Structure and function of the Arp2/3 complex

The Arp2/3 complex, a 220 kDa macromolecular assembly comprising two actin-related proteins and five other subunits, plays a key role in cellular motility by initiating the polymerization of new actin filaments. Crystal and cryo-electron microscopic structures of the Arp2/3 complex and branch junctions have clarified extensive biochemical data on the mechanism of this process.     

Structure and function of the Arp2/3 complex.

The Arp2/3 complex is a ubiquitous and essential component of the actin cytoskeleton in eukaryotic cells. It nucleates actin filaments, caps their pointed ends and cross-links them into orthogonal networks. In amoeba, vertebrates and fungi, the complex consists of actin-related proteins Arp2 and Arp3 and individual copies of five novel polypeptides. The Arps are thought to mediate pointed-end capping and nucleation. Chemical cross-linking implicates three subunits in binding the complex to the side of another actin filament.     

Interaction of cortactin and N-WASp with Arp2/3 complex

BACKGROUND: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. RESULTS: We investigated how cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of cortactin to Arp2/3 complex. CONCLUSIONS: Under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.     

Molecular dynamics simulations of Arp2/3 complex activation

Actin-related protein 2 and 3 (Arp2/3) complex forms a dendritic network of actin filaments during endocytosis and cellular locomotion by nucleating branches on the sides of preexisting actin filaments. Reconstructions of electron tomograms of branch junctions show how Arp2/3 complex anchors the branch, with Arp2 and Arp3 serving as the first two subunits of the branch. Our aim was to characterize the massive conformational change that moves Arp2 ∼30 Å from its position in crystal structures of inactive Arp2/3 complex to its position in branch junctions. Starting with the inactive crystal structure, we used atomistic-scale molecular dynamics simulations to drive Arp2 toward the position observed in branch junctions. When we applied forces to Arp2 while restraining Arp3, one block of structure (Arp2, subunit ARPC1, the globular domain of ARPC4 and ARPC5) rotated counterclockwise by 30° around a pivot point in an α-helix of ARPC4 (Glu⁸¹-Asn¹⁰⁰) to align Arp2 next to Arp3 in a second block of structure including ARPC3 and the globular domains of ARPC2. This active structure buried more surface area than the inactive conformation. The complex was stable in all simulations. In most simulations, collisions of subdomain 2 of Arp2 with Arp3 impeded the movement of Arp2.     

Turning on the Arp2/3 complex at atomic resolution

The recently published 2 A X-ray crystal structure of bovine Arp2/3 complex gives us atomic scale insight into Arp2/3-mediated actin nucleation, while cryo-EM work and functional studies begin to fill in exciting mechanistic details.     

Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit

Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2Delta Arp2/3 complex) from fission yeast. An x-ray crystal structure of the DeltaArp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2Delta Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2Delta Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.     

Characterization of Arp2/3 complex in chicken tissues

Arp2/3 protein complex consists of seven subunits (Arp2, Arp3, p41-Arc, p34-Arc, p21-Arc, p20-Arc and p16-Arc) in apparent 1:1 stoichiometry. This complex has been shown to promote the formation of Y-branch structures of F-actin in cultured cells. We generated specific antibodies against chicken Arp2, Arp3, and p34-Arc to analyze the distribution of these subunits in chicken tissues.In whole samples of brain and gizzard, antibodies against each recombinant protein reacted with single bands of predicted molecular mass based on their cDNA sequences of the antigens. Anti-p34-Arc antibody detected at least two neighboring spots in 2D-PAGE, which might suggest the existence of isoforms or modified forms. Arp2/3 complex bound to an F-actin affinity column from gizzard extract. However, Arp2/3 complex did not tightly bind major actin cytoskeleton because the complex was extracted easily when gizzard smooth muscle was homogenized in PBS. Immunoblot analysis of various tissues revealed that the amounts of Arp2/3 subunits were lower in striated muscle than in non-muscle and smooth muscle tissues. Amounts and ratio of the three subunits varied in tissues, as estimated by quantitative immunoblotting. With immunofluorescence microscopy, we also observed localization of Arp3 and p34-Arc in frozen sections of gizzard with different staining patterns around blood vessels. These results suggest that the Arp2/3 complex exists also in places where rapid actin polymerization does not occur, and that a part of the subunits may exist in different forms from the complex containing the seven subunits in some tissues.     

