The yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. 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 contain few 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 previously 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).
Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Las17p interacts with the Arp2/3 complex (see Drosophila Arp2/3 component Suppressor of profilin 2). LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Delta cells has shown that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo, independent of polymerized actin, and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins, including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton (Madania, 1999).
The Arp2/3 complex is a highly conserved cytoskeletal component that has been implicated in the nucleation of actin filament assembly. Purified Arp2/3 complex has a low intrinsic actin nucleation activity, leading to the hypothesis that an unidentified cellular activator is required for the function of this complex. Mutations in the Arp2/3 complex and in Bee1p/Las17p, a member of the Wiskott-Aldrich syndrome protein(WASP) family, lead to a loss of cortical actin structures (patches) in yeast. Bee1p has also been identified as an essential nucleation factor in the reconstitution of actin patches in vitro. WASP-like proteins interact directly with the Arp2/3 complex through a conserved carboxy-terminal domain. Bee1p and the Arp2/3 complex have been shown to co-immunoprecipitate when expressed at endogenous levels, and this interaction requires both the Arc15p and Arc19p subunits of the Arp2/3 complex. Furthermore, the carboxy-terminal domain of Bee1p greatly stimulates the nucleation activity of purified Arp2/3 complex in vitro, suggesting a direct role for WASP-family proteins in the activation of the Arp2/3 complex. Interestingly, deletion of the carboxy-terminal domain of Bee1p neither abolishes the localization of the Arp2/3 complex, as had been suggested, nor results in a severe defect in cortical actin assembly. These results indicate that the function of Bee1p is not mediated entirely through its interaction with the Arp2/3 complex, and that factors redundant with Bee1p might exist to activate the nucleation activity of the Arp2/3 complex (Winter, 1999).
WASp family proteins promote actin filament assembly by activating Arp2/3 complex and are regulated spatially and temporally to assemble specialized actin structures used in diverse cellular processes. Some WASp family members are autoinhibited until bound by activating ligands, however, regulation of the budding yeast WASp homolog (Las17/Bee1) has not yet been explored. Full-length Las17 was isolated and its biochemical activities on yeast Arp2/3 complex were characterized. Purified Las17 was not autoinhibited; in this respect, it is more similar to SCAR/WAVE than to WASp proteins. Las17 is a much stronger activator of Arp2/3 complex than its carboxyl-terminal (WA) fragment. In addition, actin polymerization stimulated by Las17-Arp2/3 is much less sensitive to the inhibitory effects of profilin compared to polymerization stimulated by WA-Arp2/3. Two SH3 domain-containing binding partners of Las17, Sla1 and Bbc1, were purified and were shown to cooperate in inhibiting Las17 activity. The two SLA1 SH3 domains required for this inhibitory activity in vitro are also required in vivo, in combination with BBC1, for cell viability and normal actin organization. It is concluded that full-length Las17 is not autoinhibited and it activates Arp2/3 complex more strongly than its WA domain alone, revealing an important role for the Las17 amino terminus in Arp2/3 complex activation. Two of the SH3 domain-containing ligands of Las17, Sla1 and Bbc1, cooperate to inhibit Las17 activity in vitro and are required for a shared function in actin organization in vivo. These results show that, like SCAR/WAVE, WASp proteins can be controlled by negative regulation through the combined actions of multiple ligands (Rodal, 2003).
What is the specific function of Las17 in cells? Localization of Las17, myosin I, and Vrp1 to cortical patches is maintained after treatment of cells with latrunculin-A, an actin monomer-sequestering agent that leads to net actin depolymerization. Therefore, these components may function upstream of actin assembly in vivo; this finding is consistent with their requirement for actin assembly in the permeabilized cell. In addition, Arp2/3 complex, myosin I, and Vrp1 are required for directional motility of cortical actin patches, which may be driven by actin polymerization. Recently, it was shown that not all actin patches contain Sla1 and that Sla1-containing patches move more slowly than Sla1-free patches. These observations are consistent with an inhibitory role for Sla1 on actin polymerization-driven patch motility. These results suggest that this inhibition may occur directly through Sla1 SH3 domain interactions with Las17 (Rodal, 2003 and references therein).
