Summary
Crossbridges in quick-frozen deep-etched blowfly flight muscles (fromSarcophaga bullata) were compared with those observed in the traditional waterbug preparation (Lethocerus) and found to be indistinguishable. Hence, blowfly was chosen as a fresher more accessible tissue for determining the effect of various fixatives and nucleotides on crossbridge structure. In rigor control, crossbridges were most regular in muscles that were stabilized before freezing by prefixation in glutaraldehyde followed by ‘hardening’ with neutralized tannic acid, so all nucleotide treatments were terminated by such fixation. MgATP (5mm) converted the rigor pattern of crossbridges into a random array of disconnected thick filament projections. Lower levels of ATP (0.1mm) caused a variable but generally lesser degree of crossbridge disconnection, as did 5mm ADP (probably because it slowly converted to ATP inside the muscle fibres). Vanadate (1–2mm) potentiated muscle relaxation in the latter two nucleotide treatments (i.e. produced a greater degree of crossbridge disconnection). Thus, differences in overall crossbridge abundance were readily apparent in chemically fixed muscles. Structural details within individual crossbridges were less well preserved, however. Chemical prefixation tended to collapse the muscle lattice, add a surface film to the filaments and thus obscure crossbridge details. Rigorous control of fixative pH largely prevented these problems and permitted recognition of the fact that inSarcophaga flight muscle, as inLethocerus muscle in rigor, the S1 ‘heads’ of crossbridges attach to the thin filaments in the expected ‘arrowhead’ configuration.
Similar content being viewed by others
References
Auber, J. &Couteaux, R. (1963) Ultrastructure de la strie z dans des muscles de diptères.J. Microsc. 2, 309–24.
Dantzig, J. A. &Goldman, Y. E. (1985) Suppression of muscle contraction by vanadate. Mechanical and ligand binding studies on glycerol-extracted rabbit fibers.J. gen. Physiol. 86, 305–27.
Elliott, G. F. (1965) Low-angle X-ray diffraction patterns from insect flight muscle.J. molec. Biol. 13, 956–8.
Goodenough, U. &Heuser, J. E. (1982) Substructure of the outer dynein arm.J. Cell Biol. 95, 798–815.
Goodno, C. C. (1979) Inhibition of myosin ATPase by vanadate ion.Proc. natn. Acad. Sci. U.S.A. 76, 2620–4.
Goodno, C. C. (1982) Myosin active-site trapping with vanadate ion.Methods Enzymol. 85, 116–23.
Goodno, C. C. &Taylor, E. W. (1982) Inhibition of actomyosin ATPase by vanadate.Proc. natn. Acad. Sci. U.S.A. 79, 21–5.
Goody, R. S., Hofmann, W., Reedy, M. K., Magid, A. &Goodno, C. (1980) Relaxation of glycerinated insect flight muscle by vanadate.J. Musc. Res. Cell Motility 1, 198–9.
Goody, R. S., Holmes, K. C., Mannherz, H. G., Barrington-Leigh, J. &Rosenbaum, G. (1975) Cross-bridge conformation as revealed by x-ray diffraction studies of insect flight muscles with ATP analogues.Biophys. J. 15, 687–705.
Heuser, J. E. (1981) Quick-freeze, deep-etch preparation of samples for 3-D electron microscopy.Trends Biochem. Sci. 6, 64–8.
Heuser, J. E. (1983) Structure of the myosin crossbridge lattice in insect flight muscle.J. molec. Biol. 169, 123–54.
Heuser, J. E. &Cooke, R. (1983) Actin-myosin intractions visualized by the quick-freeze, deep-etch replica technique.J. molec. Biol. 169, 97–122.
Heuser, J. E., Reese, T. S., Jan, L. Y., Jan, L. N., Dennis, M. J. &Evans, L. (1979) Synaptic vesicle exocytosis captured by quick-freezing and correlated with quantal transmitter release.J. Cell Biol. 81, 275–300.
Huxley, H. E. &Hanson, J. (1957) Preliminary observations on the structure of insect flight muscle. InElectron Microscopy, Proc. Stockholm. Congr. (1956), pp. 202–3. Stockholm: Almquist & Wiksell.
