Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of delta bee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that delta bee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain-containing protein implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex (Li, 1997).
Several end mutations that block the internalization step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton. END5 encodes a proline-rich protein (End5p or verprolin) required for a polarized cortical actin cytoskeleton and endocytosis. End5p interacts with actin, but its exact function is not yet known. To help elucidate End5p function, other End5p-interacting proteins were sought and the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) was identified as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilized by Las17p. The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homolog, WASp-interacting protein (WIP). Since las17 delta deletion mutants are blocked in endocytosis, it is concluded that Las17p and End5p interact and are essential for endocytosis (Naqvi, 1998).
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation (see Drosophila Arp2/3 component Suppressor of profilin 2), but its interactions with cellular components of the nucleation machinery are not well understood. Two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains are shown to bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. It is proposed that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and requires simultaneous binding of Arp2 and Arp3 (Zalevsky, 2000).
The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. Four of the members of this complex have been identified in Dictyostelium and the pirA gene, which encodes PIR121, has been disrupted. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar- mutant results in cells resembling their scar- parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, pirA- mutants were found to contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. These results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. It is suggested that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics (Blagg, 2003).
Neurons require precise targeting of their axons to form a connected network and a functional nervous system. Although many guidance receptors have been identified, much less is known about how these receptors signal to direct growth cone migration. This study used C. elegans motoneurons to study growth cone directional migration in response to a repellent UNC-6 (netrin homolog) guidance cue. The evolutionarily conserved kinase MIG-15 (NIK; Nck-interacting kinase - Drosophila homolog Misshapen) regulates motoneuron UNC-6-dependent repulsion through unknown mechanisms. Using genetics and live imaging techniques, it was shown that motoneuron commissural axon morphology defects in mig-15 mutants result from impaired growth cone motility and subsequent failure to migrate across longitudinal obstacles or retract extra processes. To identify new genes acting with mig-15, a screen was performed for genetic enhancers of the mig-15 commissural phenotype, and the ezrin/radixin/moesin ortholog ERM-1, the kinesin-1 motor UNC-116 and the actin regulator WVE-1 complex, were identified. Genetic analysis indicates that mig-15 and erm-1 act in the same genetic pathway to regulate growth cone migration and that this pathway functions in parallel to the UNC-116/WVE-1 pathway. Further, time-lapse imaging of growth cones in mutants suggests that UNC-116 might be required to stimulate protrusive activity at the leading edge, whereas MIG-15 and ERM-1 maintain low activity at the rear edge. Together, these results support a model in which the MIG-15 kinase and the UNC-116-WVE-1 complex act on opposite sides of the growth cone to promote robust directional migration (Teuliere, 2011).
Directional cell migration is critical for metazoan development. This study defines two molecular pathways that activate the Arp2/3 complex during neuroblast migration in Caenorhabditis elegans. The transmembrane protein MIG-13/Lrp12 is linked to the Arp2/3 nucleation-promoting factors WAVE (see Drosophila SCAR) or WASP (see Drosophila WASp) through direct interactions with ABL-1 (see Drosophila Abl) or SEM-5/Grb2 (see Drosophila Drk), respectively. WAVE mutations partially impaired F-actin organization and decelerated cell migration, and WASP mutations did not inhibit cell migration but enhanced migration defects in WAVE-deficient cells. Purified SEM-5 and MIG-2 synergistically stimulated the F-actin branching activity of WASP-Arp2/3 in vitro. In GFP knockin animals, WAVE and WASP were largely organized into separate clusters at the leading edge, and the amount of WASP was less than WAVE but could be elevated by WAVE mutations. These results indicate that the MIG-13-WAVE pathway provides the major force for directional cell motility, whereas MIG-13-WASP partially compensates for its loss, underscoring their coordinated activities in facilitating robust cell migration (Zhu, 2016).
