Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.     

Arp2/3 phosphorylation kickstarts cells

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.     

MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.     

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.     

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.     

MICAL2 fine-tunes Arp2/3 for actin branching

The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Remodeling of branched actin networks enables cell protrusion and sensing of the environment and is essential for cell motility. Migrating cells such as fibroblasts, immune cells, and metastatic cancer cells rely on actin dynamics to generate pushing, pulling, and squeezing forces to propel themselves. Therefore, studying the processes regulating assembly and disassembly of actin filaments is key to understanding cell locomotion in health and disease. One of the most important catalyzers of actin assembly is the Arp2/3 complex, which drives lamellipodia formation and cell protrusion. Arp2/3-generated actin networks are also important for endocytic trafficking, membrane remodeling during vesicle internalization, cargo sorting, and membrane excision (1). The seven-protein ARP2/3 complex contains two unconventional actin-related proteins (ARP2 and ARP3) and five additional subunits (ARPC1–5). Mammals express two isoforms of three of the subunits (ARP3/ARP3B, ARPC1A/ARPC1B, and ARPC5/ARPC5L), resulting in functional diversity depending on the specific isoforms incorporated into the ARP2/3 complex; however, despite some intriguing roles described in muscle development (2) and platelet function (3), little is known about the biological significance of these isoforms.The nucleation activity of ARP2/3 complex is regulated at multiple levels to ensure that new actin generation is spatially and temporally controlled. Activation is controlled by Wiskott Aldrich Syndrome Protein (WASP)–family proteins, which are themselves part of multi-protein complex machines (4). WASP-family protein complexes detect multiple inputs such as membrane phospholipids, protein–protein interactions, or post-translational modifications, and act as signaling hubs to regulate branched actin nucleation. Other proteins, such as cortactin or coronin, also modulate branch stability in an antagonistic manner (5). ARP2/3 can be post-translationally modified by phosphorylation and interaction with negative regulators, whereas actin itself is regulated by targeted oxidation of methionine residues (6). How these feedback loops that control ARP2/3 activity are coordinated with cell function is an intense area of research.Molecule interacting with CasL (MICAL) proteins have emerged as important mediators of targeted protein oxidation (6). MICAL proteins (MICAL1–3) are flavin adenine dinucleotide–binding monooxygenases capable of oxidizing target proteins (including actin), either directly or through generation of diffusible H₂O₂, which in turn oxidizes proteins in close proximity. Actin oxidation occurs on two methionine residues (Met44 and Met47), resulting in F-actin disassembly and increased cofilin-mediated F-actin severing. Although actin is the best characterized MICAL substrate, there remains the intriguing possibility of the existence of additional targets that regulate cytoskeleton dynamics.In this issue, Galloni, Carra, et al. evaluated the ability of ARP2/3 complexes, containing either ARP3 or the ARP3B isoform (i.e., isocomplexes), to promote actin assembly, and determined isoform-specific differences in their activity and molecular regulation (7). As a model system, the authors used HeLa cells infected with vaccinia virus to study actin branching, given that this virus induces actin tail nucleation in the host cells. They noticed that in cells lacking ARP3, the localization of GFP-ARP3 or GFP-ARP3B to actin tails was comparable, and both isoforms were similarly incorporated into ARP2/3 complexes (Fig. 1). However, the length of the actin tails in ARP3B-expressing cells was shorter than in ARP3-expressing counterparts. Given that ARP3 and ARP3B isocomplexes were equivalent in their ability to induce actin polymerization in vitro, these data pointed to a faster disassembly rate as the potential cause underlying shorter actin tails in ARP3B-expressing cells. Indeed, by tracking photoactivatable actin to study its dynamics, the researchers confirmed that the rate of filament disassembly was faster in ARP3B-expressing cells.Open in a separate windowFigure 1.Vaccinia virus surfs on the outside of the cell, forming an actin tail in the cytoplasm that aids its propulsion. Arp2/3 complex is involved in initiating the branched actin structures and shows slow dissociation from the branches when it is stabilized by the linker protein cortactin. When an Arp2/3 complex containing the ARP3B isoform of ARP3 forms, the dissociation is enhanced, as ARP3B is subject to oxidation by MICAL2, which is recruited to branches by coronin, causing cortactin displacement and rapid branch dissociating leading to shorter actin tails.To identify the molecular basis for the differences between ARP3 and ARP3B, the authors tested a series of ARP3 and ARP3B chimeric proteins, which revealed the importance of ARP3B amino acids 281–418 in mediating the functional differences with ARP3. In particular, Met293 was essential for ARP3B to generate short actin tails. Given that MICAL enzymes promote actin filament disassembly through oxidation of actin Met44 and Met47, Galloni, Carra, et al. decided to investigate the possibility that MICAL-induced oxidation of Met293 in ARP3B inhibits ARP3B activity. Fluorescently tagged MICAL2, but not MICAL1, was recruited to vaccinia-induced actin tails at a position relatively distant from the virus itself, similar to the actin-binding protein coronin (8). Down-regulation of MICAL2, but not MICAL1, increased actin tail stability and suppressed the short actin tail phenotype induced by ARP3B overexpression. Using an antibody raised against oxidized Met293, the researchers confirmed that ARP3B oxidation was reduced following MICAL2 knockdown. Recruitment of MICAL2 to actin tails was dependent on coronin 1C expression, and silencing of coronin 1C resulted in actin filament stabilization and reversal of ARP3B-induced actin tail shortening comparable to MICAL2 knockdown. Thus, coronin 1C recruitment of MICAL2 results in ARP3B oxidation on Met293, leading to dissociation of ARP2/3B isocomplexes and consequent actin networks destabilization.Interestingly, the authors noted that the actin nucleation promoting factor cortactin, which stabilizes ARP2/3-mediated branch points along actin filaments, was required for actin tail destabilization in ARP3B overexpressing cells but was not necessary for localization of coronin 1C or MICAL2 to actin tails. One possibility is that cortactin supports local MICAL2-mediated oxidation of ARP3B at branch points to induce filament de-branching, rather than bulk actin filament depolymerization that would result from direct actin oxidation. Since MICAL proteins are directed to specific cytoskeleton locations by interacting with Myosin 5A (9) and Myosin 15 (10), the consequences of MICAL activity on actin cytoskeleton organization and function may be fine-tuned by specific MICAL subcellular localization and interacting partners.Given that actin binds directly to the catalytic monooxygenase and calponin homology domains of MICAL proteins to increase enzyme activity and promote methionine oxidation, it is not entirely surprising that the actin-related ARP3B protein can be oxidized by MICAL2. However, the location of Met293 in ARP3B is not analogous to the Met44 or Met47 residues of actin, which raises questions regarding the mechanism of ARP3B oxidation by MICAL2. Structural modeling of the MICAL3–actin complex positions the actin loop containing Met44 and Met47 near the enzyme active site (11). ARP3B may interact with MICAL2 differently to bring Met293 close to the active site for direct oxidation, or H₂O₂ produced by MICAL2 might diffuse and oxidize highly concentrated nearby proteins. If this second possibility were true, then it is also possible that additional protein targets (e.g., coronin 1C, cortactin, additional ARP2/3 subunits) might also be oxidized on Met or Cys residues. Since the effects of MICAL1 on actin are counteracted via reduction of the oxidized Met residues by the sulfoxide reductase enzyme SelR (12), it remains to be determined if ARP3B can be similarly reactivated.     

