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J Cell Biol. 1996 Oct 1; 135(1): 169–179.
PMCID: PMC2121029

Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides


Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4- mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on- rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Barkalow K, Witke W, Kwiatkowski DJ, Hartwig JH. Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein. J Cell Biol. 1996 Jul;134(2):389–399. [PMC free article] [PubMed]
  • Agranoff BW, Murthy P, Seguin EB. Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets. J Biol Chem. 1983 Feb 25;258(4):2076–2078. [PubMed]
  • Barron-Casella EA, Torres MA, Scherer SW, Heng HH, Tsui LC, Casella JF. 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. [PubMed]
  • Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. [PubMed]
  • Bryan J. Isolation of fascin, an actin-bundling protein, and SU45, an actin-severing/capping protein from sea urchin eggs. Methods Enzymol. 1986;134:13–23. [PubMed]
  • Caldwell JE, Heiss SG, Mermall V, Cooper JA. Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin. Biochemistry. 1989 Oct 17;28(21):8506–8514. [PubMed]
  • Cano ML, Lauffenburger DA, Zigmond SH. Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution. J Cell Biol. 1991 Nov;115(3):677–687. [PMC free article] [PubMed]
  • Carlier MF, Pantaloni D. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Semin Cell Biol. 1994 Jun;5(3):183–191. [PubMed]
  • Casella JF, Torres MA. Interaction of Cap Z with actin. The NH2-terminal domains of the alpha 1 and beta subunits are not required for actin capping, and alpha 1 beta and alpha 2 beta heterodimers bind differentially to actin. J Biol Chem. 1994 Mar 4;269(9):6992–6998. [PubMed]
  • Casella JF, Maack DJ, 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]
  • Casella JF, Craig SW, Maack DJ, Brown AE. 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. [PMC free article] [PubMed]
  • Cooper JA, Walker SB, Pollard TD. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil. 1983 Apr;4(2):253–262. [PubMed]
  • Cooper JA, Blum JD, Pollard TD. Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization. J Cell Biol. 1984 Jul;99(1 Pt 1):217–225. [PMC free article] [PubMed]
  • Cooper JA, Caldwell JE, Gattermeir DJ, Torres MA, Amatruda JF, Casella JF. Variant cDNAs encoding proteins similar to the alpha subunit of chicken CapZ. Cell Motil Cytoskeleton. 1991;18(3):204–214. [PubMed]
  • Craig SW, Pardo JV. Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. Cell Motil. 1983;3(5-6):449–462. [PubMed]
  • de Rouffignac C, Quamme G. Renal magnesium handling and its hormonal control. Physiol Rev. 1994 Apr;74(2):305–322. [PubMed]
  • DiNubile MJ, Cassimeris L, Joyce M, Zigmond SH. 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. [PMC free article] [PubMed]
  • Eberle M, Traynor-Kaplan AE, Sklar LA, Norgauer J. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils? J Biol Chem. 1990 Oct 5;265(28):16725–16728. [PubMed]
  • Eddy RJ, Sauterer RA, Condeelis JS. 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]
  • Haimoto H, Kato K. S100a0 (alpha alpha) protein in cardiac muscle. Isolation from human cardiac muscle and ultrastructural localization. Eur J Biochem. 1988 Jan 15;171(1-2):409–415. [PubMed]
  • Hall AL, 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. [PMC free article] [PubMed]
  • Hartwig JH. Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol. 1992 Sep;118(6):1421–1442. [PMC free article] [PubMed]
  • Hartwig JH, Shevlin P. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages. J Cell Biol. 1986 Sep;103(3):1007–1020. [PMC free article] [PubMed]
  • Hartwig JH, Bokoch GM, Carpenter CL, Janmey PA, Taylor LA, Toker A, Stossel TP. 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. [PubMed]
  • Haus U, Trommler P, Fisher PR, Hartmann H, Lottspeich F, Noegel AA, 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. [PMC free article] [PubMed]
  • Heierhorst J, Kobe B, Feil SC, Parker MW, Benian GM, Weiss KR, Kemp BE. Ca2+/S100 regulation of giant protein kinases. Nature. 1996 Apr 18;380(6575):636–639. [PubMed]
  • Heiss SG, Cooper JA. Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. Biochemistry. 1991 Sep 10;30(36):8753–8758. [PubMed]
  • Hoock TC, Newcomb PM, Herman IM. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653–664. [PMC free article] [PubMed]
  • Hope HR, Pike LJ. Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. Mol Biol Cell. 1996 Jun;7(6):843–851. [PMC free article] [PubMed]
  • Hug C, Miller TM, Torres MA, Casella JF, Cooper JA. Identification and characterization of an actin-binding site of CapZ. J Cell Biol. 1992 Feb;116(4):923–931. [PMC free article] [PubMed]
  • Hug C, Jay PY, Reddy I, McNally JG, Bridgman PC, Elson EL, Cooper JA. Capping protein levels influence actin assembly and cell motility in dictyostelium. Cell. 1995 May 19;81(4):591–600. [PubMed]
  • Isenberg G, Aebi U, Pollard TD. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. [PubMed]
  • Ivanenkov VV, Jamieson GA, Jr, Gruenstein E, Dimlich RV. Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. J Biol Chem. 1995 Jun 16;270(24):14651–14658. [PubMed]
  • Ivanenkov VV, Dimlich RV, Jamieson GA., Jr Interaction of S100a0 protein with the actin capping protein, CapZ: characterization of a putative S100a0 binding site in CapZ alpha-subunit. Biochem Biophys Res Commun. 1996 Apr 5;221(1):46–50. [PubMed]
  • Janmey PA. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu Rev Physiol. 1994;56:169–191. [PubMed]
  • Janmey PA, Stossel TP. Gelsolin-polyphosphoinositide interaction. Full expression of gelsolin-inhibiting function by polyphosphoinositides in vesicular form and inactivation by dilution, aggregation, or masking of the inositol head group. J Biol Chem. 1989 Mar 25;264(9):4825–4831. [PubMed]
  • Janmey PA, Iida K, Yin HL, Stossel TP. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987 Sep 5;262(25):12228–12236. [PubMed]
  • Kron SJ, Drubin DG, Botstein D, Spudich JA. Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4466–4470. [PMC free article] [PubMed]
  • Kucera GL, Rittenhouse SE. Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. J Biol Chem. 1990 Apr 5;265(10):5345–5348. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Majerus PW. Inositol phosphate biochemistry. Annu Rev Biochem. 1992;61:225–250. [PubMed]
  • Marchand JB, Moreau P, Paoletti A, Cossart P, Carlier MF, Pantaloni D. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface. J Cell Biol. 1995 Jul;130(2):331–343. [PMC free article] [PubMed]
  • Moon A, Drubin DG. The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. Mol Biol Cell. 1995 Nov;6(11):1423–1431. [PMC free article] [PubMed]
  • Nachmias VT, Cassimeris L, Golla R, Safer D. Thymosin beta 4 (T beta 4) in activated platelets. Eur J Cell Biol. 1993 Aug;61(2):314–320. [PubMed]
  • Nachmias VT, Golla R, Casella JF, Barron-Casella E. Cap Z, a calcium insensitive capping protein in resting and activated platelets. FEBS Lett. 1996 Jan 15;378(3):258–262. [PubMed]
  • Otey CA, Kalnoski MH, Bulinski JC. Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell Motil Cytoskeleton. 1988;9(4):337–348. [PubMed]
  • Pantaloni D, Carlier MF. How profilin promotes actin filament assembly in the presence of thymosin beta 4. Cell. 1993 Dec 3;75(5):1007–1014. [PubMed]
  • Pollard TD. 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. [PMC free article] [PubMed]
  • Pring M, Weber A, Bubb MR. Profilin-actin complexes directly elongate actin filaments at the barbed end. Biochemistry. 1992 Feb 18;31(6):1827–1836. [PubMed]
  • Quamme GA, Dai LJ, Rabkin SW. Dynamics of intracellular free Mg2+ changes in a vascular smooth muscle cell line. Am J Physiol. 1993 Jul;265(1 Pt 2):H281–H288. [PubMed]
  • Schafer DA, Cooper JA. Control of actin assembly at filament ends. Annu Rev Cell Dev Biol. 1995;11:497–518. [PubMed]
  • Schafer DA, Korshunova YO, Schroer TA, Cooper JA. Differential localization and sequence analysis of capping protein beta-subunit isoforms of vertebrates. J Cell Biol. 1994 Oct;127(2):453–465. [PMC free article] [PubMed]
  • Spudich JA, 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]
  • Tanaka H, Yoshimura Y, Nishina Y, Nozaki M, Nojima H, Nishimune Y. Isolation and characterization of cDNA clones specifically expressed in testicular germ cells. FEBS Lett. 1994 Nov 21;355(1):4–10. [PubMed]
  • Tilney LG, DeRosier DJ, Tilney MS. How Listeria exploits host cell actin to form its own cytoskeleton. I. Formation of a tail and how that tail might be involved in movement. J Cell Biol. 1992 Jul;118(1):71–81. [PMC free article] [PubMed]
  • Walsh TP, Weber A, Higgins J, Bonder EM, Mooseker MS. Effect of villin on the kinetics of actin polymerization. Biochemistry. 1984 Jun 5;23(12):2613–2621. [PubMed]
  • Wilson DB, Neufeld EJ, Majerus PW. Phosphoinositide interconversion in thrombin-stimulated human platelets. J Biol Chem. 1985 Jan 25;260(2):1046–1051. [PubMed]
  • Witke W, Sharpe AH, Hartwig JH, Azuma T, Stossel TP, Kwiatkowski DJ. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell. 1995 Apr 7;81(1):41–51. [PubMed]
  • Zimmerle CT, Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J. 1989 Mar 1;258(2):381–387. [PMC free article] [PubMed]

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