In yeast and mammals, cortical actin is required for endocytosis, but its precise role in this process has not been defined. The SHD1 region of Sla1 binds to receptors targeted for actin-mediated endocytosis, and the carboxyl-terminal repeats of Sla1 form a complex with the endocytic proteins Pan1 (which can activate Arp2/3 complex) and End3. In the future, it will be important to test how physical interactions in Sla1-End3-Pan1, Bbc1-Vrp1-myosin I, and Sla1-receptor complexes affect Las17 activities on Arp2/3 complex (Rodal, 2003 and references therein).
Migration of cells through the reorganization of the actin cytoskeleton is essential for morphogenesis of multicellular animals. In a cell culture system, the actin-related protein (Arp) 2/3 complex functions as a nucleation core for actin polymerization when activated by the members of the WASP (Wiskott-Aldrich syndrome protein) family. However, the regulation of cell motility in vivo remains poorly understood. Homologs of the mammalian Arp2/3 complex and N-WASP in Caenorhabditis elegans play an important role in hypodermal cell migration during morphogenesis, a process known as ventral enclosure. In the absence of one of any of the C. elegans Arp2/3 complex subunits (ARX-1, ARX-2, ARX-4, ARX-5, ARX-6 or ARX-7) or of N-WASP (WSP-1), hypodermal cell migration led by actin-rich filopodia formation is inhibited during ventral enclosure owing to the reduction of filamentous actin formation. However, there is no effect on differentiation of hypodermal cells and dorsal intercalation. Disruption of the function of ARX-1 and WSP-1 in hypodermal cells also results in hypodermal cell arrest during ventral enclosure, suggesting that their function is cell autonomous. WSP-1 protein activates Arp2/3-mediated actin polymerization in vitro. Consistent with these results, the Arp2/3 complex and WSP-1 colocalize at the leading edge of migrating hypodermal cells. The stable localization of WSP-1 is dependent on the presence of Arp2/3 complex, suggesting an interaction between the Arp2/3 complex and WSP-1 in vivo (Sawa, 2003).
WASP and Ena/VASP family proteins play overlapping roles in C. elegans morphogenesis and neuronal cell migration. Specifically, these studies demonstrate that UNC-34/Ena plays a role in morphogenesis that is revealed only in the absence of WSP-1 function and that WSP-1 has a role in neuronal cell migration that is revealed only in the absence of UNC-34/Ena activity. To identify additional genes that act in parallel to unc-34/ena during morphogenesis, a screen for synthetic lethals was performed in an unc-34 null mutant background utilizing an RNAi feeding approach. This is the first reported RNAi-based screen for genetic interactors. As a result of this screen, a second C. elegans WASP family protein, wve-1, was identified that is most homologous to SCAR/WAVE proteins. Animals with impaired wve-1 function display defects in gastrulation, fail to undergo proper morphogenesis, and exhibit defects in neuronal cell migrations and axon outgrowth. Reducing wve-1 levels in either unc-34/ena or wsp-1 mutant backgrounds also leads to a significant enhancement of the gastrulation and morphogenesis defects. Thus, unc-34/ena, wsp-1, and wve-1 play overlapping roles during embryogenesis and unc-34/ena and wsp-1 play overlapping roles in neuronal cell migration. These observations show that WASP and Ena/VASP proteins can compensate for each other in vivo and provide the first demonstration of a role for Ena/VASP proteins in gastrulation and morphogenesis. In addition, these results provide the first example of an in vivo role for WASP family proteins in neuronal cell migrations and cytokinesis in metazoans (Withee, 2004).
Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells and Drosophila epithelia, but they have only been extensively studied at the leading edges of migratory fibroblasts, where they modulate the protrusion of the leading edge. They act by regulating actin-filament geometry, antagonizing the effects of actin-capping protein. Embryos lacking the C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, the data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function (Sheffield, 2007).
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).
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).
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency disease associated with eczema, hemorrhagic episodes, and recurrent severe infections. The N-terminus of the cytoplasmic WAS protein (WASP) has similarity to WH1 domains, which recognize proline-rich sequences and direct protein localization and formation of multicomponent assemblies. About one-half of the WAS-causing mutations affect the WH1 domain, but this forms only about one-fifth of the length of the protein. To understand the structural and functional effects of WAS-causing mutations within the WH1 domain, the three-dimensional model of the WASP WH1 domain was constructed based on the crystal structures of the Mena and Ev1 EVH1 (WH1) domains. Based on the model, the protein structural effects of the mutations were evaluated and putative ligand-binding regions identified. Mutations in the WASP WH1 domain were found to influence both the function and structure of the WASP. The amino acid substitutions cause general and local structural changes because of steric clashes and changes to the positions of adjacent strands and the fold of the protein. Some mutations alter the electrostatics and interactions with partners and other domains of WASP (Rong, 2000).
