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.

[1]  D. Botstein,et al.  Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[2]  L. Sklar,et al.  Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils? , 1990, The Journal of biological chemistry.

[3]  P. Fisher,et al.  The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin‐binding protein cap32/34. , 1993, The EMBO journal.

[4]  S. Craig,et al.  Cap Z06132), a Barbed End Actin-capping Protein, Is a Component of the Z-line of Skeletal Muscle Materials and Methods , 1987 .

[5]  Marie-France Carlier,et al.  How profilin promotes actin filament assembly in the presence of thymosin β4 , 1993, Cell.

[6]  S. Rabkin,et al.  Dynamics of intracellular free Mg2+ changes in a vascular smooth muscle cell line. , 1993, The American journal of physiology.

[7]  E. Neufeld,et al.  Phosphoinositide interconversion in thrombin-stimulated human platelets. , 1985, The Journal of biological chemistry.

[8]  P. Majerus,et al.  Inositol phosphate biochemistry. , 1992, Annual review of biochemistry.

[9]  E. Gruenstein,et al.  Characterization of S-100b Binding Epitopes. IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING PROTEIN, CapZ (*) , 1995, The Journal of Biological Chemistry.

[10]  J. Bryan.  Isolation of fascin, an actin-bundling protein, and SU45, an actin-severing/capping protein from sea urchin eggs. , 1986, Methods in enzymology.

[11]  G. Kucera,et al.  Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. , 1990, The Journal of biological chemistry.

[12]  Jonathan A. Cooper,et al.  Differential localization and sequence analysis of capping protein beta- subunit isoforms of vertebrates , 1994, The Journal of cell biology.

[13]  Jonathan A. Cooper,et al.  Identification and characterization of an actin-binding site of CapZ , 1992, The Journal of cell biology.

[14]  P. Janmey,et al.  Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. , 1994, Annual review of physiology.

[15]  J H Hartwig,et al.  Mechanisms of actin rearrangements mediating platelet activation , 1992, The Journal of cell biology.

[16]  T. Pollard,et al.  Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization , 1984, The Journal of cell biology.

[17]  I. Herman,et al.  Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury , 1991, The Journal of cell biology.

[18]  E. Elson,et al.  Capping protein levels influence actin assembly and cell motility in dictyostelium , 1995, Cell.

[19]  J. Higgins,et al.  Effect of villin on the kinetics of actin polymerization. , 1984, Biochemistry.

[20]  G. A. Jamieson,et al.  Interaction of S100a0 protein with the actin capping protein, CapZ: characterization of a putative S100a0 binding site in CapZ alpha-subunit. , 1996, Biochemical and biophysical research communications.

[21]  Rajasree Golla,et al.  Cap Z, a calcium insensitive capping protein in resting and activated platelets , 1996, FEBS letters.

[22]  C. Otey,et al.  Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. , 1988, Cell motility and the cytoskeleton.

[23]  J. Hartwig,et al.  The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages , 1986, The Journal of cell biology.

[24]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[25]  D. Safer,et al.  Thymosin beta 4 (T beta 4) in activated platelets. , 1993, European journal of cell biology.

[26]  Kanefusa Kato,et al.  S100a0, (αα) protein in cardiac muscle , 1988 .

[27]  Walter Witke,et al.  Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin , 1995, Cell.

[28]  M. Carlier,et al.  Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. , 1994, Seminars in cell biology.

[29]  J. Casella,et al.  Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. , 1986, The Journal of biological chemistry.

[30]  G. A. Jamieson,et al.  Interaction of S100a0Protein with the Actin Capping Protein, CapZ: Characterization of a Putative S100a0Binding Site in CapZα-Subunit , 1996 .

[31]  K. R. Weiss,et al.  Ca2+ /S100 regulation of giant protein kinases , 1996, Nature.

[32]  S. Zigmond,et al.  Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2. , 1995, Molecular biology of the cell.

[33]  J. Condeelis,et al.  A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34. , 1996, Biochimica et biophysica acta.

[34]  D. DeRosier,et al.  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 , 1992, The Journal of cell biology.

[35]  J. Cooper,et al.  Variant cDNAs encoding proteins similar to the α subunit of chicken CapZ , 1991 .

[36]  J. Condeelis,et al.  Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation , 1989, The Journal of cell biology.

[37]  D. Pantaloni,et al.  How profilin promotes actin filament assembly in the presence of thymosin beta 4. , 1993, Cell.

[38]  S. Scherer,et al.  Sequence Analysis and Chromosomal Localization of Human Cap Z , 1995, The Journal of Biological Chemistry.

[39]  H. Nojima,et al.  Isolation and characterization of cDNA clones specifically expressed in testicular germ cells , 1994, FEBS letters.

[40]  L. Pike,et al.  Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. , 1996, Molecular biology of the cell.

[41]  C T Zimmerle,et al.  Analysis of progress curves by simulations generated by numerical integration. , 1989, The Biochemical journal.

[42]  Ching‐Shih Chen,et al.  Synthesis of L-α-phosphatidyl-D-myo-inositol 3,4,5-trisphosphate, an important intracellular signalling molecule , 1994 .

[43]  D. Drubin,et al.  The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. , 1995, Molecular biology of the cell.

[44]  D. Lauffenburger,et al.  Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution , 1991, The Journal of cell biology.

[45]  Deborah A. Brown,et al.  Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface , 1992, Cell.

[46]  B. Agranoff,et al.  Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets. , 1983, The Journal of biological chemistry.

[47]  P. Janmey,et al.  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. , 1989, The Journal of biological chemistry.

[48]  Jonathan A. Cooper,et al.  Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. , 1991, Biochemistry.

[49]  J. Casella,et al.  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. , 1994, The Journal of biological chemistry.

[50]  T D Pollard,et al.  Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments , 1986, The Journal of cell biology.

[51]  G. Quamme,et al.  Renal magnesium handling and its hormonal control. , 1994, Physiological reviews.

[52]  P. Cossart,et al.  Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface , 1995, The Journal of cell biology.

[53]  P. Janmey,et al.  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. , 1987, The Journal of biological chemistry.

[54]  Jonathan A. Cooper,et al.  Control of actin assembly at filament ends. , 1995, Annual review of cell and developmental biology.

[55]  P. Janmey,et al.  Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets , 1995, Cell.

[56]  M. Bubb,et al.  Profilin-actin complexes directly elongate actin filaments at the barbed end. , 1992, Biochemistry.

[57]  S. Craig,et al.  Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. , 1983, Cell motility.

[58]  J. Condeelis,et al.  Aginactin, an agonist-regulated F-actin capping activity is associated with an Hsc70 in Dictyostelium. , 1993, Journal of Biological Chemistry.

[59]  Jonathan A. Cooper,et al.  Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin. , 1989, Biochemistry.

[60]  C Frieden,et al.  Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. , 1983, Analytical biochemistry.

[61]  J. Spudich,et al.  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. , 1971, The Journal of biological chemistry.