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. 1996 Jul 2;134(2):389–399. doi: 10.1083/jcb.134.2.389

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

PMCID: PMC2120875  PMID: 8707824

Abstract

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.

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Selected References

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  1. Amatruda J. F., Cannon J. F., Tatchell K., Hug C., Cooper J. A. Disruption of the actin cytoskeleton in yeast capping protein mutants. Nature. 1990 Mar 22;344(6264):352–354. doi: 10.1038/344352a0. [DOI] [PubMed] [Google Scholar]
  2. Amatruda J. F., Cooper J. A. Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein. J Cell Biol. 1992 Jun;117(5):1067–1076. doi: 10.1083/jcb.117.5.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barkalow K., Hartwig J. H. Actin cytoskeleton. Setting the pace of cell movement. Curr Biol. 1995 Sep 1;5(9):1000–1002. doi: 10.1016/s0960-9822(95)00200-4. [DOI] [PubMed] [Google Scholar]
  4. Barkalow K., Hartwig J. H. The role of actin filament barbed-end exposure in cytoskeletal dynamics and cell motility. Biochem Soc Trans. 1995 Aug;23(3):451–456. doi: 10.1042/bst0230451. [DOI] [PubMed] [Google Scholar]
  5. Barron-Casella E. A., Torres M. A., Scherer S. W., Heng H. H., Tsui L. C., Casella J. F. Sequence analysis and chromosomal localization of human Cap Z. Conserved residues within the actin-binding domain may link Cap Z to gelsolin/severin and profilin protein families. J Biol Chem. 1995 Sep 15;270(37):21472–21479. doi: 10.1074/jbc.270.37.21472. [DOI] [PubMed] [Google Scholar]
  6. Carlsson L., Markey F., Blikstad I., Persson T., Lindberg U. Reorganization of actin in platelets stimulated by thrombin as measured by the DNase I inhibition assay. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6376–6380. doi: 10.1073/pnas.76.12.6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Casella J. F., Cooper J. A. Purification of cap Z from chicken skeletal muscle. Methods Enzymol. 1991;196:140–154. doi: 10.1016/0076-6879(91)96015-j. [DOI] [PubMed] [Google Scholar]
  8. Casella J. F., Craig S. W., Maack D. J., Brown A. E. Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle. J Cell Biol. 1987 Jul;105(1):371–379. doi: 10.1083/jcb.105.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casella J. F., Flanagan M. D., Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature. 1981 Sep 24;293(5830):302–305. doi: 10.1038/293302a0. [DOI] [PubMed] [Google Scholar]
  10. Casella J. F., Maack D. J., Lin S. Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. J Biol Chem. 1986 Aug 15;261(23):10915–10921. [PubMed] [Google Scholar]
  11. DiNubile M. J., Cassimeris L., Joyce M., Zigmond S. H. Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2. Mol Biol Cell. 1995 Dec;6(12):1659–1671. doi: 10.1091/mbc.6.12.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eddy R. J., Sauterer R. A., Condeelis J. S. Aginactin, an agonist-regulated F-actin capping activity is associated with an Hsc70 in Dictyostelium. J Biol Chem. 1993 Nov 5;268(31):23267–23274. [PubMed] [Google Scholar]
  13. Fox J. E. Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib. J Clin Invest. 1985 Oct;76(4):1673–1683. doi: 10.1172/JCI112153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fox J. E., Phillips D. R. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature. 1981 Aug 13;292(5824):650–652. doi: 10.1038/292650a0. [DOI] [PubMed] [Google Scholar]
  15. Hall A. L., Schlein A., Condeelis J. Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells. J Cell Biochem. 1988 Jul;37(3):285–299. doi: 10.1002/jcb.240370304. [DOI] [PubMed] [Google Scholar]
  16. Hall A. L., Warren V., Dharmawardhane S., Condeelis J. Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation. J Cell Biol. 1989 Nov;109(5):2207–2213. doi: 10.1083/jcb.109.5.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hartwig J. H., Bokoch G. M., Carpenter C. L., Janmey P. A., Taylor L. A., Toker A., Stossel T. P. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell. 1995 Aug 25;82(4):643–653. doi: 10.1016/0092-8674(95)90036-5. [DOI] [PubMed] [Google Scholar]