All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which these proteins activate Arp2/3 complex-induced actin polymerization. In this study, the Arp2/3 complex-mediated actin polymerization activity was characterized of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulate the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, has a more potent activity than other VCA proteins. The chimeric protein experiments reveal that the V motif is more important to the activation potency than the CA region; two V motifs are required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein was overexpressed, one in which the VCA region of N-WASP is replaced with WAVE1 VCA, microspike formation is suppressed. Interestingly, when the N-WASP VCA region is replaced with WAVE1 VCA, this chimeric protein having two V motifs can induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation (Yamaguchi, 2000).
The protein N-WASP regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate [PIP(2)] yields potent polymerization. Regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive 'closed' conformation. In this state, both the Cdc42- and PIP(2)-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals (Prehoda, 2000).
Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic [VCA] or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. A focus was placed on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduces actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP leads to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP is much smaller than that of wild-type N-WASP. Partial deletion of acidic residues does not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion (Suetsugu, 2001a).
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 was found to associate 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, 2001b).
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).
Cdc42 is a small GTPase of the Rho family which regulates the formation of actin filaments to generate filopodia. Although there are several proteins such as PAK, ACK and WASP (Wiskott-Aldrich syndrome protein) that bind Cdc42 directly, none of these can account for the filopodium formation induced by Cdc42. Before it can induce filopodium formation, Cdc42 must bind a WASP-related protein, N-WASP, that is richest in neural tissues but is expressed ubiquitously. N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP, which is expressed in hematopoietic cells, does not, despite the structural similarities between WASP and N-WASP. In a cell-free system, addition of active Cdc42 significantly stimulates the actin-depolymerizing activity of N-WASP, creating free barbed ends from which actin polymerization can then take place. This activation seems to be caused by exposure of N-WASP's actin-depolymerizing region induced by Cdc42 binding (Miki, 1998).
Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. A mechanism is described that links one Rho family member, Cdc42, to actin polymerization. N-WASP, a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization (Rohatgi, 1999).
Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). This activation reaction has been dissected and it has been found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2) terminus and the COOH terminus must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed the delineation of a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP, and second, as an indirect activator of Cdc42 (Rohatgi, 2000).
WASP was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. A detailed analysis on the domain-function relationship of WASP has been carried out, with the following results: (1) the C-terminal verprolin-cofilin-acidic domain is essential for the regulation of actin cytoskeleton; (2) the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clusters especially around the nucleus as wild type WASP without inducing actin clustering. (3) Quite unexpectedly, a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site (Kato, 1999).
Native WASp was purified from bovine thymus and its ability to stimulate actin nucleation by Arp2/3 complex was studied. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate [PIP(2)] micelles allow WASp to activate actin nucleation by Arp2/3 complex, and this is further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrate around lipid micelles and vesicles, providing that Cdc42 is GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction (Higgs, 2000).
The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. The autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents, is reported. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context (Kim, 2000).
An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway is described. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome (Ho, 2004).
Sequence analysis reveals that Toca-1 is structurally related to proteins of the PCH (pombe Cdc15 h) family; these proteins have been implicated recently in a wide variety of actin-dependent processes, including cytokinesis, membrane trafficking, and cellular morphogenesis. This protein family is conserved throughout eukaryotic evolution and includes human formin binding protein 17 (FBP17), human Cdc42-interacting protein 4 (CIP4), human syndapins, D. melanogaster Cip4, C. elegans CE27939, S. cerevisiae Bzz1p, and S. pombe Cdc15. Members of this protein family are defined by a common domain structure that includes a FER/CIP4 homology (FCH) domain at the N terminus and one or two Src homology 3 (SH3) domains at the C terminus. The FCH domain is found in a large number of proteins involved in signal transduction, but its function is largely unknown. In addition, many PCH proteins are also predicted to contain coiled-coil domains. In the case of Toca-1, FBP17, CIP4, and D. melanogaster Cip4, one of these coiled-coil regions has homology to a domain called HR1 (protein kinase C-related kinase homology region 1), which was originally identified as a Rho-interactive module in several RhoA binding proteins. The functional conservation of Toca-1 across species is highlighted by the finding that Toca-1 homologs from X. tropicalis and D. melanogaster can complement the MCAP2B activity in an in vitro assay system (Ho, 2004).
