Summary
It is well established that caldesmon binds to actin (K b–107-108 m −1) and to tropomyosin (K b106 m −1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 106 m −1 in low salt buffer, but is weakened by increasing [KCl] reaching 105 mM−1 in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg2+ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen ‘frayed’ from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous ‘whiskers’ are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (Kb=106 m −1) which is independent of [Ca2+] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the ‘contractile domain’ in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.
Similar content being viewed by others
References
Clarke, M. L. &Tregear, R. T. (1982) Tension maintenance and crossbridge detachment.FEBS Lett. 143, 217–19.
Cooke, P. H., Fay, F. S. &Craig, R. (1989) Myosin filaments isolated from skinned amphibian smooth muscle cells are side polar.J. Muscle Res. Cell Motil. 10, 206–20.
Craig, R. &Megerman, J. (1977) Assembly of smooth muscle myosin into side polar filaments.J. Cell Biol. 75, 990–6.
Cross, R. A., Hodge, T. P. &Kendrick-Jones, J. (1991) Self assembly mechanism of non-sarcomeric myosin II.J. Cell. Sci. (Suppl)14, 17–21.
Furst, D. O., Cross, R. A., DeMey, R. A. &Small, J. V. (1986) Caldesmon is an elongated, flexible molecule localised in the actomyosin domains of smooth muscle.EMBO J. 5, 251–7.
Galaszkiewicz, B., Mossakowska, M., Osinska, H. &Dabrowska, R. (1985) Polymerisation of g-actin by caldesmon.FEBS Lett. 184, 144–9.
Haeberle, J. R., Trybus, K. M. &Warshaw, D. M. (1991) Caldesmon-dependent regulation of thin filament mobility.Biophys. J,59, 58a.
Hai, C. &Murphy, R. A. (1989) Ca2+, crossbridge phosphorylation and contraction.Ann. Rev. Physiol. 51, 285–98.
Hemric, M. E. &Chalovich, J. M. (1988) Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin.J. Biol. Chem. 263, 1878–85.
Hemric, M. E. &Chalovich, J. M. (1990) Characterisation of caldesmon binding to myosin.J. Biol. Chem. 265, 19672–8.
Ikebe, M. &Reardon, S. (1988) Binding of caldesmon to smooth muscle myosin.J. Biol. Chem. 263, 3055–8.
Kamm, K. E. &Stull, J. T. (1989) Regulation of smooth muscle contractile elements by second messengers.Ann. Rev. Physiol. 51, 299–313.
Lash, J. A., Sellers, J. R. &Hathaway, D. R. (1986) The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin.J. Biol. Chem. 261, 16155–60.
Lehman, W., Moody, C. J. &Craig, R. (1990) Caldesmon and the structure of vertebrate smooth muscle thin filaments.Ann. N.Y. Acad. Sci. 599, 75–84.
Mabuchi, K. &Wang, C-L. A. (1991) Electron microscopic studies of chicken gizzard caldesmon and its complex with calmodulin.J. Muscle Res. Cell Motil. 13, 146–51.
Margossian, S. S. &Lowey, S. (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle.Methods Enzymol. 85, 55–71.
Marston, S. B., Redwood, C. S. &Lehman, W. (1988) Reversal of caldesmon function by anti-caldesmon antibodies confirms its role in the calcium regulation of vascular smooth muscle thin filaments.Biochem. Biophys. Res. Commun. 155, 197–202.
Marston, S. B. (1989a) A tight binding interaction between smooth muscle native thin filaments and heavy meromyosin in the presence of MgATP.Biochem. J. 259, 303–6.
Marston, S. B. (1989b) What is latch? New ideas about tonic contraction in smooth muscle.J. Muscle Res. Cell Motil. 10, 97–100.
Marston, S. B. (1990) Stoichiometry and stability of caldesmon in native thin filaments from sheet aorta smooth muscle.Biochem. J. 272, 305–10.
Marston, S. B. &Smith, C. W. (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. &Smith, C. W. (1985) The thin filaments of smooth muscles.J. Muscle Res. Cell Motil. 6, 669–708.
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.
Moody, C. J., Marston, S. B. &Smith, C. W. J. (1985) Bundling of actin filaments by aorta caldesmon is not related to its regulatory function.FEBS Lett. 191, 107–12.
Pinter, K. &Marston, S. B. (1992) Phosphorylation of sheep aorta caldesmon.J. Muscle Res. Cell Motil. 13 (Abstract of the XXth European Muscle meeting).
Pritchard, K. &Marston, S. B. (1988) The control of calciumregulated thin filaments from vascular smooth muscle by calmodulin and other calcium-binding proteins.Biochem. Soc. Trans. 16, 355–6.
Pritchard, K. &Marston, S. B. (1989) Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.Biochem. J. 257, 839–43.
Redwood, C. A., Kendrick-Jones, J. &Marston, S. B. (1992) Probing caldesmon structure function by expression and mutagenesis of caldesmon cDNA.J. Muscle Res. Cell Motil. 14 (Abstract of the XXth European Muscle meeting).
Sellers, J. R., Pato, M. D. &Adelstein, R. S. (1981) Reversible phosphorylation of smooth muscle myosin, heavy meromyosin and platelet myosin.J. Biol. Chem. 256, 13137–42.
Small, J. V. &Squire, J. M. (1972) The contractile apparatus of smooth muscle.J. Mol. Biol. 67, 117–49.
Smith, C. W., 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.
Stafford, W. F., Jancso, A. &Graceffa, P. (1990) Caldesmon from rabbit liver: molecular weight and length by analytical ultracentrifugation.Arch. Biochem. Biophys. 281, 66–9.
Sutherland, C. &Walsh, M. P. (1989) Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin.J. Biol. Chem. 264, 578–83.
Taggart, M. J. &Marston, S. B. (1988) The effects of vascular smooth muscle caldesmon on force production by desensitised skeletal muscle fibres.FEBS Lett. 242, 171–4.
Velaz, L., Hemric, M. E., Benson, 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.
Velaz L., Ingraham, R. H. Chalovich, J. M. (1990) Dissociation of the effect of caldesmon on the ATPase activity and on the binding of smooth heavy meromyosin to actin by partial digestion of caldesmon.J. Biol. Chem. 265, 2929–34.
Walsh, M. P. &Sutherland, C. (1989) A model for caldesmon in latch-bridge formation in smooth muscle.Adv. Exp. Med. Biol. 255, 337–46.
Yanagisawa, M., Hamada, Y., Katsuragawa, Y., Imamura M., Mikawa, T. &Masaki, T. (1987) Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence.J. Mol. Biol. 198, 143–57.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Marston, S., Pinter, K. & Bennett, P. Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments. J Muscle Res Cell Motil 13, 206–218 (1992). https://doi.org/10.1007/BF01874158
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01874158