Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex

Members of the family of WASP-family Verprolin homologous proteins (WAVEs) activate the Arp2/3 complex to induce actin polymerization. The WAVE family comprises three proteins, namely, WAVE1, WAVE2 and WAVE3. Among them, WAVE2 is crucial for activation of the Arp2/3 complex for the formation of branched actin filaments in lamellipodia. Activation of mitogen-activated protein (MAP) kinase signalling results in the phosphorylation of the WAVE family proteins; however, which of the three WAVE proteins is phosphorylated is unclear. We found that in vitro WAVE2 is directly phosphorylated by a MAP kinase, i.e. extracellular signal-regulated kinase (ERK) 2. The proline-rich region and the verprolin, cofilin and acidic (VCA) region of WAVE2 were phosphorylated. Interestingly, the phosphorylated VCA region had a higher affinity for the Arp2/3 complex. However, the phosphorylation of the VCA region resulted in reduced induction of Arp2/3-mediated actin polymerization in vitro. The role of the phosphorylation of the proline-rich region was not determined.     

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.     

Requirement of the basic region of N-WASP/WAVE2 for actin-based motility

WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Delta basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Delta basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Protein complexes regulating Arp2/3-mediated actin assembly

Key steps in regulating actin dynamics are the de novo nucleation and elongation of actin filaments, which can be catalysed by a limited number of proteins and protein complexes. Among these, Arp2/3 complex and formins are the best studied. Arp2/3-complex activity is controlled through signalling-dependent association with nucleation-promoting factors, such as the WASP/WAVE family proteins. A common theme for these molecules, which is well established for WAVEs but is only just beginning to emerge for WASPs, is that they act as coincident detectors of a variety of signalling pathways through the formation of large multi-molecular complexes.     

Exo70 stimulates the arp2/3 complex for lamellipodia formation and directional cell migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1, 2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in?vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.     

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.     

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.     

Under lock and key: Spatiotemporal regulation of WASP family proteins coordinates separate dynamic cellular processes

WASP family proteins are nucleation promoting factors that bind to and activate the Arp2/3 complex in order to stimulate nucleation of branched actin filaments. The WASP family consists of WASP, N-WASP, WAVE1-3, WASH, and the novel family members WHAMM and JMY. Each of the family members contains a C-terminus responsible for their nucleation promoting activity and unique N-termini that allow for them to be regulated in a spatiotemporal manner. Upon activation they reorganize the cytoskeleton for different cellular functions depending on their subcellular localization and regulatory protein interactions. Emerging evidence indicates that WASH, WHAMM, and JMY have functions that require the coordination of both actin polymerization and microtubule dynamics. Here, we review the mechanisms of regulation for each family member and their associated in vivo functions including cell migration, vesicle trafficking, and neuronal development.     

Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex.

WAVE/SCAR protein was identified as a protein which has similarity to WASP and N-WASP, especially in its C terminal. Recently, WAVE/SCAR protein has been shown to cooperate with the Arp2/3 complex, a nucleation core for actin polymerization in vitro. However, in spite of its general function, WAVE/SCAR expression is mainly restricted to the brain, suggesting the existence of related molecule(s). We here identified two human WAVE/SCAR homologues, which cover other organs. We named the original WAVE1 and newly identified ones WAVE2 and WAVE3. WAVE2 had a very wide distribution with strong expression in peripheral blood leukocytes and mapped on chromosome Xp11.21, next to the WASP locus. WAVE3 and WAVE1 had similar distributions. WAVE3 was strongly expressed in brain and mapped on chromosome 13q12. WAVE1 was mapped on chromosome 6q21-22. Ectopically expressed WAVE2 and WAVE3 induced actin filament clusters in a similar manner with WAVE1. These actin cluster formations were suppressed by deletion of their C-terminal VPH (verproline homology)/WH2 (WASP homology 2) domain. Further, WAVE2 and WAVE3 associate with the Arp2/3 complex as does WAVE1. Our identification of WAVE homologues suggests that WAVE family proteins have general function for regulating the actin cytoskeleton in many tissues.     

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.     

Regulation of actin dynamics by WASP family proteins

Rapid reorganization of the actin cytoskeleton underlies morphological changes and motility of cells. WASP family proteins have received a great deal of attention as the signal-regulated molecular switches that initiate actin polymerization. The first member, WASP, was identified as the product of a gene of which dysfunction causes the human hereditary disease Wiskott-Aldrich syndrome. There are now five members in this protein family, namely WASP, N-WASP, WAVE/Scar1, 2, and 3. WASP and N-WASP have functional and physical associations with Cdc42, a Rho family small GTPase involved in filopodium formation. In contrast, there is evidence that links the WAVE/Scar proteins with another Rho family protein, Rac, which is a regulator of membrane ruffling. All WASP family members have a VCA domain at the C-terminus through which Arp2/3 complex is activated to nucleate actin polymerization. Analyses of model organisms have just begun to reveal unexpected functions of WASP family proteins in multicellular organisms.     

Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2

Background  

The Scar/WAVE family of proteins mediates signals to actin assembly by direct activation of the Arp2/3 complex. These proteins have been characterised as major regulators of lamellipodia formation downstream of Rac activation and as members of large protein complexes.     

IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the actin-related protein2/3 complex activator Scar/WAVE that regulates actin and microtubule organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.     

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super‐resolution imaging, we revealed the nanoscale organization and dynamics of branched F‐actin regulators in spines. Branched F‐actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger‐like protrusions. This spatial segregation differs from lamellipodia where both branched F‐actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin‐like protein‐2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F‐actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free‐diffusion on the membrane. Enhanced Rac1 activation and Shank3 over‐expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F‐actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.     

Caldesmon inhibits Arp2/3-mediated actin nucleation

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

Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway

Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion.     

Hierarchical regulation of WASP/WAVE proteins

Members of the Wiskott-Aldrich syndrome protein (WASP) family control actin dynamics in eukaryotic cells by stimulating the actin nucleating activity of the Arp2/3 complex. The prevailing paradigm for WASP regulation invokes allosteric relief of autoinhibition by diverse upstream activators. Here we demonstrate an additional level of regulation that is superimposed upon allostery: dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly by this system. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3 domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between different family members. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.     

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.     

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.