The following view of the Cdc42 signaling pathway is proposed. Formation of PIP2 on membranes (such as a vesicle surface) leads to the recruitment and activation of Cdc42. Prenylated Cdc42 inserts into the membrane and forms high avidity sites that recruit Toca-1 and the N-WASP-WIP complex. Activation of N-WASP then could proceed through one of two paths: both Cdc42 and Toca-1 could cooperate to activate the N-WASP-WIP complex, or Toca-1 could function indirectly by relieving the inhibition of N-WASP by WIP. Toca-1 is ideally positioned to be an important regulatory node for the Cdc42 pathway. The function of Toca-1 suggests a specific mechanism by which PCH family proteins can influence actin nucleation in a wide variety of cellular processes such as vesicle motility and cytokinesis. Important future questions include the precise biochemical mechanism by which the N-WASP-WIP complex is activated by Toca-1 and Cdc42, as well as investigation into the regulation of Toca-1 itself by other signals (Ho, 2004).
Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system, a proline-rich WASP-interacting protein (WIP; see Drosophila Verprolin 1) has been identified that coimmunoprecipitates with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and includes actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increases polymerized actin content and induces the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function (Ramesh, 1997).
Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. Recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASP), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASP-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. The presence of profilin in Nck precipitates has been demonstrated suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin (Anton, 1998).
WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologs, the function of WIP was analyzed in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome (Vadula, 1999).
Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signaling cascades to actin polymerization. The amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP, is responsible for N-WASP recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella, WIP is recruited by N-WASP. These observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signaling pathways to recruit the N-WASP-WIP complex. It is proposed that the N-WASP-WIP complex has a pivotal function in integrating signaling cascades that lead to actin polymerization (Moreau, 2000).
Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant [Cdc42(V12)] and by N-WASP. These results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella (Martinez-Quiles, 2001).
A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, the exact nature of its binding site has not been known. The N-WASP WH1 binding motif in WIP has now been identified and characterized in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif is conserved in WIP, CR16, WICH, and yeast verprolin (Zett, 2002).
F-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. Adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation (Sasahara, 2003).
The importance of the SH3 domain of Src family kinase Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, mass spectrometry was used to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). Focus was also placed on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, it was confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. Hck and ELMO1 interact in intact cells and ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. These results suggest that these proteins may be novel activators/effectors of Hck (Scott, 2002).
The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP), the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs) was examined. The results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. It was also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, these results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment (Chou, 2006).
SH2/SH3 adaptor proteins play a critical role in tyrosine kinase signaling pathways, regulating essential cell functions by increasing the local concentration or altering the subcellular localization of downstream effectors. The SH2 domain of the Nck adaptor can bind tyrosine-phosphorylated proteins, while its SH3 domains can modulate actin polymerization by interacting with effectors such as WASp/Scar family proteins. Although several studies have implicated Nck in regulating actin polymerization, its role in living cells is not well understood. An antibody-based system was used to experimentally modulate the local concentration of Nck SH3 domains on the plasma membrane of living cells. Clustering of fusion proteins containing all three Nck SH3 domains induced localized polymerization of actin, including the formation of actin tails and spots, accompanied by general cytoskeletal rearrangements. All three Nck SH3 domains were required, as clustering of individual SH3 domains or a combination of the two N-terminal Nck SH3 domains failed to promote significant local polymerization of actin in vivo. Changes in actin dynamics induced by Nck SH3 domain clustering required the recruitment of N-WASp, but not WAVE1, and were unaffected by downregulation of Cdc42. Therefore, high local concentrations of Nck SH3 domains are sufficient to stimulate localized, Cdc42-independent actin polymerization in living cells. This study provides strong evidence of a pivotal role for Nck in directly coupling ligand-induced tyrosine phosphorylation at the plasma membrane to localized changes in organization of the actin cytoskeleton through a signaling pathway that requires N-WASp (Rivera, 2004).
WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton (Wu, 1998).
Bruton's tyrosine kinase (Btk: see Btk family kinase at 29A) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. One of the major phosphoproteins associated with Btk in the preB cell line NALM6 has been identified as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. WASP is transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed an evaluation of the catalytic specificity of Btk, the exact nature of which is still unknown (Baba, 1999).
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).
Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. The SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp; Grb2 stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to the active N-WASp monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway (Carlier, 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 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 SH2–SH3 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 machinery—the 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).
