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.     

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.     

Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.     

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.     

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.     

Rate of treadmilling of actin filaments in vitro

Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h.     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments

Regulated assembly of actin-filament networks provides the mechanical force that pushes forward the leading edge of motile eukaryotic cells and intracellular pathogenic bacteria and viruses. When activated by binding to actin filaments and to the WA domain of Wiskott-Aldrich-syndrome protein (WASP)/Scar proteins, the Arp2/3 complex nucleates new filaments that grow from their barbed ends. The Arp2/3 complex binds to the sides and pointed ends of actin filaments, localizes to distinctive 70 degrees actin-filament branches present in lamellae, and forms similar branches in vitro. These observations have given rise to the dendritic nucleation model for actin-network assembly, in which the Arp2/3 complex initiates branches on the sides of older filaments. Recently, however, an alternative mechanism for branch formation has been proposed. In the 'barbed-end nucleation' model, the Arp2/3 complex binds to the free barbed end of a filament and two filaments subsequently grow from the branch. Here we report the use of kinetic and microscopic experiments to distinguish between these models. Our results indicate that the activated Arp2/3 complex preferentially nucleates filament branches directly on the sides of pre-existing filaments.     

Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.     

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.     

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.     

Actin-filament stochastic dynamics mediated by ADF/cofilin

BACKGROUND: The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. RESULTS: In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. CONCLUSIONS: Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells.     

Comparison of filamin A-induced cross-linking and Arp2/3 complex-mediated branching on the mechanics of actin filaments.

We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 microm actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.     

ADP-ribosylated actin caps the barbed ends of actin filaments

The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin

The actin filament network immediately under the plasma membrane at the leading edge of rapidly moving cells consists of short, branched filaments, while those deeper in the cortex are much longer and are rarely branched. Nucleation by the Arp2/3 complex activated by membrane-bound factors (Rho-family GTPases and PIP(2)) is postulated to account for the formation of the branched network. Tropomyosin (TM) binds along the sides of filaments and protects them from severing proteins and pointed-end depolymerization in vitro. Here, we show that TM inhibits actin filament branching and nucleation by the Arp2/3 complex activated by WASp-WA. Tropomyosin increases the lag at the outset of polymerization, reduces the concentration of ends by 75%, and reduces the number of branches by approximately 50%. We conclude that TM bound to actin filaments inhibits their ability to act as secondary activators of nucleation by the Arp2/3 complex. This is the first example of inhibition of branching by an actin binding protein. We suggest that TM suppresses the nucleation of actin filament branches from actin filaments in the deep cortex of motile cells. Other abundant actin binding proteins may also locally regulate the branching nucleation by the Arp2/3 complex in cells.     

Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1

BACKGROUND: Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo. RESULTS: Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo. CONCLUSIONS: We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.     

Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex.

One of the earliest events in the process of cell motility is the massive generation of free actin barbed ends, which elongate to form filaments adjacent to the plasma membrane at the tip of the leading edge. Both cofilin and Arp2/3 complex have been proposed to contribute to barbed end formation during cell motility. Attempts to assess the functions of cofilin and Arp 2/3 complex in vivo indicate that both cofilin and Arp2/3 complex contribute to actin polymerization: cofilin by severing and Arp2/3 by nucleating and branching. In order to determine if the activities of cofilin and Arp2/3 complex interact, we employed a light microscope-based assay to visualize actin polymerization directly in the presence of both proteins. The results indicate that cofilin generates barbed ends to increase the mass of freshly polymerized F-actin but does not directly affect the activity of Arp2/3 complex. However, while ADP, ADP-Pi, and newly polymerized ATP-filaments are all capable of supporting Arp2/3-mediated branching, newly polymerized F-actin supports most of the Arp2/3-induced branch formation. The results suggest that, in vivo, cofilin contributes to barbed end formation by inducing the initial increase in the number of barbed ends leading to increased ATP-F-actin, which in turn supports higher levels of dendritic nucleation by active Arp2/3 complex.