Summary
An improved rotary shadowing technique enabled us to visualize chicken gizzard caldesmon (CaD) and its complexes with one or two covalently linked calmodulin (CaM) molecules by electron microscopy. Using a monoclonal antibody against an epitope in the N-terminal region of CaD (anti-N), we can now identify the end of the molecule that is involved in binding to another protein molecule. Thus in the 1:1 complex of CaD and CaM, the CaM molecule was almost always associated with the C-terminus of CaD, indicating preferential CaM-binding to the C-terminal region. We have also studied binding of CaD to filamentous actin (F-actin), using an EM technique that avoids spraying or freeze drying and thereby preserves the structure of F-actin. Only one end of CaD appeared to bind to F-actin, leaving the rest of the molecule projecting away from the filament. While the majority of anti-N bound at the free end of CaD, some antibody molecules were found on F-actin. These findings suggest that either end of CaD can bind to F-actin. Experiments using a monoclonal antibody against the C-terminus of CaD (anti-C) supported this idea. When the native thin filaments that contain endogenous CaD were incubated with anti-N, almost all the bound antibodies were found on the filaments, indicating that the N-terminal regions of CaD interact with actin, and that the binding affinity of the N-terminal region of CaD for actin is higher in vivo than that in vitro, either because the properties of CaD have been altered during purification, or because of the presence of some other component(s) associated with the native filaments.
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ADAM, L. P., HAEBERLE, J. R. & HATHAWAY, D. R. (1989) Phosphorylation of caldesmon in arterial smooth muscle. J. Biol. Chem. 264, 7698–703.
ADAMS, S., DASGUPTA, G., CHALOVICH, J. M. & REISLER, E. (1990) Immunochemical evidence for the binding of caldesmon to the NH2-terminal segment of actin. J. Biol. Chem. 265, 19652–7.
AARTEGI, A., FATTOUM, A. & KASSAB, R. (1990a) Crosslinking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin. J. Biol. Chem. 265, 2231–7.
AARTEGI, A., FATTOUM, A., DERANCOURT, J. & KASSAB, R. (1990b) Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actincalmodulin-binding region. J. Biol. Chem. 265, 15231–8.
ARETSCHER, A. (1984) Smooth muscle caldesmon. J. Biol. Chem. 259, 12873–80.
DEDMAN, J. R. & KAETZEL, M. A. (1983) Calmodulin purification and fluorescence labelling. Methods Enzymol. 102, 1–8.
ELLIOT, A. & OFFER, G. (1978) Shape and flexibility of the myosin molecule. J. Mol. Biol. 123, 505–19.
FUJII, T., IMAI, M., ROSENFELD, G. C. & ARYAN, J. (1987) Domain mapping of chicken gizzard caldesmon. J. Biol. Chem. 262, 2757–63.
FÜRST, D. O., CROSS, R. A., MEY, J. D. & SMALL, J. V. (1986) Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle. EMBO J. 5, 251–7.
GRACEFFA, P. & JANSCÓ, A. (1991) Disulfide cross-linking of caldesmon to actin. J. Biol. Chem. 266, 20305–10.
GRACEFFA, P., WANG, C.-L. A. & STAFFORD, W. F. (1988) Caldesmon: molecular weight and subunit composition by analytical ultracentrifugation. J. Biol. Chem. 263, 14196–202.
HALL, C. E. (1956) Visualization of individual macromolecules with the electron microscope. Proc. Natl. Acad. Sci. USA 42, 801–6.
HORIUCHI, K. Y., MIYATA, H. & CHACKO, S. (1986) Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins. Biochem. Biophys. Res. Common. 136, 962–8.
LEHMAN, W., CRAIG, R., LUI, J. & MOODY, C. (1989) Caldesmon and the structure of smooth muscle thin filaments: immunolocalization of caldesmon on the thin filaments. J. Muscle Res. Cell Motility 10, 101–12.
LESZYK, J., MORNET, D., AUDEMARD, E. & COLLINS, J. H. (1989) Amino acid sequence of a 15 kilodalton actin-binding fragment of turkey gizzard caldesmon: similarity with dystrophin, tropomyosin and the tropomyosin-binding region of troponin T. Biochem. Biophys. Res. Commun. 160, 210–6.
LEVINE, B. A., MOIR, A. J. G., AUDEMARD, E., MORNET, D., PATCHELL, V. B. & PERRY, S. V. (1990) Structural study of gizzard caldesmon and its interaction with actin: binding involves residues of actin also recognized by myosin subfragment 1. Eur. J. Biochem. 193, 687–96.
LIN, J. J-C., LIN, J. L-C., DAVIS-NANTHAKUMAR, E. J. & LOURIM, D. (1988) Monoclonal antibodies against caldesmon, a Ca2+/calmodulin- and actin-binding protein of smooth muscle and non-muscle cells. Hybridoma 7, 273–88.
