Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo ¹H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Mechanical Distortion of Single Actin Filaments Induced by External Force: Detection by Fluorescence Imaging

Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a “bio-nano strain gauge” that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys³⁷⁴ and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by ∼6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys³⁷⁴ or rhodamine-phalloidin showed only an ∼2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys³⁷⁴ approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.

The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.     

Effect of polymerization on the subdomain 3/4 loop of yeast actin

The Holmes F-actin model predicts a polymerization-dependent conformation change of a subdomain 3/4 loop with a hydrophobic tip (residues 266-269), allowing interaction with a hydrophobic surface on the opposing strand of the filament producing filament stabilization. We introduced cysteines in place of Val(266), Leu(267), and Leu(269) in yeast actin to allow attachment of pyrene maleimide. Pyrene at each of these positions produced differing fluorescence spectra in G-actin. Polymerization decreased the fluorescence for the 266 and 267 probes and increased that for the 269 probe. The direction of the fluorescence change was mirrored with a smaller and less hydrophobic probe, acrylodan, when attached to 266 or 269. Following polymerization, increased acrylamide quenching was observed for pyrene at 266 or 267 but not 269. The 267 probe was the least accessible of the three in G- and F-actin. F-actin quenching was biphasic for the 265, 266, and 269 but not 267 probes, suggesting that in F-actin, the pyrene samples multiple environments. Finally, in F-actin the probe at 266 interacts with one at Cys(374) on a monomer in the opposing strand, producing a pyrene excimer band. These results indicate a polymerization-dependent movement of the subdomain 3/4 loop partially consistent with Holmes' model.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ～5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca²⁺-dependent, but not completely reversible, as on removal of Ca²⁺ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca²⁺ in a micromolar range, with half maximum activation at about 3×10⁻⁶ M. The protein becomes inactivated when Ca²⁺ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.     

19F nuclear magnetic resonance studies of selectively fluorinated derivatives of G- and F-actin

G-Actin is a globular protein (Mr 42 300) known to have three cysteine residues that are at least partially exposed and chemically reactive (Cys-10, -284, and -374). When G-actin was reacted with 3-bromo-1,1,1-trifluoropropanone, three resolvable 19F resonances were observed in the 19F NMR spectrum. This fluorinated G-actin derivative remained fully polymerizable, and its 31P NMR spectrum was not significantly different from that of unmodified G-actin, indicating that the chemical modification did not denature the actin and the modified residues do not interfere with the extent of polymerization or the binding of adenosine 5'-triphosphate. One of the three 19F resonances was assigned to fluorinated Cys-374 on the basis of its selective reaction with N-ethylmaleimide. This resonance was dramatically broadened after polymerization of fluorinated G-actin, while the other two resonances were not markedly broadened or shifted. Thus, Cys-10 and -284 are not involved in or appreciably affected by the polymerization of G-actin, while the mobility of the 19F label at Cys-374 is markedly reduced.     

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Intermolecular dynamics and function in actin filaments

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.     

Bacterial lipopolysaccharide induces actin reorganization,intercellular gap formation,and endothelial barrier dysfunction in pulmonary vascular endothelial cells: Concurrent F-actin depolymerization and new actin synthesis

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: (1) transendothelial ¹⁴C-albumin flux, (2) F-actin organization with fluorescence microscopy, (3) F-actin quantitation by spectrofluorometry, and (4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in ¹⁴C-albumin flux (P < 0.001) and intercellular gap formation at ≥ 2–6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (±SE) G-actin (μg/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization. © 1993 Wiley-Liss, Inc.     

维生素E和伊那普利对高糖改变肾小球系膜细胞肌动蛋白组装的影响

研究了维生素E(VE)和伊那普利(EN)对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白组装的影响。结果证明,MC在HG培养时,F-actin失去粗大束状外观呈不规则网状,显示F-actin部分去组装。与正常浓度葡萄糖(NG)培养的MC相比,HG引起F-actin荧光强度降低,G-actin荧光强度升高和F/G-actin荧光强度比值下降。VE和EN加入培养后,HG引起的F-actin部分去组装及F-和G-actin荧光强度的变化均恢复正常,提示,VE和EN可防止HG引起的MC actin去组装。     

The molecular dynamics of actin measured by a spin probe attached to lysine

Rabbit skeletal muscle G-actin was labeled with a spin probe, 3-(5-fluoro-2,4-dinitroanilino)proxyl. Tryptic digestion of the labeled actin followed by ultrafiltration and ion-exchange column chromatography indicated that the label was attached to residue Lys-61. This residue is found within a 9-kDa N-terminal segment that is easily degraded by proteolytic enzymes. The rate of reduction of the nitroxide bond by ascorbate was measured to determine the accessibility of the probe to small molecules in the solvent. These experiments showed that label bound to G-actin was relatively inaccessible to ascorbate, suggesting that it is buried within the protein structure. Polymerization further decreased the accessibility of the probe. Replacing bound Ca2+ with Mn2+ decreased the observed intensity of the electron paramagnetic resonance signal, indicating the spin label is about 2 nm distant from the metal binding site on the actin molecule. Labels attached to G-actin displayed an absorption spectrum characteristic of rotational motion with a correlation time (tau c) of 7 X 10(-9) s, which is faster than that for the whole molecule. Labels attached to F-actin had a value of tau c, measured using saturation transfer electron paramagnetic resonance, of 2 X 10(-5) s, which shows that the probe has a greater degree of mobility than the filament. The binding of heavy meromyosin or troponin-tropomyosin to labeled actin resulted in a further increase in the rotational correlation times, with the greatest decrease in mobility (tau c = 1 X 10(-4) s) observed when both were bound. Together the above results suggest that the 9-kDa segment of actin is mobile relative to the rest of the molecule and that this mobility can be influenced by the binding of heavymeromyosin or troponin-tropomyosin.     