WAVE/SCAR protein has similarity to WASP and N-WASP, especially in its C terminal. WAVE/SCAR protein cooperates 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). Two human WAVE/SCAR homologs have been identified that cover other organs. The original has been named WAVE1 and newly identified ones WAVE2 and WAVE3. WAVE2 has a very wide distribution with strong expression in peripheral blood leukocytes and maps on chromosome Xp11.21, next to the WASP locus. WAVE3 and WAVE1 have similar distributions. WAVE3 was strongly expressed in brain and maps on chromosome 13q12. WAVE1 maps on chromosome 6q21-22. Ectopically expressed WAVE2 and WAVE3 induces actin filament clusters in a manner similar to WAVE1. These actin cluster formations are suppressed by deletion of the C-terminal VPH (verproline homology)/WH2 (WASP homology 2) domain. Further, WAVE2 and WAVE3 associate with the Arp2/3 complex as does WAVE1. This identification of WAVE homologs suggests that WAVE family proteins have general function for regulating the actin cytoskeleton in many tissues (Suetsugu, 1999).
Characterization of multiprotein complexes involved in actin remodeling and cytoskeleton reorganization is essential to understand the basic mechanisms of cell motility and migration. To identify proteins implicated in these processes, the mouse Wave1/Scar gene, a member of the Wiskott-Aldrich syndrome protein (WASP) family, has been isolated. The mouse Wave1 gene was physically localized on chromosome 10 and spans over 12 Kb comprising eight exons and seven introns. The mouse Wave1 complementary DNA encodes a predicted 559 amino acid protein, with a SCAR homology domain, a basic domain, a proline-rich region, a WASP homology domain and an acidic domain conserved in the orthologous proteins. The Wave1 transcription initiation site maps 210 base pairs upstream of the ATG translational start site. The presumptive proximal promoter contains putative consensus binding sites for E2 basic helix-loop-helix transcription factors, hepatocyte nuclear factor-3beta, S8 homeodomain protein, zinc finger transcription factor MZF-1, and an interferon-stimulated response element. Northern analysis has demonstrated a strong expression of a unique (approximately 2.6 Kb) Wave1 transcript in brain tissue, and in situ hybridization shows restricted expression to Purkinje cells from the cerebellum and pyramidal cells from the hippocampus. Characterization and expression analyses of the murine Wave1 gene provide the basis toward functional studies in mouse models of the role of Wave1 in neuronal cytoskeleton organization (Benachenhou, 2002).
Scar/WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. WAVE-1 has been identified in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin because the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, a previously unrecognized function is proposed for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl (Westphal, 2000).
WASP family proteins induce actin polymerization through a C-terminal verprolin homology, cofilin homology, and acidic (VCA) region by activating the Arp2/3 complex. The N-WASP VCA region is the most potent activator of the Arp2/3 complex. In addition, full-length WAVE1 and a WAVE1 VCA fragment show differential activity. The mechanisms underlying these differences are poorly understood. The activities of various N-WASP and WAVE1 VCA mutant proteins were examined with several types of fusion moieties. When fused to GST, maltose-binding protein, or the WAVE1 proline-rich domain, N-WASP VCA and WAVE1 VCA mutant proteins with two V motifs show stronger activities than wild-type WAVE1 VCA with one V motif, demonstrating the importance of two V motifs for strong VCA activity. A WAVE1 VCA fragment tagged with six histidines (His) shows markedly reduced activity compared to GST-fused VCA, whereas His-tagged N-WASP VCA shows similar activity to GST-fused VCA. An additional V motif fails to enhance WAVE1 VCA activity in the His-tagged form. Thus, the WAVE1 VCA fragment may exist in an unfavorable conformation to activate the Arp2/3 complex, implying the existence of a structural difference between WAVE1 and N-WASP VCAs in addition to the number of V motifs (Yamaguchi, 2002).
