Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 May 1;315(Pt 3):753–759. doi: 10.1042/bj3150753

Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity.

R Dabrowska 1, H Hinssen 1, B Gałazkiewicz 1, E Nowak 1
PMCID: PMC1217271  PMID: 8645154

Abstract

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.

Full Text

The Full Text of this article is available as a PDF (597.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Bretscher A. Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties. J Biol Chem. 1984 Oct 25;259(20):12873–12880. [PubMed] [Google Scholar]
  3. Bryan J., Coluccio L. M. Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes. J Cell Biol. 1985 Oct;101(4):1236–1244. doi: 10.1083/jcb.101.4.1236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chacko S., Miyata H., Horiuchi K. Y. Modulation of actomyosin ATPase by thin filament-associated proteins. Prog Clin Biol Res. 1987;245:143–158. [PubMed] [Google Scholar]
  5. Cooper J. A., Pollard T. D. Effect of capping protein on the kinetics of actin polymerization. Biochemistry. 1985 Jan 29;24(3):793–799. doi: 10.1021/bi00324a039. [DOI] [PubMed] [Google Scholar]
  6. Crosbie R., Adams S., Chalovich J. M., Reisler E. The interaction of caldesmon with the COOH terminus of actin. J Biol Chem. 1991 Oct 25;266(30):20001–20006. [PMC free article] [PubMed] [Google Scholar]
  7. Czuryło E. A., Venyaminov SYu, Dabrowska R. Studies on secondary structure of caldesmon and its C-terminal fragments. Biochem J. 1993 Jul 15;293(Pt 2):363–368. doi: 10.1042/bj2930363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dabrowska R., Goch A., Gałazkiewicz B., Osińska H. The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta. 1985 Sep 27;842(1):70–75. doi: 10.1016/0304-4165(85)90295-8. [DOI] [PubMed] [Google Scholar]
  9. Doi Y., Frieden C. Actin polymerization. The effect of brevin on filament size and rate of polymerization. J Biol Chem. 1984 Oct 10;259(19):11868–11875. [PubMed] [Google Scholar]
  10. Gałazkiewicz B., Belagyi J., Dabrowska R. The effect of caldesmon on assembly and dynamic properties of actin. Eur J Biochem. 1989 May 15;181(3):607–614. doi: 10.1111/j.1432-1033.1989.tb14767.x. [DOI] [PubMed] [Google Scholar]
  11. Gałazkiewicz B., Mossakowska M., Osińska H., Dabrowska R. Polymerization of G-actin by caldesmon. FEBS Lett. 1985 May 6;184(1):144–149. doi: 10.1016/0014-5793(85)80671-2. [DOI] [PubMed] [Google Scholar]
  12. Gusev N. B., Pritchard K., Hodgkinson J. L., Marston S. B. Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments. J Muscle Res Cell Motil. 1994 Dec;15(6):672–681. doi: 10.1007/BF00121074. [DOI] [PubMed] [Google Scholar]
  13. Hartwig J. H., Kwiatkowski D. J. Actin-binding proteins. Curr Opin Cell Biol. 1991 Feb;3(1):87–97. doi: 10.1016/0955-0674(91)90170-4. [DOI] [PubMed] [Google Scholar]
  14. Hinssen H., Small J. V., Sobieszek A. A Ca2+-dependent actin modulator from vertebrate smooth muscle. FEBS Lett. 1984 Jan 23;166(1):90–95. doi: 10.1016/0014-5793(84)80051-4. [DOI] [PubMed] [Google Scholar]
  15. Horiuchi K. Y., Miyata H., Chacko S. Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins. Biochem Biophys Res Commun. 1986 May 14;136(3):962–968. doi: 10.1016/0006-291x(86)90426-2. [DOI] [PubMed] [Google Scholar]
  16. Houk T. W., Jr, Ue K. The measurement of actin concentration in solution: a comparison of methods. Anal Biochem. 1974 Nov;62(1):66–74. doi: 10.1016/0003-2697(74)90367-4. [DOI] [PubMed] [Google Scholar]
  17. Ishikawa R., Yamashiro S., Matsumura F. Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon. J Biol Chem. 1989 Oct 5;264(28):16764–16770. [PubMed] [Google Scholar]
