The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.     

Effect of cytochalasin B on formation and properties of muscle F-actin

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(α-aminoethyl ether)-N,N′-tetraacetic acid and reduced by MgCl₂ at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

Effect of cytochalasin B on formation and properties of muscle F-actin.

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(alpha-aminoethyl ether)-N,N'-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

A monovalent cation-sensitive actin-binding factor in a myeloid leukemia cell line

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.     

Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex

The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.     

Study of a protein complex from the brain which reduces actin viscosity. II. The complex inhibits elongation of actin filaments at the end preferable for polymerization and fragments the filaments

Functional properties of the protein complex from bovine brain that shortens actin filaments are described. In the presence of Ca2+ complex shortens actin filaments and increases the initial rate of actin polymerization. In the absence of free calcium ions the complex loses its accelerating effect on actin polymerization, but still possesses actin filament shortening activity. Neither phalloidin nor tropomyosin prevent the shortening of actin filaments induced by the protein complex. Therefore the protein complex causes the fragmentation of actin filament. The data on actin polymerization in the presence of F-actin nuclei have indicated that the protein complex inhibits the elongation step of actin polymerization. The analysis of elongation in the presence of both the protein complex and cytochalasin D has demonstrated that the inhibition occurs on the fast-growing end of actin filaments.     

Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1- pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre- activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.     

Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G- actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G- actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.     

Cytochalasin B slows but does not prevent monomer addition at the barbed end of the actin filament

We used Limulus sperm acrosomal actin bundles to examine the effect of 2 microM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 microM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 microM) in solutions containing 75 mM KCl, 5 mM MgCl2, 10 mM Imidazole, pH 7.2, and 2 microM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants were 8- to 10-fold smaller at the barbed end in the presence of CB along with an accompanying twofold increase in critical concentration at that end. Over the time course of experimentation there was little evidence for potentiation by CB of the nucleation step of assembly. CB did not sever actin filaments; instead its presence increased the susceptibility of actin filaments to breakage from the gentle shear forces incurred during sample preparation. Under these experimental conditions, the assembly rate constants and critical concentration at the pointed end were the same in both the presence and the absence of CB.     

Transformation of actin-encapsulating liposomes induced by cytochalasin D.

Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction.     

Isolation and characterization of hog thyroid actin

When the thyroglobulin content is subtracted, actin represents approximately 4.6% of the total protein content in the hog thyroid gland. Actin has been isolated from acetone-dehydrated slices and purified to homogeneity by gel filtration, DEAE-cellulose chromatography and two polymerization-depolymerization cycles. Purified actin (Mr = 42000) contains the beta and gamma species with a 2 to 1 stoichiometry. In the presence of 0.1 M KCl and 2 mM MgCl2 thyroid actin polymerized into 6 nm diameter filaments; under these conditions the critical concentration was 30 micrograms/ml and the intrinsic viscosity 4.7 dl/g.     

Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages.

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Interactions of actin, myosin, and an actin-binding protein of rabbit pulmonary macrophages. III. Effects of cytochalasin B

Low concentrations (greater than or equal to 10(-7) M) of cytochalasin B reversibly inhibit the temperature-dependent gelation of actin by an actin-binding protein. The cytochalasin B concentrations which maximally inhibit actin gel formation are 10-fold lower than the concentrations which maximally impair phagocytosis by intact macrophages. Cytochalasin B also prevents the polymerization of monomeric actin in sucrose extracts of macrophages in the absence but not the presence of 0.1 M CKl. 10(-6) M cytochalasin B dissolves macrophage extract gels and gels comprised of purified actin and actin-binding protein by dissociating actin-binding protein from actin filaments. This concentration of cytochalasin B, however, does not depolymerize the actin filatments.     

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.     

Effect of capping protein on the kinetics of actin polymerization

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.     

Mechanism of Cdc42-induced Actin Polymerization in Neutrophil Extracts

Cdc42, activated with GTPγS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool.Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 μm in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 μm. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.