.1083/jcb.95.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Silberstein L., Lowey S. Isolated and distribution of myosin isoenzymes in chicken pectoralis muscle. J Mol Biol. 1981 May 15;148(2):153–189. doi: 10.1016/0022-2836(81)90510-6. [DOI] [PubMed] [Google Scholar]
  37. Sjöström M., Squire J. M. Fine structure of the A-band in cryo-sections. The structure of the A-band of human skeletal muscle fibres from ultra-thin cryo-sections negatively stained. J Mol Biol. 1977 Jan 5;109(1):49–68. doi: 10.1016/s0022-2836(77)80045-4. [DOI] [PubMed] [Google Scholar]
  38. Squire J. M. General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments. J Mol Biol. 1973 Jun 25;77(2):291–323. doi: 10.1016/0022-2836(73)90337-9. [DOI] [PubMed] [Google Scholar]
  39. Takahashi K. Topography of the myosin molecule as visualized by an improved negative staining method. J Biochem. 1978 Mar;83(3):905–908. doi: 10.1093/oxfordjournals.jbchem.a131988. [DOI] [PubMed] [Google Scholar]
  40. Trinick J. A. End-filaments: a new structural element of vertebrate skeletal muscle thick filaments. J Mol Biol. 1981 Sep 15;151(2):309–314. doi: 10.1016/0022-2836(81)90517-9. [DOI] [PubMed] [Google Scholar]
  41. Trinick J., Lowey S. M-protein from chicken pectoralis muscle: isolation and characterization. J Mol Biol. 1977 Jun 25;113(2):343–368. doi: 10.1016/0022-2836(77)90146-2. [DOI] [PubMed] [Google Scholar]
  42. Turner D. C., Wallimann T., Eppenberger H. M. A protein that binds specifically to the M-line of skeletal muscle is identified as the muscle form of creatine kinase. Proc Natl Acad Sci U S A. 1973 Mar;70(3):702–705. doi: 10.1073/pnas.70.3.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
  44. Wallimann T., Turner D. C., Eppenberger H. M. Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle. J Cell Biol. 1977 Nov;75(2 Pt 1):297–317. doi: 10.1083/jcb.75.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang K., McClure J., Tu A. Titin: major myofibrillar components of striated muscle. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3698–3702. doi: 10.1073/pnas.76.8.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]
  47. Winkelmann D. A., Lowey S., Press J. L. Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell. 1983 Aug;34(1):295–306. doi: 10.1016/0092-8674(83)90160-5. [DOI] [PubMed] [Google Scholar]
  48. Wrigley N. G. The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J Ultrastruct Res. 1968 Sep;24(5):454–464. doi: 10.1016/s0022-5320(68)80048-6. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES