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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors

Arp2/3 complex initiates the growth of branched actin-filament networks by inducing actin polymerization from the sides of pre-existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three-dimensional structures at ~2 nm resolution of Arp2/3 complex with three different bound NPFs: N-WASp, Scar-VCA and cortactin. All of these structures adopt a conformation with the two actin-related proteins in an actin-filament-like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin-filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.     

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。     

New mechanisms and functions of actin nucleation

In cells the de novo nucleation of actin filaments from monomers requires actin-nucleating proteins. These fall into three main families--the Arp2/3 complex and its nucleation promoting factors (NPFs), formins, and tandem-monomer-binding nucleators. In this review, we highlight recent advances in understanding the molecular mechanism of actin nucleation by both well-characterized and newly identified nucleators, and explore current insights into their cellular functions in membrane trafficking, cell migration and division. The mechanisms and functions of actin nucleators are proving to be more complex than previously considered, with extensive cooperation and overlap in their cellular roles.     

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.     

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.     

Relative actin nucleation promotion efficiency by WASP and WAVE proteins in endothelial cells

The mammalian genome encodes multiple Wiskott-Aldrich syndrome protein (WASP)/WASP-family Verprolin homologous (WAVE) proteins. Members of this family interact with the actin related protein (Arp) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full-length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors.     

Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities

Background: Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?Results: We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.Conclusion: The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.     

A teamwork promotion of formin-mediated actin nucleation by Bud6 and Aip5 in Saccharomyces cerevisiae

Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucleation-promoting factors (NPFs) into functional protein complexes. Selective inter- and intramolecular interactions between the nucleation complex constituents enable diverse modes of complex assembly in initiating actin polymerization on demand. Budding yeast has two formins, Bni1 and Bnr1, which are teamed up with different NPFs. However, the selective pairing between formin NFs and NPFs into the nucleation core for actin polymerization is not completely understood. By examining the functions and interactions of NPFs and NFs via biochemistry, genetics, and mathematical modeling approaches, we found that two NPFs, Aip5 and Bud6, showed joint teamwork effort with Bni1 and Bnr1, respectively, by interacting with the C-terminal intrinsically disordered region (IDR) of formin, in which two NPFs work together to promote formin-mediated actin nucleation. Although the C-terminal IDRs of Bni1 and Bnr1 are distinct in length, each formin IDR orchestrates the recruitment of Bud6 and Aip5 cooperatively by different positioning strategies to form a functional complex. Our study demonstrated the dynamic assembly of the actin nucleation complex by recruiting multiple partners in budding yeast, which may be a general feature for effective actin nucleation by formins.     

Control of actin assembly and disassembly at filament ends

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

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.     

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.