  18. Ishikawa R., Yamashiro S., Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989 May 5;264(13):7490–7497. [PubMed] [Google Scholar]
  19. Janmey P. A., Chaponnier C., Lind S. E., Zaner K. S., Stossel T. P., Yin H. L. Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking. Biochemistry. 1985 Jul 2;24(14):3714–3723. doi: 10.1021/bi00335a046. [DOI] [PubMed] [Google Scholar]
  20. Kabsch W., Vandekerckhove J. Structure and function of actin. Annu Rev Biophys Biomol Struct. 1992;21:49–76. doi: 10.1146/annurev.bb.21.060192.000405. [DOI] [PubMed] [Google Scholar]
  21. Katayama E., Ikebe M. Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament. Biophys J. 1995 Jun;68(6):2419–2428. doi: 10.1016/S0006-3495(95)80424-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koepf E. K., Burtnick L. D. Interaction of plasma gelsolin with tropomyosin. FEBS Lett. 1992 Aug 31;309(1):56–58. doi: 10.1016/0014-5793(92)80738-3. [DOI] [PubMed] [Google Scholar]
  23. Kouyama T., Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981;114(1):33–38. [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lehrer S. S., Betteridge D. R., Graceffa P., Wong S., Seidel J. C. Comparison of the fluorescence and conformational properties of smooth and striated tropomyosin. Biochemistry. 1984 Apr 10;23(8):1591–1595. doi: 10.1021/bi00303a001. [DOI] [PubMed] [Google Scholar]
  26. Lehrer S. S., Morris E. P. Comparison of the effects of smooth and skeletal tropomyosin on skeletal actomyosin subfragment 1 ATPase. J Biol Chem. 1984 Feb 25;259(4):2070–2072. [PubMed] [Google Scholar]
  27. MacLean-Fletcher S., Pollard T. D. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem Biophys Res Commun. 1980 Sep 16;96(1):18–27. doi: 10.1016/0006-291x(80)91175-4. [DOI] [PubMed] [Google Scholar]
  28. Makuch R., Kołakowski J., Dabrowska R. The importance of C-terminal amino acid residues of actin to the inhibition of actomyosin ATPase activity by caldesmon and troponin I. FEBS Lett. 1992 Feb 10;297(3):237–240. doi: 10.1016/0014-5793(92)80546-s. [DOI] [PubMed] [Google Scholar]
  29. Marston S. B., Smith C. W. The thin filaments of smooth muscles. J Muscle Res Cell Motil. 1985 Dec;6(6):669–708. doi: 10.1007/BF00712237. [DOI] [PubMed] [Google Scholar]
  30. Marston S. Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle. Biochem J. 1990 Dec 1;272(2):305–310. doi: 10.1042/bj2720305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McLaughlin P. J., Gooch J. T., Mannherz H. G., Weeds A. G. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature. 1993 Aug 19;364(6439):685–692. doi: 10.1038/364685a0. [DOI] [PubMed] [Google Scholar]
  32. Pope B. J., Wagner P. D., Weeds A. G. Heterogeneity of myosin heavy chains in subfragment-1 isoenzymes rabbit skeletal myosin. J Mol Biol. 1977 Jan 25;109(3):470–473. doi: 10.1016/s0022-2836(77)80024-7. [DOI] [PubMed] [Google Scholar]
  33. Smith C. W., Pritchard K., Marston S. B. The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin. J Biol Chem. 1987 Jan 5;262(1):116–122. [PubMed] [Google Scholar]
  34. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  35. Stepkowski D., Szczesna D., Wrotek M., Kakol I. Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin. Biochim Biophys Acta. 1985 Oct 18;831(3):321–329. doi: 10.1016/0167-4838(85)90114-1. [DOI] [PubMed] [Google Scholar]
  36. Strzelecka-Gołaszewska H., Hinssen H., Sobieszek A. Influence of an actin-modulating protein from smooth muscle on actin-myosin interaction. FEBS Lett. 1984 Nov 19;177(2):209–216. doi: 10.1016/0014-5793(84)81285-5. [DOI] [PubMed] [Google Scholar]
  37. Vandekerckhove J. Actin-binding proteins. Curr Opin Cell Biol. 1990 Feb;2(1):41–50. doi: 10.1016/s0955-0674(05)80029-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES