Interactions with Actin Monomers,Actin Filaments,and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.     

Dissection of Arp2/3 complex actin nucleation mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces cerevisiae

Actin nucleation by the Arp2/3 complex is under tight control, remaining inactive until stimulation by nucleation-promoting factors (NPFs). Although multiple NPFs are expressed in most cell types, little is known about how they are coordinated and whether they perform similar or distinct functions. We examined genetic relationships among the four S. cerevisiae NPFs. Combining las17delta with pan1-101 or myo3delta myo5delta was lethal at all temperatures, whereas combining pan1-101 with myo3delta myo5delta showed no genetic interaction and abp1delta partially suppressed las17delta. These data suggest that NPFs have distinct and overlapping functions in vivo. We also tested genetic interactions between each NPF mutant and seven different temperature-sensitive arp2 alleles and purified mutant Arp2/3 complexes to compare their activities. Two arp2 alleles with mutations at the barbed end were severely impaired in nucleation, providing the first experimental evidence that Arp2 nucleates actin at its barbed end in vitro and in vivo. Another arp2 allele caused partially unregulated ("leaky") nucleation in the absence of NPFs. Combining this mutant with a partially unregulated allele in a different subunit of Arp2/3 complex was lethal, suggesting that cells cannot tolerate high levels of unregulated activity. Genetic interactions between arp2 alleles and NPF mutants point to Abp1 having an antagonistic role with respect to other NPFs, possibly serving to attenuate their stronger activities. In support of this model, Abp1 binds strongly to Arp2/3 complex, yet has notably weak nucleation-promoting activity and inhibits Las17 activity on Arp2/3 complex in a dose-responsive manner.     

Activation of nucleation promoting factors for directional actin filament elongation: Allosteric regulation and multimerization on the membrane

Nucleation promoting factors (NPFs) activate the Arp2/3 complex to produce branched actin filaments. Branched actin filaments are observed in most organelles, and specific NPFs, such as WASP, N-WASP, WAVEs, WASH, and WHAMM, exist for each organelle. Interestingly, Arp2/3 and NPFs are both inactive by themselves, and thus require activation. The exposure of the Arp2/3 activating region, the VCA fragment, is recognized to be a key event in the activation of the NPFs. Together, small GTPase binding, phosphorylation, SH3 binding, and membrane binding promote VCA exposure synergistically. The increase in the local concentration of NPF by multimerization is thought to occur with the combination of such activators, to maximally activate the NPF and confine the region of actin polymerization. The mechanism of uni-directional filament extension beneath the membrane also is discussed.     

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.     

Making muscles- Arp,two, three

In Drosophila embryos, muscle fiber formation via myoblast fusion relies on essential contributions made by the conserved Arp2/3 microfilament nucleation machinery. Two key nucleation promoting factors (NPFs), SCAR and WASp, have been shown to mediate this aspect of Arp2/3 function. We have used these unique circumstances, to study the requirements and coordination of distinct NPF activities, within a common developmental setting. Our results suggest that, although operating within close spatial and temporal proximity, the two regulators of actin polymerization are used in a step-wise manner and perform separate functional roles. Our approach also allows us to assess the involvement of the Arp2/3 machinery in formation of a distinct, fusion-associated actin structure.     

Cytoskeletal regulation: coordinating actin and microtubule dynamics in membrane trafficking

The Arp2/3 complex is essential for actin nucleation and filament elongation in a variety of intracellular processes. This functional versatility is exerted through the regulation of its activity by nucleation-promoting factors (NPFs). The discovery of a new NPF, WHAMM, reveals unexpected connections between the actin and microtubule cytoskeletons and membrane dynamics during ER-to-Golgi transport.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

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.     

The structural basis of actin filament branching by the Arp2/3 complex

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.     

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

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

WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond

Arp2/3 complex mediates the nucleation of actin filaments in multiple subcellular processes, and is activated by nucleation-promoting factors (NPFs) from the Wiskott-Aldrich Syndrome family. In exciting new developments, this family has grown by three members: WASH, WHAMM and JMY, which extend the repertoire of dynamic membrane structures that are remodeled following Arp2/3 activation in vivo. These novel NPFs share an actin- and Arp2/3-interacting WCA module, and combine Arp2/3 activation with additional biochemical functions, including capping protein inhibition, microtubule engagement or Arp2/3-independent actin nucleation, none of which had been previously associated with canonical WCA-harboring proteins. Uncovering the physiological relevance of these unique activities will require concerted efforts from multiple disciplines, and is sure to impact our understanding of how the cytoskeleton controls so many dynamic subcellular events.     

Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments

The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical “or gate” capable integrating diverse upstream signals.     

Abl2/Abl-related Gene Stabilizes Actin Filaments,Stimulates Actin Branching by Actin-related Protein 2/3 Complex,and Promotes Actin Filament Severing by Cofilin

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.     

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.     

WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport

The Arp2/3 complex is an actin nucleator that plays a critical role in many cellular processes. Its activities are regulated by nucleation-promoting factors (NPFs) that function primarily during plasma membrane dynamics. Here we identify a mammalian NPF called WHAMM (WASP homolog associated with actin, membranes, and microtubules) that localizes to the cis-Golgi apparatus and tubulo-vesicular membrane transport intermediates. The modular organization of WHAMM includes an N-terminal domain that mediates Golgi membrane association, a coiled-coil region that binds microtubules, and a WCA segment that stimulates Arp2/3-mediated actin polymerization. Overexpression and depletion studies indicate that WHAMM is important for maintaining Golgi structure and facilitating anterograde membrane transport. The ability of WHAMM to interact with microtubules plays a role in membrane tubulation, while its capacity to induce actin assembly promotes tubule elongation. Thus, WHAMM is an important regulator of membrane dynamics functioning at the interface of the microtubule and actin cytoskeletons.     

Actin filament nucleation and elongation factors – structure–function relationships

The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tβ4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs–Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been “outsourced” to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.     

肌动蛋白相关蛋白2/3复合体的结构、功能与调节

微丝参与了细胞形态维持及细胞运动等多种重要的细胞过程。微丝由肌动蛋白单体组装而成 ,肌动蛋白相关蛋白 2 / 3(Arp2 /Arp3,Arp2 / 3)复合体在微丝形成过程中起重要作用。Arp2 / 3复合体由 7个亚单位组成 ,在细胞内受到多种核化促进因子的调节 ,并与这些因子协同作用来调节肌动蛋白的核化。Arp2 / 3复合体结构、功能及调节的研究对于阐明微丝形成机制及细胞骨架与某些信号分子的关系有重要意义。     

Identification of a Novel Regulatory Sequence of Actin Nucleation Promoting Factor Encoded by Autographa californica Multiple Nucleopolyhedrovirus

Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.     

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.     

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex

Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery.