Functional surfaces on the p35/ARPC2 subunit of Arp2/3 complex required for cell growth, actin nucleation, and endocytosis

The Arp2/3 complex is comprised of seven evolutionarily conserved subunits and upon activation by WASp or another nucleation promoting factor nucleates the formation of actin filaments. These events are critical for driving a wide range of cellular processes, including motility, endocytosis, and intracellular trafficking. However, an in depth understanding of the Arp2/3 complex activation and nucleation mechanism is still lacking. Here, we used a mutagenesis approach in Saccharomyces cerevisiae to dissect the structural and functional roles of the p35/ARPC2 subunit. Using integrated alleles that target conserved and solvent-exposed residues, we identified surfaces on p35/ARPC2 required for cell growth, actin organization, and endocytosis. In parallel, we purified the mutant Arp2/3 complexes and compared their actin assembly activities both in the presence and in the absence of WASp. The majority of alleles with defects mapped to one face of p35/ARPC2, where there was a close correlation between loss of actin nucleation and endocytosis. A second site required for nucleation and endocytosis was identified near the contact surface between p35/ARPC2 and p19/ARPC4. A third site was identified at a more distal conserved surface, which was critical for endocytosis but not nucleation. These findings pinpoint the key surfaces on p35/ARPC2 required for Arp2/3 complex-mediated actin assembly and cellular function and provide a higher resolution view of Arp2/3 structure and mechanism.     

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.     

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.     

Activation of the Arp2/3 complex by the actin filament binding protein Abp1p

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.     

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.     

The verprolin-like central (vc) region of Wiskott-Aldrich syndrome protein induces Arp2/3 complex-dependent actin nucleation.

Wiskott-Aldrich Syndrome protein (WASp) and related proteins stimulate actin filament nucleation by Arp2/3 complex. The isolated C-terminal VCA domain of WASp (containing Verprolin-like, Central and Acidic regions) is constitutively active but autoinhibited in the full-length protein. This study compared the ability of parts of VCA fused to the C terminus of glutathione S-transferase (GST) to bind actin and Arp2/3 complex in vitro and to activate actin polymerization in vitro and in cells. Fluorescence anisotropy measurements showed that GST-CA and GST-A bound Arp2/3 complex with K(d) values of 0.11 microm and 1.0 microm, respectively, whereas GST-VC displayed almost undetectable binding (K(d) > 1 mm). However, GST-VC activated actin nucleation through Arp2/3 complex in vitro, though requiring 70-fold higher concentration than GST-VCA while neither GST-CA nor GST-A activated Arp2/3 complex in vitro, though both GST-CA and GST-A inhibited Arp2/3 complex activation by WASp VCA. None of these constructs bound WASp from macrophage lysates. Both GST-VC and GST-CA induced actin accumulations when microinjected into primary human macrophages or human endothelial vein cells. However, only microinjection of GST-VC led to a significant increase of cellular polymerized actin. Additionally, endogenous Arp2/3 complex, but not WASp, colocalized with these GST-VC-induced actin accumulations. These data suggest that WASp constructs lacking the A region, previously thought to be indispensable for actin nucleation, are able to bind and activate Arp2/3 complex in vitro and in vivo.     

Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the Arp2/3 complex

The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.     

Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex

We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP(2)) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction.     

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by WASp (Wiskott Aldrich syndrome protein). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of the Arp2/3 complex have yet to be resolved. In particular, some of the individual subunits in the complex have not been assigned clear functional roles, including p40/ARPC1. Here, we dissected the structure and function of Saccharomyces cerevisiae p40/ARPC1, which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on p40/ARPC1 required for function in vivo: one site contacts p19/ARPC4, one contacts p15/ARPC5, and one site resides in an extended structural “arm” of p40/ARPC1. Using a novel strategy, we purified the corresponding lethal mutant Arp2/3 complexes from yeast and compared their actin nucleation activities. Lethal mutations at the contact with p19/ARPC4 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with p15/ARPC5 led to unregulated (“leaky”) nucleation in the absence of WASp. Lethal mutations in the extended arm drastically reduced nucleation, and the same mutations disrupted the ability of the purified p40/ARPC1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/ARPC1 performs at least three distinct, essential functions in regulating Arp2/3 complex-mediated actin assembly: 1) suppression of spontaneous nucleation by the Arp2/3 complex, which requires proper contacts with p15/ARPC5; 2) propagation of WASp activation signals via contacts with p19/ARPC2; and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.     

Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages

The Yersinia outer surface protein invasin binds to β1 integrins on target cells and has been shown to trigger phagocytic uptake by macrophages. Here, we investigated the role of the actin regulator Wiskott–Aldrich syndrome protein (WASp), its effector the Arp2/3 complex and the Rho-GTPases CDC42Hs, Rac and Rho in invasin/β1 integrin-triggered phagocytosis. During uptake of invasin-coated latex beads, the α5β1 integrin, WASp and the Arp2/3 complex were recruited to the developing actin-rich phagocytic cups in primary human macrophages. Blockage of β1 integrins by specific antibodies, inhibition of Arp2/3 function by microinjection of inhibitors or the use of WASp knockout macrophages inhibited phagocytic cup formation and uptake. Furthermore, microinjection of the dominant negative GTPase mutants N17CDC42Hs, N17Rac or the Rho-specific inhibitor C3-transferase into macrophages greatly attenuated invasin-induced formation of cups. These data suggest that during invasin-triggered phagocytosis β1 integrins activate actin polymerization via CDC42Hs, its effector WASp and the Arp2/3 complex. The contribution of Rac and Rho to phagocytic cup formation also suggests a complex interplay between different Rho GTPases during phagocytosis of pathogens.     

Nucleotide- and Activator-Dependent Structural and Dynamic Changes of Arp2/3 Complex Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.     

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.     

Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6-9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.     

Negative regulation of yeast WASp by two SH3 domain-containing proteins

BACKGROUND: WASp family proteins promote actin filament assembly by activating Arp2/3 complex and are regulated spatially and temporally to assemble specialized actin structures used in diverse cellular processes. Some WASp family members are autoinhibited until bound by activating ligands; however, regulation of the budding yeast WASp homolog (Las17/Bee1) has not yet been explored. RESULTS: We isolated full-length Las17 and characterized its biochemical activities on yeast Arp2/3 complex. Purified Las17 was not autoinhibited; in this respect, it is more similar to SCAR/WAVE than to WASp proteins. Las17 was a much stronger activator of Arp2/3 complex than its carboxyl-terminal (WA) fragment. In addition, actin polymerization stimulated by Las17-Arp2/3 was much less sensitive to the inhibitory effects of profilin compared to polymerization stimulated by WA-Arp2/3. Two SH3 domain-containing binding partners of Las17, Sla1 and Bbc1, were purified and were shown to cooperate in inhibiting Las17 activity. The two SLA1 SH3 domains required for this inhibitory activity in vitro were also required in vivo, in combination with BBC1, for cell viability and normal actin organization. CONCLUSIONS: Full-length Las17 is not autoinhibited and activates Arp2/3 complex more strongly than its WA domain alone, revealing an important role for the Las17 amino terminus in Arp2/3 complex activation. Two of the SH3 domain-containing ligands of Las17, Sla1 and Bbc1, cooperate to inhibit Las17 activity in vitro and are required for a shared function in actin organization in vivo. Our results show that, like SCAR/WAVE, WASp proteins can be controlled by negative regulation through the combined actions of multiple ligands.     

Structure of Actin-related protein 8 and its contribution to nucleosome binding

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8–Arp4–actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3–H4 tetramers over H2A–H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3–H4)₂ over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.     

SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.     

A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo

BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family. RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex. CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.     

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.     

Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.     

Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.