Summary
Rabbit psoas muscle fibres, relaxed and in rigor, have been freeze substituted for electron microscopy. Fourier transforms and average density maps of micrographs of transverse sections have been obtained and compared to X-ray diffraction data. The Fourier amplitudes from rigor and relaxed muscle are comparable to equatorial data from X-ray diffraction of muscle if there is more disorder in the electron micrographs which can be described by a ‘temperature’ factor. The phases of reflections out to the 3,2 have been determined; those reflections at the same radius and therefore not separable in the X-ray patterns, such as the 2,1 and the 1,2, are separated in the transforms of sections through the A band. In transforms from both rigor and relaxed muscle they have the same phase. In rigor muscle they have different amplitudes. All the phases are positive or negative showing that the lattice is centrosymmetric at the resolution obtained. The phases obtained generally support those suggested by model building studies using X-ray diffraction data.
In rigor muscle, areas where the cross-bridges are regularly attached are clearly seen in thin transverse sections. A handedness to this structure is indicated by a lack of mirror symmetry, in both the Fourier transform of thick sections, and in the averaged density map. This correlates well with the arrangement where the myosin head is bound as in the acto-S1 structure but only to actin monomers within a limited azimuthal range.
Similar content being viewed by others
References
Brenner, B. & Yu, L. C. (1985) Equatorial X-ray diffraction from single skinned rabbit psoas fibers at various degrees of activation. Biophys. J. 48, 829–34.
Brenner, B., Yu, L. C. & Podolsky, R. J. (1984) X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths. Biophys. J. 46, 299–306.
Eastwood, A. B., Wood, D. S., Block, K. L. & Sorenson, M. M. (1979) Chemically skinned mammalian skeletal muscle. 1 The structure of skinned rabbit psoas. Tissue & Cell. 11, 553–66.
Handley, D. A., Alexander, J. T. & Chien, S. (1981) The design and use of a simple device for rapid quench-freezing of biological samples. J. Microsc. 121, 273–82.
Haselgrove, J. C., Stewart, M. & Huxley, H. (1976) Cross-bridge movement during muscle contraction. Nature 261, 606–8.
Hayat, M. A. (1989) Principles and techniques of electron microscopy. London: The Macmillan Press Ltd.
Henderson, R., Baldwin, J. M., Downing, K. H., Lepault, J. & Zemlin, F. (1986) Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 A resolution. Ultramicroscopy 19, 147–78.
Heuser, J. E. & Cooke, R. (1983) Actin-myosin interactions visualised by the quick-freeze, deep-etch replica technique. J. Mol. Biol. 169, 97–122.
Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L. & Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, 275–300.
Hirose, K. & Wakabayashi, T. (1988) Thin filaments of rabbit skeletal muscle are in helical register. J. Mol. Biol. 204, 797–801.
Hirose, K. & Wakabayashi, T. (1993) Structural changes of cross-bridges of rabbit skeletal muscle during isometric contraction. J. Muscle Res. Cell Motil. 14, 432–45.
Hirose, K., Lenart, T. D., Murray, J. M., Franzini-Armstrong, C. & Goldman, Y. E. (1993) Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP. Biophys. J. 65, 397–408.
Hirose, K., Franzini-Armstrong, C., Goldman, Y. E. & Murray, J. M. (1994) Structural changes in muscle crossbridges accompanying force generation. J. Cell Biol. 127, 763–78.
Huxley, H. E. (1968) Structural difference between resting and rigor muscle; evidence from intensity changes in the low-angle equatorial X-ray diagram. J. Mol. Biol. 37, 507–20.
Huxley, H., Popp, D., Ouynag, G. & Sosa, H. (1992) Cross-bridges in frozen contracting muscle. Faseb J. 6, 300.
Ip, W. & Heuser, J. (1983) Direct visualization of the myosin cross-bridge helices on relaxed rabbit psoas thick filaments. J. Mol. Biol. 171, 105–9.
Irving, T. C. & Millman, B. M. (1989) Changes in thick filament structure during compression of the filament lattice in relaxed frog sartorius muscle. J. Muscle Res. Cell Motil. 10, 385–96.
Kensler, R. W. & Stewart, M. (1993) The relaxed cross-bridge pattern in isolated rabbit psoas muscle thick filaments. J. Cell Sci. 105, 841–8.
LI, Q. (1993) X-ray diffraction studies on muscle contraction. PhD. Thesis. University of Guelph.
Lowy, J., Popp, D. & Stewart, A. A. (1991) X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60, 812–24.
Milligan, R. A. & Flicker, P. F. (1987) Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J. Cell Biol 105, 29–39.
Milligan, R. A., Whittaker, M. & Safer, D. (1990) Molecular structure of F-actin and location of surface binding sites. Nature 348, 217–21.
Naylor, G. R. S. & Podolsky, R. J. (1981) X-ray diffraction of strained muscle fibres in rigor. Proc. Natl Acad. Sci. U.S.A. 78, 5559–63.
Padron, R. & Craig, R. (1989) Disorder induced in nonoverlap cross-bridges by loss of adenosine triphosphate. Biophys. J. 56, 927–33.
Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C. & Milligan, R. A. (1993a) Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 58–65.
Rayment, I., Rypniewski, W. R., Schmidt-Base, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993b) Three-dimensional structure of myosin subfragment-1: A molecular motor. Science 261, 50–8.
Reedy, M. K. & Reedy, M. C. (1985) Rigor cross-bridge structure in tilted single filament layers and flared-X formations for insect flight muscle. J. Mol. Biol. 185, 145–76.
Sosa, H., Ouyang, G. & Huxley, H. (1993) Electron microscope study of contracting muscle fibers during fast mechanical transients. Biophys. J. 64, 26.
Sosa, H., Popp, D., Ouyang, G. & Huxley, H. E. (1994) Ultrastructure of skeletal muscle fibres studied by a plunge quick freeze method: myofilament lengths. Biophys. J. 67, 283–92.
Squire, J. M. & Harford, J. J. (1988) Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states. J. Muscle Res. Cell Motil. 9, 344–58.
Squire, J. M. & Luther, P. K. (1980) Three-dimensional structure of the vertebrate muscle A-band. II. The myosin filament superlattice. J. Mol. Biol. 141, 409–39.
Taylor, K. A., Reedy, M. C., Córdova, L. & Reedy, M. K. (1989) Three-dimensional reconstruction of insect flight muscle. I. The rigor myac layer. J. Cell Biol. 109, 1085–102.
Trus, B. L., Steven, A. C., Mcdowall, A. W., Unser, M., Dubochet, J. & Podolsky, R. J. (1989) Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated sections. Biophys. J. 55, 713–24.
Tsukita, S. & Yano, M. (1985) Actomyosin structure in contracting muscle detected by rapid freezing. Nature 317, 182–4.
Wray, J. S. (1987) Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8, 62.
Yagi, N. (1992) Effects of N-ethylmaleimide on the structure of skinned frog skeletal muscles. J. Muscle Res. Cell Motil. 13, 457–63.
Yu, L. C. (1989) Analysis of equatorial X-ray diffraction patterns from skeletal muscle. Biophys. J. 55, 433–40.
Yu, L. C. & Brenner, B. (1986) High resolution equatorial X-ray diffraction from single skinned rabbit psoas fibres. Biophys. J. 49, 133–5.
Yu, L. C. & Brenner, B. (1989) Structures of actomyosin cross-bridges in relaxed and rigor muscle fibers. Biophys. J. 55, 411–53.
Yu, L. C., Steven, A. C., Naylor, G. R. S., Gamble, R. C. & Podolsky, R. J. (1985) Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation. Biophys. J. 47, 311–21.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hawkins, C.J., Bennett, P.M. Evaluation of freeze substitution in rabbit skeletal muscle. Comparison of electron microscopy to X-ray diffraction. J Muscle Res Cell Motil 16, 303–318 (1995). https://doi.org/10.1007/BF00121139
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00121139