Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments

Actin depolymerizing factor (ADF)/cofilin enhances turnover of actin filaments by severing and depolymerizing filaments. A number of proteins functionally interact with ADF/cofilin to modulate the dynamics of actin filaments. Actin-interacting protein 1 (AIP1) has emerged as a conserved WD-repeat protein that specifically enhances ADF/cofilin-induced actin dynamics. Interaction of AIP1 with actin was originally characterized by a yeast two-hybrid system. However, biochemical studies revealed its unique activity on ADF/cofilin-bound actin filaments. AIP1 alone has negligible effects on actin filament dynamics, whereas in the presence of ADF/cofilin, AIP1 enhances filament fragmentation by capping ends of severed filaments. Studies in model organisms demonstrated that AIP1 genetically interacts with ADF/cofilin and participates in several actin-dependent cellular events. The crystal structure of AIP1 revealed its unique structure with two seven-bladed beta-propeller domains. Thus, AIP1 is a new class of actin regulatory proteins that selectively enhances ADF/cofilin-dependent actin filament dynamics.     

Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.     

Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks

BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.     

Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two actin-binding sites on human cofilin. One target site was chosen because we previously showed that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments.     

Enhancement of actin-depolymerizing factor/cofilin-dependent actin disassembly by actin-interacting protein 1 is required for organized actin filament assembly in the Caenorhabditis elegans body wall muscle

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal beta-propeller, whereas one residue is located in the C-terminal beta-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.     

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.     

Interaction of maize actin-depolymerising factor with actin and phosphoinositides and its inhibition of plant phospholipase C.

We have previously reported the isolation of three Zea mays genes that encode actin-depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of Acanthamoeba actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP₂), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP₂ is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

Actin depolymerization factor/cofilin activation regulates actin polymerization and tension development in canine tracheal smooth muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.     

AIP1/WDR1 supports mitotic cell rounding

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin

The ADF (actin-depolymerizing factor)/cofilin family is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1), much less is known about its reactivation through dephosphorylation. Here we report Slingshot (SSH), a family of phosphatases that have the property of F actin binding. In Drosophila, loss of ssh function dramatically increased levels of both F actin and phospho-cofilin (P cofilin) and disorganized epidermal cell morphogenesis. In mammalian cells, human SSH homologs (hSSHs) suppressed LIMK1-induced actin reorganization. Furthermore, SSH and the hSSHs dephosphorylated P cofilin in cultured cells and in cell-free assays. Our results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.     

Ser6 in the maize actin‐depolymerizing factor, ZmADF3, is phosphorylated by a calcium‐stimulated protein kinase and is essential for the control of functional activity

Maize actin-depolymerizing factor, ZmADF, binds both G- and F-actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal-responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium-stimulated protein kinase present in a 40–70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3–4) does not bind G- or F-actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.     

Putting a new twist on actin: ADF/cofilins modulate actin dynamics.

The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.     

Determining the differences in actin binding by human ADF and cofilin.

The actin-depolymerizing factor (ADF)/cofilin family of proteins play an essential role in actin dynamics and cytoskeletal re-organization. Human tissues express two isoforms in the same cells, ADF and cofilin, and these two proteins are more than 70% identical in amino acid sequence. We show that ADF is a much more potent actin-depolymerizing agent than cofilin: the maximum level of depolymerization at pH 8 by ADF is about 20 microM compared to 5 microM for cofilin, but little depolymerization occurs at pH 6.5 with either protein. However, we find little difference between the two proteins in their binding to filaments, their severing activities or their activation of subunit release from the pointed ends of filaments. Likewise, they show no significant differences in their affinities for monomeric actin: both bind 15-fold more tightly to actin.ADP than to actin.ATP. Complexes between actin.ADP and ADF or cofilin associate with both barbed and pointed ends of filaments at similar rates (close to those of actin.ATP and much higher than those of actin.ADP). This explains why high concentrations of both proteins reverse the activation of subunit release at pointed ends. The major difference between the two proteins is that the nucleating activity of cofilin-actin.ADP complexes is twice that of ADF-actin.ADP complexes and this, in turn, is twice that of actin.ATP alone. It is this weaker nucleating potential of ADF-actin.ADP that accounts for the much higher steady-state depolymerizing activity. The pH-sensitivity is due to the nucleating activity of complexes being greater at pH 6.5 than at pH 8. Sequence analysis of mammalian and avian isoforms shows a consistent pattern of charge differences in regions of the protein associated with F-actin-binding that may account for the differences in activity between ADF and cofilin.     

A protein kinase from neutrophils that specifically recognizes Ser-3 in cofilin

Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (>/=70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin.     

The ADF/cofilin family: actin-remodeling proteins

The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.