Insertions within the Actin Core of Actin-related Protein 3 (Arp3) Modulate Branching Nucleation by Arp2/3 Complex

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a “cryptic dimer” that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The β4/β5 insert mediates interactions of Arp2/3 complex with actin filaments and “dampers” the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/β15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/β15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.     

Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1–Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.     

Critical conformational changes in the Arp2/3 complex are induced by nucleotide and nucleation promoting factor

Actin nucleation and branching by the Arp2/3 complex is tightly regulated by activating factors. However, the mechanism of Arp2/3 complex activation remains unclear. We used fluorescence resonance energy transfer (FRET) to probe the conformational dynamics of the Arp2/3 complex accompanying its activation. We demonstrate that nucleotide binding promotes a substantial conformational change in the complex, with distinct conformations depending on the bound nucleotide. Nucleotide binding to each Arp is critical for activity and is coupled to nucleation promoting factor (NPF) binding. The binding of Wiskott-Aldrich syndrome protein (WASP) family NPFs induces further conformational reorganization of the Arp2/3 complex, and the ability to promote this conformational reorganization correlates with activation efficiency. Using an Arp2/3 complex that is fused to the actin binding domain of WASP, we confirm that the NPF-induced conformational change is critical for activation, and that the actin and Arp2/3 binding activities of WASP are separable, but are independently essential for activity.     

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.     

Molecular Analysis of Arp2/3 Complex Activation in Cells

Many forms of cellular motility are driven by the growth of branched networks of actin filaments, which push against a membrane. In the dendritic nucleation model, Arp2/3 complex is critical, binding to the side of an existing mother filament, nucleating a new daughter filament, and thus creating a branch. Spatial and temporal regulation of Arp2/3 activity is critical for efficient generation of force and movement. A diverse collection of Arp2/3 regulatory proteins has been identified. They bind to and/or activate Arp2/3 complex via an acidic motif with a conserved tryptophan residue. We tested this model for Arp2/3 regulator function in vivo, by examining the roles of multiple Arp2/3 regulators in endocytosis in living yeast cells. We measured the molecular composition of the actin network in cells with mutations that removed the acidic motifs of the four Arp2/3 regulators previously shown to influence the proper function of the actin network. Unexpectedly, we did not find a simple or direct correlation between defects in patch assembly and movement and changes in the composition and dynamics of dendritic nucleation proteins. Taken together our data does not support the simple hypothesis that the primary role for Arp2/3 regulators is to recruit and activate Arp2/3. Rather our data suggests that these regulators may be playing more subtle roles in establishing functional networks in vivo.     

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.     

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.     

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.     

Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein.

The Arp2/3 complex is a highly conserved cytoskeletal component that has been implicated in the nucleation of actin filament assembly. Purified Arp2/3 complex has a low intrinsic actin nucleation activity, leading to the hypothesis that an unidentified cellular activator is required for the function of this complex. We showed previously that mutations in the Arp2/3 complex and in Bee1p/Las17p, a member of the Wiskott-Aldrich syndrome protein(WASP) family, lead to a loss of cortical actin structures (patches) in yeast. Bee1p has also been identified as an essential nucleation factor in the reconstitution of actin patches in vitro. Recently, it was reported that WASP-like proteins might interact directly with the Arp2/3 complex through a conserved carboxy-terminal domain. Here, we have shown that Bee1p and the Arp2/3 complex co-immunoprecipitate when expressed at endogenous levels, and that this interaction requires both the Arc15p and Arc19p subunits of the Arp2/3 complex. Furthermore, the carboxy-terminal domain of Bee1p greatly stimulated the nucleation activity of purified Arp2/3 complex in vitro, suggesting a direct role for WASP-family proteins in the activation of the Arp2/3 complex. Interestingly, deletion of the carboxy-terminal domain of Bee1p neither abolished the localization of the Arp2/3 complex, as had been suggested, nor resulted in a severe defect in cortical actin assembly. These results indicate that the function of Bee1p is not mediated entirely through its interaction with the Arp2/3 complex, and that factors redundant with Bee1p might exist to activate the nucleation activity of the Arp2/3 complex.     

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.     

Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex

The Wiskott-Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol(-1) drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 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.     

Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation

The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective in ATP hydrolysis. Arp2 ATP hydrolysis and Arp2/3-dependent actin nucleation occur almost simultaneously. However, ATP hydrolysis is not required for nucleation. In addition, Arp2 ATP hydrolysis is not required for the release of a WASP-like activator from y-branches. ATP hydrolysis by Arp2, and possibly Arp3, is essential for efficient y-branch dissociation in vitro. In living cells, both Arp2 and Arp3 ATP-hydrolysis mutants exhibit defects in endocytic internalization and actin-network disassembly. Our results suggest a critical feature of dendritic nucleation in which debranching and subsequent actin-filament remodelling and/or depolymerization are important for endocytic vesicle morphogenesis.     

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.     

Activation of Arp2/3 complex-mediated actin polymerization by cortactin

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.     

Arp2/3 Complex Inhibition Prevents Meiotic Maturation in Porcine Oocytes

The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.     

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.     

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.     

Distinct roles for Arp2/3 regulators in actin assembly and endocytosis

The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.