Growth factors initiate cytoskeletal rearrangements tightly coordinated with nuclear signaling events. It was hypothesized that the angiogenic growth factor VEGF may utilize oxidants that are site-directed to a complex critical to both cytoskeletal and mitogenic signaling. The WASP-family verprolin homologous protein-1 (WAVE1) has been identified as a binding partner for the NADPH oxidase adapter p47phox within membrane ruffles of VEGF-stimulated cells. Within 15 min of VEGF stimulation, p47phox coprecipitated with WAVE1, with the ruffle and oxidase agonist Rac1, and with the Rac1 effector PAK1. VEGF also increases p47phox phophorylation, oxidant production, and ruffle formation, all of which are dependent upon PAK1 kinase activity. The antioxidant MnTBAP and ectopic expression of either the p47-binding WAVE1 domain or the WAVE1-binding p47phox domain decreases VEGF-induced ruffling, while the active mutant p47(S303,304,328D) stimulates oxidant production and formation of circular dorsal ruffles. Both kinase-dead PAK1(K298A) and MnTBAP decrease c-Jun NH4-terminal kinase (JNK) activation by VEGF, whereas dominant-negative JNK does not block ruffle formation, suggesting a bifurcation of mitogenic and cytoskeletal signaling events at or distal to the oxidase but proximal to JNK. Thus WAVE1 may act as a scaffold to recruit the NADPH oxidase to a complex involved with both cytoskeletal regulation and downstream JNK activation (Wu, 2003).
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. Tandem mass spectrometry has been used to identify an Abi1-based complex containing WAVE2 (see Drosophila Abi), 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 (Innocenti, 2004).
SCAR (also known as WAVE) is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization. In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 (Nck-associated protein 1) proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed, but recent data suggest that it is essential for SCAR function. This study shows that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA- cells move and chemotax surprisingly effectively. These results show that the members of the complex have unexpectedly diverse biological roles (Ibarra, 2006)
Dynamic cell movements and rearrangements are essential for the generation of the mammalian body plan, although relatively little is known about the genes that coordinate cell movement and cell fate. WAVE complexes are regulators of the actin cytoskeleton that couple extracellular signals to polarized cell movement. Mouse embryos that lack Nap1, a regulatory component of the WAVE complex, arrest at midgestation and have defects in morphogenesis of all three embryonic germ layers. WAVE protein is not detectable in Nap1 mutants, and other components of the WAVE complex fail to localize to the surface of Nap1 mutant cells; thus loss of Nap1 appears to inactivate the WAVE complex in vivo. Nap1 mutants show specific morphogenetic defects: they fail to close the neural tube, fail to form a single heart tube (cardia bifida), and show delayed migration of endoderm and mesoderm. Other morphogenetic processes appear to proceed normally in the absence of Nap1/WAVE activity: the notochord, the layers of the heart, and the epithelial-to-mesenchymal transition (EMT) at gastrulation appear normal. A striking phenotype seen in approximately one quarter of Nap1 mutants is the duplication of the anteroposterior body axis. The axis duplications arise because Nap1 is required for the normal polarization and migration of cells of the Anterior Visceral Endoderm (AVE), an early extraembryonic organizer tissue. Thus, the Nap1 mutant phenotypes define the crucial roles of Nap1/WAVE-mediated actin regulation in tissue organization and establishment of the body plan of the mammalian embryo (Rakeman, 2006; full text of article).
The cytoskeletal regulators that mediate the change in the neuronal cytoskeletal machinery from one that promotes oriented motility to one that facilitates differentiation at the appropriate locations in the developing neocortex remain unknown. Nck-associated protein 1 (Nap1), an adaptor protein thought to modulate actin nucleation, is selectively expressed in the developing cortical plate, where neurons terminate their migration and initiate laminar-specific differentiation. Loss of Nap1 function disrupts neuronal differentiation. Premature expression of Nap1 in migrating neurons retards migration and promotes postmigratory differentiation. Nap1 gene mutation in mice leads to neural tube and neuronal differentiation defects. Disruption of Nap1 retards the ability to localize key actin cytoskeletal regulators such as WAVE1 to the protrusive edges where they are needed to elaborate process outgrowth. Thus, Nap1 plays an essential role in facilitating neuronal cytoskeletal changes underlying the postmigratory differentiation of cortical neurons, a critical step in functional wiring of the cortex (Yokota, 2007).
Developing neurons must respond to a wide range of extracellular signals during the process of brain morphogenesis. One mechanism through which immature neurons respond to such signals is by altering cellular actin dynamics. A recently discovered link between extracellular signaling events and the actin cytoskeleton is the WASP/WAVE (Wiscott-Aldrich Syndrome protein/WASP-family verprolin-homologous protein) family of proteins. Through a direct interaction with the Arp2/3 (actin-related protein) complex, this family functions to regulate the actin cytoskeleton by mediating signals from cdc42 as well as other small GTPases. To evaluate the role of WASP/WAVE proteins in the process of neuronal morphogenesis, a retroviral gene trap was used to generate a line of mice bearing a disruption in the WAVE1 gene. Using a heterologous reporter gene, it was found that WAVE1 expression becomes increasingly restricted to the CNS over the course of development. Homozygous disruption of the WAVE1 gene results in postnatal lethality. In addition, these animals have severe limb weakness, a resting tremor, and notable neuroanatomical malformations without overt histopathology of peripheral organs. No alterations were detected in neuronal morphology in vivo or the ability of embryonic neurons to form processes in vitro. These data indicate that WAVE1, although important for the general development of the CNS, is not essential for the formation and extension of neuritic processes (Dahl, 2003).
The Scar/WAVE family of scaffolding proteins organize molecular networks that relay signals from the GTPase Rac to the actin cytoskeleton. The WAVE-1 isoform is a brain-specific protein expressed in a variety of areas including the regions of the hippocampus and the Purkinje cells of the cerebellum. Targeted disruption of the WAVE-1 gene generates mice with reduced anxiety, sensorimotor retardation, and deficits in hippocampal-dependent learning and memory. These sensorimotor and cognitive deficits are analogous to the symptoms of patients with 3p-syndrome mental retardation who are haploinsufficient for WRP/MEGAP, a component of the WAVE-1 signaling network. Thus WAVE-1 is required for normal neural functioning (Soderling, 2003).
The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. The WAVE2 gene was first inactivated by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survive until approximately embryonic day 12.5 and display growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells display normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibit severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement (Yan, 2003).
WAVE2 function has been disrupted in mice. WAVE2 is expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos show hemorrhages and die at about embryonic day 10. Deficiency in WAVE2 has no significant effect on vasculogenesis, but it decreases sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity forms in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries is severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo (Yamazaki, 2003).
Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge; WAVE2, but not WAVE1, is essential essential for this directed migration. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM (Suetsugu, 2003).
Under uniform stimuli that result in random migration, coordination of the formation of peripheral and dorsal ruffles does not seem to be required, and thus the two types of ruffles develop and appear separately. Formation of both types of ruffles appears to be regulated by Rac, because both peripheral and dorsal ruffle formation are suppressed by DN Rac and Wortmannin, a PI-3 kinase inhibitor. Further, PIP3 also accumulates at dorsal ruffles prior to membrane extension. Therefore, activation of Rac through PIP3 binding Rac GEFs including Vav, Sos, and SWAP-70 is thought to occur also in dorsal ruffles. However, constitutive activation of Rac results in only induction of peripheral ruffles. Since WAVE2 is essential in peripheral ruffle formation under PDGF treatment, constitutively active Rac induction of peripheral ruffles is impaired only in WAVE2-deficient cells. This result suggests that WAVE2 is the primary effecter of Rac in formation of ruffles. Consistently, the adaptor molecule IRSp53, which links WAVE2 and Rac, specifically binds to WAVE2, not to WAVE1. In the actin polymerization assay using WAVE2-deficient cell lysate, Rac-induced actin polymerization is impaired in WAVE2-deficient cell lysate, not in wild-type cell lysate, also indicating that WAVE2 is the primary effecter of Rac (Suetsugu, 2003).
Because constitutive activation of Rac does not cause dorsal ruffles, some additional signals are required for dorsal ruffle formation. The regulation of WRP, a RacGAP that binds to WAVE1 and inactivates Rac, might be involved in dorsal ruffles. Activation of c-Abl and recruitment of c-Cbl are involved in dorsal ruffle formation. c-Abl associates with WAVE1. Abi1 also interacts with WAVE1 and is involved in dorsal ruffle formation. These signals may be required for dorsal ruffle formation through unknown regulation of WAVE1 (Suetsugu, 2003).
Rac is a Rho-family small GTPase that induces the formation of membrane ruffles. However, it is poorly understood how Rac-induced reorganization of the actin cytoskeleton, which is essential for ruffle formation, is regulated. A novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP family Verprolin-homologous protein (WAVE), has been identified as a regulator of actin reorganization downstream of Rac. Ectopically expressed WAVE induces the formation of actin filament clusters that overlap with the expressed WAVE itself. In this actin clustering, profilin, a monomeric actin-binding protein that has been suggested to be involved in actin polymerization, has been shown to be essential. The expression of a dominant-active Rac mutant induces the translocation of endogenous WAVE from the cytosol to membrane ruffling areas. Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce actin reorganization specifically suppresses the ruffle formation induced by Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon that is also known to be dependent on rapid actin reorganization. The deltaVPH WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells. Taken together, it is concluded that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling (Miki, 1998).
Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42, which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. IRSp53, a substrate for insulin receptor with unknown function, has been demonstrated to be the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, it was found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex (Miki, 2000).
Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. This study reports a mechanism by which Rac and the adapter protein Nck (Drosophila homolog: Dreadlocks) activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologs of human PIR121 (p53-inducible messenger RNA with a relative molecular mass [M(r)] of 140,000), Nap125 (NCK-associated protein with an M(r) of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. It is therefore proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation (Eden, 2002).
IRSp53 binds both Rac and WAVE2, inducing formation of Rac/IRSp53/WAVE2 complex that is important for membrane ruffling. However, recent reports that note a specific interaction between IRSp53 and Cdc42 but not Rac, have led to a re-examination of the binding of IRSp53 to Rac. Immunoprecipitation analysis and pull-down assay reveal that full-length IRSp53 binds Rac much less efficiently than the N-terminal fragment, which may be caused by intramolecular interaction. Interestingly, the intramolecular interaction is interrupted by the binding of WAVE2 and full-length IRSp53 associates with Rac in the presence of WAVE2. IRSp53 induces spreading and neurite formation of N1E-115 cells, which presumably reflect functional cooperation with Rac (Miki, 2002).
WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Tandem mass spectrometry has been used to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, it is proposed that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac (Soderling, 2002).
The insulin receptor tyrosine kinase substrate p53 (IRSp53) links Rac and WAVE2 and has been implicated in lamellipodia protrusion. Recently, however, IRSp53 has been reported to bind to both Cdc42 and Mena to induce filopodia. To shed independent light on IRSp53 function, the localizations and dynamics of IRSp53 and WAVE2 were determined in B16 melanoma cells. In cells spread well on a laminin substrate, IRSp53 localizes at the tips of both lamellipodia and filopodia. The same localization is observed in living cells with IRSp53 tagged with enhanced green florescence protein (EGFP-IRSp53), but only during protrusion. From the transfection of deletion mutants the N-terminal region of IRSp53, which binds active Rac, was shown to be responsible for its localization. Although IRSp53 has been reported to regulate filopodia formation with Mena, EGFP-IRSp53 shows the same localization in MVD7 Ena/VASP (vasodilator stimulated phosphoprotein) family deficient cells. WAVE2 tagged with DsRed1 colocalizes with EGFP-IRSp53 at the tips of protruding lamellipodia and filopodia and, in double-transfected cells, the IRSp53 signal in filopodia decreases before that of WAVE2 during retraction. These results suggest an alternative modulatory role for IRSp53 in the extension of both filopodia and lamellipodia, through WAVE2 (Nakagawa, 2003).
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. This study identified a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and has shown that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration (Dang, 2013).
The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex is not known. Full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament -- a dendritic nucleation mechanism. The Arp2/3 complex, either with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex (Machesky, 1999).
Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. A light microscopy assay reveals many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favor branched nucleation; phosphate release from aged actin filaments favors dissociation of Arp2/3 complex from the pointed ends of filaments, and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favor the selective disassembly of proximal regions of the network (Blanchoin, 2000).