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.     

Caldesmon inhibits Arp2/3-mediated actin nucleation

The Arp2/3 complex greatly accelerates actin polymerization, which is thought to play a major role in cell motility by inducing membrane protrusions including ruffling movements. Membrane ruffles contain a variety of actin-binding proteins, which would modulate Arp2/3-dependent actin polymerization. However, their exact roles in actin polymerization remain to be established. Because caldesmon is present in membrane ruffles, as well as in stress fibers, it may alter Arp2/3-mediated actin polymerization. We have found that caldesmon greatly retards Arp2/3-induced actin polymerization. Kinetic analyses have revealed that caldesmon inhibits the nucleation process, whereas it does not largely reduce elongation. Caldesmon is found to inhibit binding of Arp2/3 to F-actin, which apparently reduces the ability of F-actin as a secondary activator of Arp2/3-mediated nucleation. We also have found that the inhibition of the binding between actin and caldesmon either by Ca(2+)/calmodulin or by phosphorylation with cdc2 kinase reverses the inhibitory effect of caldesmon on Arp2/3-induced actin polymerization. Our results suggest that caldesmon may be a key protein that modulates membrane ruffling and that this may involve changes in caldesmon phosphorylation and/or intracellular calcium concentrations during signal transduction.     

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.     

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.     

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.     

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.     

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.     

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.