Implications of oxidovanadium(IV) binding to actin

Oxidovanadium(IV), a cationic species (VO²⁺) of vanadium(IV), binds to several proteins, including actin. Upon titration with oxidovanadium(IV), approximately 100% quenching of the intrinsic fluorescence of monomeric actin purified from rabbit skeletal muscle (G-actin) was observed, with a V₅₀ of 131 μM, whereas for the polymerized form of actin (F-actin) 75% of quenching was obtained and a V₅₀ value of 320 μM. Stern-Volmer plots were used to estimate an oxidovanadium(IV)-actin dissociation constant, with K_d of 8.2 μM and 64.1 μM VOSO₄, for G-actin and F-actin, respectively. These studies reveal the presence of a high affinity binding site for oxidovanadium(IV) in actin, producing local conformational changes near the tryptophans most accessible to water in the three-dimensional structure of actin. The actin conformational changes, also confirmed by ¹H NMR, are accompanied by changes in G-actin hydrophobic surface, but not in F-actin. The ¹H NMR spectra of G-actin treated with oxidovanadium(IV) clearly indicates changes in the resonances ascribed to methyl group and aliphatic regions as well as to aromatics and peptide-bond amide region. In parallel, it was verified that oxidovanadium(IV) prevents the G-actin polymerization into F-actin. In the 0-200 μM range, VOSO₄ inhibits 40% of the extent of polymerization with an IC₅₀ of 15.1 μM, whereas 500 μM VOSO₄ totally suppresses actin polymerization. The data strongly suggest that oxidovanadium(IV) binds to actin at specific binding sites preventing actin polymerization. By affecting actin structure and function, oxidovanadium(IV) might be responsible for many cellular effects described for vanadium.     

Kinetic studies of a hepatoma alkaline phosphatase

To help interpret the electron spin resonance (esr) spectra of spin-labeled actin, the positions of attachment of the spin labels, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide to rabbit skeletal muscle actin have been determined. For this purpose spin-labeled peptides released by tryptic digestion of the spin-labeled actin were isolated by chromatography and identified from their positions of elution and amino acid composition.With purified F-actin that had not undergone structural changes both labels reacted exclusively with the sulfhydryl group of the C-terminal sequence. But if the actin was stored in the F-form in the absence of ATP it evidently underwent a structural alteration because reaction was then at another sulfhydryl group, in the N-terminal sequence, and the actin had an irregular appearance in the electron microscope. ADP and tripolyphosphate were as effective as ATP in preventing this alteration. A maximum of 1 equiv of spin label was bound, irrespective of the site of labeling, and the two sites appeared to be mutually exclusive, possibly because they are adjacent. With G-actin, and with actin denatured by guanidine hydrochloride, there was also reaction at other sites. The shapes of the esr spectra of F-actin that contained Mg²⁺, Ca²⁺, or Mn²⁺ did not depend on whether labeling was at the C- or N-terminal positions, although F-actin labeled in the latter position contained a small proportion of highly mobile label, possibly a result of denaturation. The reduction in the size of the esr signal of labeled G-actin on replacing Mg²⁺ with Mn²⁺ did not appear to be dependent on the position of labeling.     

A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca²⁺. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca²⁺ than with it. Ca²⁺-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Binding of 1,N6-ethanoadenosine triphosphate to actin

G-actin is known to bind one molecule of ATP. Its polymerization to F-actin is accompanied by the splitting off of the terminal phosphate of the bound nucleotide. We have found that the fluorescent 1,N⁶-ethanoadenosine triphosphate (?ATP) can substitute for ATP in G-actin and that G-actin containing bound ?ATP possesses essentially full polymerizability. The binding of this ATP analog has been studied by following the inactivation of the ?ATP·G-actin complex. The binding constant (4?5.7 × 10⁶ M^?1) obtained in the absence of EDTA is about 50% of that for ATP, while the binding constant obtained in the presence of EDTA (0.9?3.0 × 10⁵ M^?1) is comparable to those for ATP and ADP. These findings suggest that ?ATP can be used as a structural probe for actin. The fluorescence lifetime of ?ATP bound to G·actin is 36 nsec. The rotational relaxation time of ?ATP·G-actin is near 60 nsec. at 20°C.     

Cofilin (ADF) affects lateral contacts in F-actin

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.