WAVE1/Scar1, a WASP-family protein that functions downstream of Rac in membrane ruffling, can induce part of the reorganization of the actin cytoskeleton without Arp2/3 complex. WAVE1 has been reported toassociate and activate Arp2/3 complex at its C-terminal region, which is rich in acidic residues. The deletion of the acidic residues abolishes the interaction with and the activation ability of Arp2/3 complex. The expression of the mutant WAVE1 lacking the acidic residues (DeltaA), however, induces actin-clustering in cells as the wild-type WAVE1 does. In addition, this actin-clustering can not be suppressed by the coexpression of the Arp2/3 complex-sequestering fragment (CA-region) derived from N-WASP, which clearly inhibits Rac-induced membrane ruffling. This study therefore demonstrates that WAVE1 reorganizes the actin cytoskeleton not only through Arp2/3 complex but also through another unidentified mechanism that may be important but has been neglected thus far (Sasaki, 2000).
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 were compared; 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, it was 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. It is concluded that 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 (Zalevsky, 2001).
Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerization at the tips of these processes via molecular complexes that remain to be characterized. A green fluorescent protein (GFP) fusion of the Wiskott-Aldrich syndrome protein family member Scar1/WAVE1 is shown to be specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1-GFP is recruited only to protruding lamellipodia and is absent from filopodia. The localization of Scar is facilitated by the finding that the inhibition of lamellipodia formation by ectopical expression of Scar, can be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerization specifically engaged in the protrusion of lamellipodia (Hahne, 2001).
WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. The localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, is observed in protruding lamellipodia. Rat fibroblast cell line 3Y1 protrudes lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 are concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena are observed to localize at the lamellipodium edge. Interestingly, Mena tends to concentrate at the microspike tips but WAVE does not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, is much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, is localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE is distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42 (Nakagawa, 2001).
The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). The branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 is activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there is no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which associates with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhances recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility (Suetsugu, 2001).
The formation and extension of filopodia in response to an extracellular stimulus by guidance cues determine the path of growth cone advance. Actin-filament bundling and actin polymerization at the tips supply the driving force behind the formation and elongation. This study tries to clarify how signals in response to extracellular cues are transformed to induce filopodial generation and extension. Observations on the formation process of filopodia at growth cones in the neuroblastoma cell line NG108 show that WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein) isoforms play crucial and distinct roles in this process. WAVE1 is continuously distributed along the leading edge only and is not found in the filopodia. WAVE2 and WAVE3 discretely localize at the initiation sites of microspikes on the leading edge and also concentrate at the tips of protruding filopodia. WAVE isoforms localize at the filopodial tips through SHD (SCAR homology domain), next to its leucine zipper-like motif. Furthermore, time-lapse observations of filopodial formation in living cells show that WAVE2 and WAVE3 are continuously expressed at the tips of filopodia during elongation. These results indicate that WAVE2 or WAVE3 may guide the actin bundles into the filopodia and promote actin assembly at the tips (Nozumi, 2003).
Although many of the regulators of actin assembly are known, how these components act together to organize cell shape and movement is not understood. To address this question, the spatial dynamics were analyzed of a key actin regulator-the Scar/WAVE complex-which plays an important role in regulating cell shape in both metazoans and plants. The Hem-1/Nap1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that exhibit many of the properties of morphogenesis in motile cells, such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. It is proposed that cell motility results from the collective behavior of multiple self-organizing waves (Weiner, 2007; full text of article).
Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. This study analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable (Steffen, 2006).
Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein) -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. This study reports a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 [p53-inducible messenger RNA with a relative molecular mass (Mr) of 140,000], Nap125 (NCK-associated protein with an Mr of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. It is therefore proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation (Eden, 2002).
Members of the Rho family of small GTPases, such as Cdc42 and Rac1, and of the Src homology (SH) domain-containing SH2SH3 adapter protein family, such as NCK, link extracellular signals and actin nucleation through pathways that include the WASP family of proteins and the actin nucleation machinerythe Arp2/3 complex. All WASP family members contain a conserved verprolin-homology, cofilin-homology, acidic (VCA) domain that directly binds and activates the Arp2/3 complex. The Arp2/3 complex, in turn, catalyses the nucleation of actin filaments. To prevent undesirable spontaneous actin nucleation in the absence of input signals, the activity of the WASP proteins is tightly regulated. For example, N-WASP is found predominantly in an autoinhibited conformation in which the carboxy-terminal VCA domain is occluded through interaction with the amino terminus of the protein. When Cdc42 binds to the Cdc42/Rac1 interactive binding (CRIB) domain of N-WASP or when NCK binds to the polyproline region of N-WASP, this autoinhibition is relieved and the VCA domain is unmasked. Phosphatidylinositol(4,5)bisphosphate (PIP2) can further activate N-WASP in cooperation with NCK or Cdc42 by binding to a basic region of N-WASP (Eden, 2002).
The WAVE proteins (WAVE1, WAVE2 and WAVE3 in mammals and orthologues in Drosophila and Dictyostelium are similar in structure to N-WASP. They all have a C-terminal VCA domain, a polyproline region and a basic region. Unlike N-WASP, WAVE proteins do not contain a CRIB domain, and direct binding of WAVE1 to Rac1 has not been detected. But much evidence suggests that WAVE1 functions downstream of Rac1. WAVE1 is translocated from the cytoplasm to membrane ruffles induced by Rac1, and dominant-negative WAVE1 abolishes the formation of Rac1-dependent lamellipodia and Rac1-dependent neurite extensions. The mechanism of regulation of WAVE1 is likely to be fundamentally different from that of N-WASP: whereas N-WASP is autoinhibited, recombinant WAVE1 is constitutively active in stimulating the actin nucleation activity of Arp2/3. Therefore, WAVE1 activity is either inhibited in trans by other cellular regulators or regulated by post-translational modifications (Eden, 2002).
Although WAVE1 had been implicated as the downstream target of Rac1, no regulatory linkage had been found previously. Consequently, the important Rac1-dependent pathway for actin nucleation has not been described. Similarly, although the association of NCK and Rac1 with NAP125 and PIR121 has been observed in several screens, their role as regulators of actin nucleation has not been shown. These results indicate that WAVE1, like N-WASP and WASP, mediates signals from NCK and the Rho GTPases. The activation mechanisms of WAVE1 and N-WASP are very different: N-WASP is autoinhibited, whereas WAVE1 is trans-inhibited. The action of Rac1 and NCK is to disassemble the trans-inhibited WAVE1 complex, which releases the active WAVE1 protein in association with HSPC300. Consistent with this model, Rac1 and WAVE1 do not colocalize in the lamellipodium: WAVE1 is localized at the extreme edge of the lamellipodium, whereas Rac1 is distributed diffusely over the lamellipodium (Eden, 2002).
Although the predominant regulation of WAVE1 activity described here is relief of trans-inhibition, an additional positive regulation by proteins that bind WAVE1 directly in an activator-independent manner is also possible. For example, IRSp53 has been reported to bind WAVE2 directly and enhance activation of Arp2/3 by recombinant WAVE2. Preliminary data show that HSPC300, which remains associated with WAVE1 after activation, may also have a stimulating function on actin polymerization. The activation and dissociation of the WAVE1 complex process releases a subcomplex of NAP125 and PIR121, and this subcomplex may be free to interact with other cellular components. In this way, a Rac1 or NCK signal might potentially coordinate several cellular processes -- similar to pathways that are activated by the alpha- and the gamma-subunits in heterotrimeric G-protein signalling (Eden, 2002).
A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. This study tests the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores (Rawe, 2004).
Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2, and HSPC300. However, there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets. This study analyzes the roles of the unique Dictyostelium Abi (see Drosophila Abi). abiA null mutants show less severe defects in motility than do scar null cells, indicating (unexpectedly) that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways (Pollitt, 2008).
The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. This study identifies a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. It is concluded that the Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.
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