  18. Hartwig J. H. Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol. 1992 Sep;118(6):1421–1442. doi: 10.1083/jcb.118.6.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haus U., Trommler P., Fisher P. R., Hartmann H., Lottspeich F., Noegel A. A., Schleicher M. The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34. EMBO J. 1993 Oct;12(10):3763–3771. doi: 10.1002/j.1460-2075.1993.tb06054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heiss S. G., Cooper J. A. Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. Biochemistry. 1991 Sep 10;30(36):8753–8758. doi: 10.1021/bi00100a006. [DOI] [PubMed] [Google Scholar]
  21. Hug C., Miller T. M., Torres M. A., Casella J. F., Cooper J. A. Identification and characterization of an actin-binding site of CapZ. J Cell Biol. 1992 Feb;116(4):923–931. doi: 10.1083/jcb.116.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  23. Janmey P. A., Stossel T. P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 1987 Jan 22;325(6102):362–364. doi: 10.1038/325362a0. [DOI] [PubMed] [Google Scholar]
  24. Kovacsovics T. J., Bachelot C., Toker A., Vlahos C. J., Duckworth B., Cantley L. C., Hartwig J. H. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem. 1995 May 12;270(19):11358–11366. doi: 10.1074/jbc.270.19.11358. [DOI] [PubMed] [Google Scholar]
  25. Kurth M. C., Bryan J. Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin. J Biol Chem. 1984 Jun 25;259(12):7473–7479. [PubMed] [Google Scholar]
  26. Kurth M. C., Wang L. L., Dingus J., Bryan J. Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions. J Biol Chem. 1983 Sep 25;258(18):10895–10903. [PubMed] [Google Scholar]
  27. 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]
  28. Lassing I., Lindberg U. Polyphosphoinositide synthesis in platelets stimulated with low concentrations of thrombin is enhanced before the activation of phospholipase C. FEBS Lett. 1990 Mar 26;262(2):231–233. doi: 10.1016/0014-5793(90)80197-q. [DOI] [PubMed] [Google Scholar]
  29. Lind S. E., Janmey P. A., Chaponnier C., Herbert T. J., Stossel T. P. Reversible binding of actin to gelsolin and profilin in human platelet extracts. J Cell Biol. 1987 Aug;105(2):833–842. doi: 10.1083/jcb.105.2.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lind S. E., Yin H. L., Stossel T. P. Human platelets contain gelsolin. A regulator of actin filament length. J Clin Invest. 1982 Jun;69(6):1384–1387. doi: 10.1172/JCI110578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  32. Nachmias V. T., Golla R., Casella J. F., Barron-Casella E. Cap Z, a calcium insensitive capping protein in resting and activated platelets. FEBS Lett. 1996 Jan 15;378(3):258–262. doi: 10.1016/0014-5793(95)01474-8. [DOI] [PubMed] [Google Scholar]
  33. Pollard T. D. Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol. 1986 Dec;103(6 Pt 2):2747–2754. doi: 10.1083/jcb.103.6.2747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sauterer R. A., Eddy R. J., Hall A. L., Condeelis J. S. Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae. J Biol Chem. 1991 Dec 25;266(36):24533–24539. [PubMed] [Google Scholar]
  35. Schafer D. A., Hug C., Cooper J. A. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J Cell Biol. 1995 Jan;128(1-2):61–70. doi: 10.1083/jcb.128.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schafer D. A., Korshunova Y. O., Schroer T. A., Cooper J. A. Differential localization and sequence analysis of capping protein beta-subunit isoforms of vertebrates. J Cell Biol. 1994 Oct;127(2):453–465. doi: 10.1083/jcb.127.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schafer D. A., Mooseker M. S., Cooper J. A. Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation. J Cell Biol. 1992 Jul;118(2):335–346. doi: 10.1083/jcb.118.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sultan C., Plantavid M., Bachelot C., Grondin P., Breton M., Mauco G., Lévy-Toledano S., Caen J. P., Chap H. Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate. J Biol Chem. 1991 Dec 15;266(35):23554–23557. [PubMed] [Google Scholar]
  39. Toker A., Bachelot C., Chen C. S., Falck J. R., Hartwig J. H., Cantley L. C., Kovacsovics T. J. Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase. J Biol Chem. 1995 Dec 8;270(49):29525–29531. doi: 10.1074/jbc.270.49.29525. [DOI] [PubMed] [Google Scholar]
  40. Winokur R., Hartwig J. H. Mechanism of shape change in chilled human platelets. Blood. 1995 Apr 1;85(7):1796–1804. [PubMed] [Google Scholar]
  41. Witke W., Sharpe A. H., Hartwig J. H., Azuma T., Stossel T. P., Kwiatkowski D. J. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell. 1995 Apr 7;81(1):41–51. doi: 10.1016/0092-8674(95)90369-0. [DOI] [PubMed] [Google Scholar]

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