Kobayashi, T., Martensen, T., Nath, J. &Flavin, M. (1978) Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility.Biochem. Biophys. Res. Commun. 81, 1313–18.
Macara, I. G. (1980) Vanadium—an element in search of a role.Trends Biochem. Sci. 4, 92–4.
Magid, A. &Goodno, C. C. (1982) Inhibition of cross-bridge force by vanadate ion.Biophys. J. 37, 107a.
Marston, S. B., Rodger, C. D. &Tregear, R. T. (1976) Changes in muscle crossbridges when β,γ-imido-ATP binds to myosin.J. molec. Biol. 104, 263–76.
McDowall, A. W., Hofman, W., Lepault, J., Adrian, M. &Dubochet, J. (1984) Cryo-electron microscopy of vitrified insect flight muscle.J. molec. Biol. 178, 105–11.
Pringle, J. W. S. (1977) The availability of insect fibrillar muscle. InInsect Flight Muscle (edited byTregear, R. T.), pp. 337–44. Amsterdam: North Holland.
Pringle, J. W. S. (1978) Stretch activation of muscle: function and mechanism.Proc. R. Soc. (Lond.) Ser. B 201, 107–30.
Rayns, D. G. (1972) Myofilaments and cross bridges as demonstrated by freeze-fracturing and etching.J. Ultra. Res. 40, 103–21.
Reedy, M. K. (1968) Ultrastructure of insect flight muscle. I. Screw sense and structural grouping in the rigor cross-bridge lattice.J. molec. Biol. 31, 155–76.
Reedy, M. K., Bahr, G. F. &Fischman, D. A. (1972) How many myosins per cross-bridge? I. Flight muscle myofibrils from the blowfly,Sarcophaga bullata.Cold Spring Harbor Symp. quant. Biol. 37, 397–421.
Reedy, M. K., Goody, R. S., Hoffman, W. &Rosenbaum, G. (1983a) Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. I. X-ray diffraction monitoring during preparation for electron microscopy of muscle fibres fixed in rigor, in ATP and in AMPPNP.J. Musc. Res. Cell Motility 4, 25–53.
Reedy, M. K. &Reedy, M. C. (1985) Rigor crossbridge structure in tilted single filament layers and flared-X formations from insect flight muscle.J. molec. Biol. 185, 145–76.
Reedy, M. C., Reedy, M. K. &Goody, R. S. (1983b) Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed in rigor, in ATP and in AMPPNP.J. Musc. Res. Cell Motility 4, 55–81.
Rome, E. (1968) X-ray diffraction studies of the filament lattice of striated muscle in various bathing media.J. molec. Biol. 37, 331–44.
Rome, E. (1972) Relaxation of glycerinated muscle: Low-angle x-ray diffraction studies.J. molec. Biol. 65, 331–45.
Sale, W. S. &Gibbons, I. R. (1979) Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin-sperm flagella.J. Cell Biol. 82, 291–8.
Squire, J. M. (1973) General model of myosin filament structure. III. Molecular packing arrangements in myosin filaments.J. molec. Biol. 77, 291–323.
Squire, J. M. (1977) The structure of insect thick filaments. InInsect Flight Muscle (edited byTregear, R. T.), pp. 91–112. Amsterdam: North Holland.
Squire, J. M. (1981)The Structural Basis of Muscle Contraction, New York: Plenum Press.
Taylor, K. A., Reedy, M. C., Cordova, L. &Reedy, M. K. (1984) Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections.Nature, Lond. 310, 285–91.
White, D. C. S. (1970) Rigor contraction and the effect of various phosphate compounds on glycerinated insect flight and vertebrate muscle.J. Physiol. (Lond.) 208, 583–605.
Worthington, C. F. (1961) X-ray diffraction studies on the large-scale molecular structure of insect muscle.J. molec. Biol. 3, 618–33.
Wray, J. S. (1979) Filament geometry and the activation of insect flight muscles.Nature, Lond. 280, 325–6.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Heuser, J.E. Crossbridges in insect flight muscles of the blowfly (Sarcophaga bullata). J Muscle Res Cell Motil 8, 303–321 (1987). https://doi.org/10.1007/BF01568887
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01568887