Summary
Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca2+ channel.
Similar content being viewed by others
Abbreviations
- SP:
-
spanning protein
- SR:
-
sarcoplasmic reticulum
- TC:
-
terminal cisternae
- T-tubule:
-
transverse tubule
- HMW:
-
high molecular weight
- ELISA:
-
enzyme linked immunosorbent assay
- PMSF:
-
phenylmethyl sulphonylfluoride
- SDS:
-
sodium dodecyle sulphate
References
Bianchi, C. P. (1968) Pharmacological actions on excitation-contraction coupling in striated muscle.Fed. Proc. 27, 126–131.
Brunschwig, J-P., Brandt, N. Caswell, A. H. &Lukeman, D. S. (1982) Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting.J. Cell Biol. 93, 533–542.
Burnette, W. N. (1981) Western blotting: electrophoretic transfer of protein from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose to radiographic detection with antibody and radioiodinated protein A.Anal. Bioch. 112, 195–203.
Cadwell, J. J. S. &Caswell, A. H. (1982). Identification of a constituent of junctional feet linking terminal cisternae to transverse tubules in skeletal muscle.J. Cell Biol. 93, 543–550.
Campbell, K. P., Knudson, C. M., Imagawa, T., Loung, A. T., Sutko, J. L., Kohl, S. D., Raab, C. R. &Madson, L. (1987) Identification and characterization of the high affinity [3H] ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel.J. Biol. Chem. 262, 6460–6463.
Caswell, A. H. &Brunschwig, J-P. (1984) Identification and extraction of proteins that compose the triad junction of skeletal muscle.J. Cell Biol,99, 929–939.
Caswell, A. H., Lau, Y. H. &Brunschwig, J-P. (1976) Ouabain-binding vesicles from skeletal muscle.Arch. Biochem. Biophys. 176, 417–430.
Chandler, W. K., Rakowski, R. F. &Schneider, M. F. (1976) Effects of glycerol treatment and maintained depolarization on charge movement in skeletal muscle.J. Physiol. (Lond.) 254, 285–316.
Costantin, L. L. &Podolsky, R. J. (1967) Depolarization of the internal membrane system in the activation of frog skeletal muscle.J. Gen. Physiol. 50, 1101–1124.
Eisenberg, B. R. &Eisenberg, R. S. (1982) The T-SR junction in contracting single skeletal muscle fibers.J. Gen. Physiol,79, 1–19.
Endo, M., Tanaka, M. &Ogawa, Y. (1970) Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres.Nature (Lond.) 228, 34–36.
Ford, L. E. &Podolsky, R. J. (1970) Regenerative calcium release within muscle cells.Science 167, 58–59.
Franzini-Armstrong, C. (1970) Studies of the triad. I. Structure of the junction in frog twitch fibers.J. Cell Biol. 47, 488–499.
Inui, M., Saito, A. &Fleischer, S. (1987) Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle.J. Biol. Chem. 262, 1740–1747.
Kawamoto, R. M., Brunschwig, J-P., Kim, K. C. &Caswell, A. H. (1986) Isolation, characterization, and localization of the spanning protein from skeletal muscle triads.J. Cell Biol. 103, 1405–1414.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage-T4.Nature (Lond.) 277, 680–685.
Lai, F. A., Erickson, H., Block, B. A. &Meissner, G. (1987) Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum.Bioch. Biophys. Res. Comm. 143, 704–709.
Schneider, M. F. &Chandler, W. K. (1973) Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling.Nature 242, 244–46.
Somlyo, A. V. (1979) Bridging structures spanning the junctional gap at the triad of skeletal muscle.J. Cell Biol. 80, 743–750.
Towbin, H., Staehelin, T. &Gordon, J. (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some application.Proc. Natn. Acad. Sci. 76, 4350–4354.
Vergara, J., Tsien, R. Y. &Delay, M. (1985) Inositol 1,4,5-triphosphate: a possible chemical link in excitation-contraction coupling in muscle.Proc. Natl. Acad. Sci. USA 82, 6352–6356.
Volpe, P., Salviati, G., Divirgilio, F. &Pozzan, T. (1985) Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle.Nature (Lond.) 316, 347–349.
Zorzato, F., Margreth, A. &Volpe, P. (1986) Direct photoaffinity labelling of junctional sarcoplasmic reticulum with [14C] Doxorubicin.J. Biol. Chem. 261, 13252–13257.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kawamoto, R.M., Brunschwig, J.P. & Caswell, A.H. Localization by immunoelectron microscopy of spanning protein of triad junction in terminal cisternae/triad vesicles. J Muscle Res Cell Motil 9, 334–343 (1988). https://doi.org/10.1007/BF01773877
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01773877