Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.     

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (~75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.     

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.     

Cofilin increases the torsional flexibility and dynamics of actin filaments

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.     

Actin filament severing by cofilin

Cofilin is essential for cell viability and for actin-based motility. Cofilin severs actin filaments, which enhances the dynamics of filament assembly. We investigated the mechanism of filament severing by cofilin with direct fluorescence microscopy observation of single actin filaments in real time. In cells, actin filaments are likely to be attached at multiple points along their length, and we found that attaching filaments in such a manner greatly increased the efficiency of filament severing by cofilin. Cofilin severing increased and then decreased with increasing concentration of cofilin. Together, these results indicate that cofilin severs the actin filament by a mechanism of allosteric and cooperative destabilization. Severing is more efficient when relaxation of this cofilin-induced instability of the actin filament is inhibited by restricting the flexibility of the filament. These conclusions have particular relevance to cofilin function during actin-based motility in cells and in synthetic systems.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis.

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.     

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.     

Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d- cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co- localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Mapping the interaction of cofilin with subdomain 2 on actin

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.     

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.     

Clusters of a Few Bound Cofilins Sever Actin Filaments

Cofilin is an essential actin filament severing protein that accelerates the assembly dynamics and turnover of actin networks by increasing the number of filament ends where subunits add and dissociate. It binds filament subunits stoichiometrically and cooperatively, forming clusters of contiguously-bound cofilin at sub-saturating occupancies. Filaments partially occupied with cofilin sever at boundaries between bare and cofilin-decorated segments. Imaging studies concluded that bound clusters must reach a critical size (C_c) of 13–100 cofilins to sever filaments. In contrast, structural and modeling studies suggest that a few or even a single cofilin can sever filaments, possibly with different severing rate constants. How clusters grow through the cooperative incorporation of additional cofilin molecules, specifically if they elongate asymmetrically or uniformly from both ends and if they are modulated by filament shape and external force, also lacks consensus. Here, using hydrodynamic flow to visualize individual actin filaments with TIRF microscopy, we found that neither flow-induced filament bending, tension, nor surface attachment conditions substantially affected the kinetics of cofilin binding to actin filaments. Clusters of bound cofilin preferentially extended toward filament pointed ends and displayed severing competency at small sizes (C_c < 3), with no detectable severing dependence on cluster size. These data support models in which small clusters of cofilin introduce local, but asymmetric, structural changes in actin filaments that promote filament severing with a rate constant that depends weakly on the size of the cluster.     

Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 μm, corresponding to a flexural rigidity of 0.040 pN μm². Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 μm and the filament flexural rigidity to 0.0091 pN μm². That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.     

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.     

A Highlights from MBoC Selection: Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.     

Cofilin is an essential component of the yeast cortical cytoskeleton

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin- like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

Aip1 and cofilin promote rapid turnover of yeast actin patches and cables: a coordinated mechanism for severing and capping filaments

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin-binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to "prune" tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Delta mutants.