LIN, J. J-C., DAVIS-NANTHAKUMAR, E. J., JIN, J-P., LOURIM, D., NOVY, R. E. 081 & LIN, J. L-C. (1991) Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca2+/calmodulin and to actin or actin-tropomyosin filaments. Cell Motil. Cytoskeleton 20, 95–108.
LYNCH, W. P., RISEMAN, V. M. & ARETSCHER, A. (1987) Smooth muscle caldesmon is an extended flexible monomeric protein in solution that can readily undergo reversible intra- and intermolecular sulfhydryl crosslinking. A mechanism for caldesmon's F-actin bundling activity. J. Biol. Chem. 262, 7429–37.
MABUCHI, K. (1990) Melting of myosin and tropomyosin: electron microscopic observations. J. Struct. Biol. 103, 249–56.
MABUCHI, K. (1991) Heavy-meromyosin-decorated actin filaments: a simple method to preserve actin filaments for rotary shadowing. J. Struct. Biol. 107, 22–8.
MABUCHI, K. & WANG, C-L. A. (1991) Electron microscopic studies of chicken gizzard caldesmon and its complex with calmodulin. J. Muscle Res. Cell. Motil. 12, 145–51.
MARSTON, S. B. & SMITH, C. W. J. (1984) Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle. J. Muscle Res. Cell Motil. 5, 559–75.
MARSTON, S. B. & LEHMAN, W. (1985) Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments. Biochem. J. 231, 517–22.
MARSTON, S. B. & REDWOOD, C. S. (1991) The molecular anatomy of caldesmon. Biochem. J. 279, 1–16.
MARTIN, F., HARRICANE, M-C., AUDEMARD, E., PONS, F. & MORNET, D. (1991) Conformational change of turkey gizzard caldesmon induced by specific chemical modification with carbodiimide. Eur. J. Biochem. 195, 335–42.
MOMMAERTS, W. F. H. M. (1952) The molecular transformation of actin. 1. Globular actin. J. Biol. Chem. 198, 445–75.
MOODY, C., LEHMAN, W. & CRAIG, R. (1990) Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments. J. Muscle Res. Cell Motility 11, 176–85.
NGAI, P. K. & WALSH, M. P. (1984) Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. J. Biol. Chem. 259, 13656–9.
SANDERS, C. & SMILLIE, L. B. (1984) Chicken gizzard tropomyosin: head-to-tail assembly and interaction with F-actin and troponin. Can. J. Biochem. Cell Biol. 62, 443–48.
SMITH, C. W. J. & MARSTON, S. B. (1985) Disassembly and reconstitution of the Ca2+-sensitive thin filaments of vascular smooth muscle. FEBS Lett. 184, 115–19.
SMITH, C. W. J., PRITCHARD, K. & MARSTON, S. B. (1987) The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin. J. Biol. Chem. 262, 116–22.
SOBUE, K., MURAMOTO, K., FUJITA, M. & KAKIUCHI, S. (1981) Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc. Natl. Acad. Sci. USA 78, 5652–5.
SOBUE, K. & SELLERS, J. R. (1991) Caldesmon, a novel regulatory protein in smooth muscle and non-muscle actomyosin systems. J. Biol. Chem. 266, 12115–8.
SZPACENKO, A. & DABROWSKA, R. (1986) Functional domain of caldesmon. FEBS Lett. 202, 182–6.
TYLER, J. M. & ARANTON, D. (1980) Rotary shadowing of extended molecules dried from glycerol. J. Ultrastruct. 71, 95–102.
VELAZ, L., HEMRIC, M. E., AENSON, C. E. & CHALOVICH, J. M. (1989) The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin. J. Biol. Chem. 264, 9602–10.
WANG, C-L. A. (1988) Photocrosslinking between calmodulin and/or actin with chicken gizzard caldesmon. Biochem. Biophys. Res. Commun. 156, 1033–8.
WANG, C-L. A., WANG, L-W. C. XU, S., LU, R. C., SAAVEDRA-ALANIS, V. & ARYAN, J. (1991a) Localization of the calmodulin- and the actin-binding sites of caldesmon. J. Biol. Chem. 266, 9166–72.
WANG, C-L. A., CHALOVICH, J. M., GRACEFFA, P., LU, R. C., MABUCHI, K. & STAFFORD, W. S. (1991b) A long helix from the central region of smooth muscle caldesmon. J. Biol. Chem. 266, 13958–63.
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Mabuchi, K., Lin, J.JC. & Wang, CL.A. Electron microscopic images suggest both ends of caldesmon interact with actin filaments. J Muscle Res Cell Motil 14, 54–64 (1993). https://doi.org/10.1007/BF00132180
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DOI: https://doi.org/10.1007/BF00132180