Integration of signals to the Arp2/3 complex

The Arp2/3 complex is necessary for nucleating the formation of branched networks of actin filaments at the cell cortex, and an increasing number of proteins able to activate the Arp2/3 complex have been described. The Wiskott-Aldrich syndrome protein (WASP) family and cortactin comprise the large majority of the known activators. WASPs bind to Arp2/3 via an acidic (A) domain, and a WH2 domain appears to bring an actin monomer to Arp2/3, promoting the nucleation of the new filament. Cortactin also binds the Arp2/3 complex via an A domain; however, it also binds to actin filaments, which helps activate the Arp2/3 complex and stabilise the newly created branches between the filaments.     

Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state.     

Arp2/3 complex-independent actin regulatory function of WAVE

We report that WAVE1/Scar1, a WASP-family protein that functions downstream of Rac in membrane ruffling, can induce part of the reorganization of the actin cytoskeleton without Arp2/3 complex. WAVE1 has been reported to associate and activate Arp2/3 complex at its C-terminal region that is rich in acidic residues. The deletion of the acidic residues abolished the interaction with and the activation ability of Arp2/3 complex. The expression of the mutant WAVE1 lacking the acidic residues (DeltaA), however, induced actin-clustering in cells as the wild-type WAVE1 did. In addition, this actin-clustering could not be suppressed by the coexpression of the Arp2/3 complex-sequestering fragment (CA-region) derived from N-WASP, which clearly inhibits Rac-induced membrane ruffling. This study therefore demonstrates that WAVE1 reorganizes the actin cytoskeleton not only through Arp2/3 complex but also through another unidentified mechanism that may be important but has been neglected thus far.     

End versus side branching by Arp2/3 complex

We investigate the issue of end versus side branching of actin filaments by Arp2/3 complex, using a combination of analytic theory, polymerization assays, and quantitative modeling. The analytic theory shows that the effect of capping protein on the initial stages of actin polymerization in the presence of Arp2/3 complex depends strongly on whether new Arp2/3 complex-induced branches grow from the sides or ends of existing filaments. Motivated by these results, we measure and quantitatively model the kinetics of actin polymerization in the presence of activated Arp2/3 complex, for a range of concentrations of capping protein. Our model includes the most important types of events involving actin and actin-binding proteins, and can be adjusted to include end branching, side branching, or both. The side-branching model gives a better fit to the experimental data than the end-branching model. An end-plus-side model including both types of branching gives a moderate improvement in the quality of the fit. Another side-branching model, based on aging of subunits' capacity for branch formation, gives a significantly better fit than the end-plus-side model. We discuss implications for actin polymerization in cells.     

Acceleration of yeast actin polymerization by yeast Arp2/3 complex does not require an Arp2/3-activating protein

The Arp2/3 complex creates filament branches leading to an enhancement in the rate of actin polymerization. Work with Arp complexes from different sources indicated that it was inactive by itself, required an activating factor such as the Wiskott-Aldrich syndrome protein (WASP), and might exhibit a preference for ATP or ADP-P(i) actin. However, with yeast actin, P(i) release is almost concurrent with polymerization, eliminating the presence of an ADP-P(i) cap. We thus investigated the ability of the yeast Arp2/3 complex (yArp2/3) to facilitate yeast actin polymerization in the presence and absence of the Arp2/3-activating factor Las17p WA. yArp2/3 significantly accelerates yeast actin but not muscle actin polymerization in the absence of Las17p WA. The addition of Las17p WA further enhances yeast actin polymerization by yArp2/3 and allows the complex to now assist muscle actin polymerization. This actin isoform difference is not observed with bovine Arp2/3 complex, because the neural WASP VCA fragment is required for polymerization of both actins. Observation of individual branching filaments showed that Las17p WA increased the persistence of filament branches. Compared with wild type actin, the V159N mutant actin, proposed to be more ATP-like in behavior, exhibited an enhanced rate of polymerization in the presence of the yArp2/3 complex. yArp2/3 caused a significant rate of P(i) release prior to observation of an increase in filament mass but while branched structures were present. Thus, yeast F-actin can serve as a primary yArp2/3-activating factor, indicating that a newly formed yeast actin filament has a topology, unlike that of muscle actin, that is recognized specifically by yArp2/3.     

Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex

Members of the family of WASP-family Verprolin homologous proteins (WAVEs) activate the Arp2/3 complex to induce actin polymerization. The WAVE family comprises three proteins, namely, WAVE1, WAVE2 and WAVE3. Among them, WAVE2 is crucial for activation of the Arp2/3 complex for the formation of branched actin filaments in lamellipodia. Activation of mitogen-activated protein (MAP) kinase signalling results in the phosphorylation of the WAVE family proteins; however, which of the three WAVE proteins is phosphorylated is unclear. We found that in vitro WAVE2 is directly phosphorylated by a MAP kinase, i.e. extracellular signal-regulated kinase (ERK) 2. The proline-rich region and the verprolin, cofilin and acidic (VCA) region of WAVE2 were phosphorylated. Interestingly, the phosphorylated VCA region had a higher affinity for the Arp2/3 complex. However, the phosphorylation of the VCA region resulted in reduced induction of Arp2/3-mediated actin polymerization in vitro. The role of the phosphorylation of the proline-rich region was not determined.     

The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics.     

Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.     

A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.     

Arp2/3 complex activity in filopodia of spreading cells

Background  

Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices.     

The Arp2/3 complex: a multifunctional actin organizer

The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes. The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement. Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization. First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes. Second, it can nucleate and cross-link actin filaments in vitro. Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function. Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years.     

Molecular dynamics simulation and coarse-grained analysis of the Arp2/3 complex

A molecular dynamics investigation and coarse-grained analysis of inactivated actin-related protein (Arp) 2/3 complex is presented. It was found that the nucleotide binding site within Arp3 remained in a closed position with bound ATP or ADP, but opened when simulation with no nucleotide was performed. In contrast, simulation of the isolated Arp3 subunit with bound ATP, showed a fast opening of the nucleotide binding cleft. A homology model for the missing subdomains 1 and 2 of Arp2 was constructed, and it was also found that the Arp2 binding cleft remained closed with bound nucleotide. Within the nucleotide binding cleft a distinct opening and closing period of 10 ns was observed in many of the simulations of Arp2/3 as well as isolated Arp3. Substitution studies were employed, and several alanine substitutions were found to induce a partial opening of the ATP binding cleft in Arp3 and Arp2, whereas only a single substitution was found to induce opening of the ADP binding cleft. It was also found that the nucleotide type did not cause a substantial change on interfacial contacts between Arp3 and the ArpC2, ArpC3 and ArpC4 subunits. Nucleotide-free Arp3 had generally less stable contacts, but the overall contact architecture was constant. Finally, nucleotide-dependent coarse-grained models for Arp3 are developed that serve to further highlight the structural differences induced in Arp3 by nucleotide hydrolysis.     

Involvement of Arp2/3 complex in MCP-1-induced chemotaxis

The migrating monocyte shows dynamic actin polymerization in response to MCP-1. We investigated the involvement of the actin-related protein 2 and 3 complex (Arp2/3 complex) during chemotaxis of a human monocyte cell line (THP-1). To clarify whether the Arp2/3 complex directly polymerizes actin in response to MCP-1 stimulation, THP-1 cells were transfected with complementary DNA constructs encoding ScarWA. In ScarWA-transfected cells, neither recruitment of Arp2/3 complex at the leading edge nor actin polymerization was detected. Indeed, migration induced by MCP-1 was almost completely blocked. At the same time, transfection also interfered with the recruitment of integrin beta-1 at the leading edge and reduced affinity binding to fibronectin. Immunoprecipitation with an anti-Arp2 antibody showed that integrin beta-1 and WASP were co-precipitated under the condition of MCP-1 stimulation. These results indicate that interaction between the Arp2/3 complex and WASP stimulates actin polymerization and integrin beta-1-mediated adhesion during MCP-1-induced chemotaxis of THP-1 cells.