To identify potential trans-inhibitory proteins for WAVE1 activation, proteins were sought that interact with WAVE1. All of the detectable WAVE1 protein in soluble bovine brain extracts elutes in size-exclusion chromatography as a complex with an Mr of 500K. This complex was purified to about 90% purity as judged by SDS polyacrylamide gel electrophoresis and Coomassie blue staining. Three proteins co-fractionated and were subsequently co-immunoprecipitated with WAVE1 (Eden, 2002).
The proteins were identified unambiguously by mass spectrometry as the bovine orthologues of the following human proteins: WAVE1; PIR121; Nap125, and HSPC300, which encodes a protein with an Mr of 9K. To test whether WAVE1 activity is inhibited in trans by the other proteins in the complex, the ability of the complex to activate Arp2/3 was tested. Whereas recombinant WAVE1 activates the Arp2/3 complex, the native WAVE1 complex does not. Rac1 is fully effective in relieving the inhibition of WAVE1 in the complex (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 novel adaptor protein has been identified that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif; this protein has been termed WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It binds Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhances N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induces marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induces microspike formation when coexpressed with WISH. The contribution of WISH to a rapid actin polymerization induced by brain extract in vitro was examined. Arp2/3 complex is essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increases actin polymerization as does Cdc42. However, WISH unexpectedly can activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation (Fukuoka, 2001).
Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. A member of the family, designated as RhoT, has been identified that is closely related to Tc10. Tc10 is highly expressed in muscular tissues and brain and is induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. RhoT is predominantly expressed in heart and uterus and is induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generates actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produces much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 does not do this by itself. Tc10 and RhoT as well as Cdc42 bind to the N-terminal CRIB-motif-containing portion of N-WASP and activates N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT is prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, is prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake (Abe, 2003).
Several microbial pathogens including enteropathogenic E. coli (EPEC) exploit mammalian tyrosine-kinase signaling cascades to recruit Nck adaptor proteins and activate N-WASP-Arp2/3-mediated actin assembly. To promote localized actin 'pedestal formation', EPEC translocates the bacterial effector protein Tir into the plasma membrane, where it is tyrosine-phosphorylated and binds Nck. Enterohemorrhagic E. coli (EHEC) also generates Tir-dependent pedestals, but in the absence of phosphotyrosines and Nck recruitment. To identify additional EHEC effectors that stimulate phosphotyrosine-independent actin assembly, EHEC mutants containing specific deletions in putative pathogenicity-islands (class of genomic islands, which are acquired by horizontal gene transfer) were systematically generated. Among 0.33 Mb of deleted sequences, only one ORF was critical for pedestal formation. It lies within prophage-U, and encodes a protein similar to the known effector EspF. This proline-rich protein, EspFU, is the only EHEC effector of actin assembly absent from EPEC. Whereas EHEC Tir cannot efficiently recruit N-WASP or trigger actin polymerization, EspFU associates with Tir, binds N-WASP, and potently stimulates Nck-independent actin assembly (Campellone, 2004).
N-WASP regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. Heat shock protein 90 (HSP90) regulates N-WASP-induced actin polymerization in cooperation with phosphorylation of N-WASP. HSP90 binds directly to N-WASP, but binding alone does not affect the rate of N-WASP/Arp2/3 complex-induced in vitro actin polymerization. An Src family tyrosine kinase, v-Src, phosphorylates and activates N-WASP. HSP90 increases the phosphorylation of N-WASP by v-Src, leading to enhanced N-WASP-dependent actin polymerization. In addition, HSP90 protects phosphorylated and activated N-WASP from proteasome-dependent degradation, resulting in amplification of N-WASP-dependent actin polymerization. Association between HSP90 and N-WASP is increased in proportion to activation of N-WASP by phosphorylation. HSP90 is colocalized and associated with active N-WASP at podosomes in 3Y1/v-Src cells and at growing neurites in PC12 cells, whose actin structures are clearly inhibited by blocking the binding of HSP90 to N-WASP. These findings suggest that HSP90 induces efficient activation of N-WASP downstream of phosphorylation signal by Src family kinases and is critical for N-WASP-dependent podosome formation and neurite extension (Par, 2005).
Cells can retain information about previous stimuli to produce distinct future responses. The biochemical mechanisms by which this is achieved are not well understood. The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho-family GTPase Cdc42, whose activation leads to stimulation of the actin nucleating assembly, Arp2/3 complex. Efficient phosphorylation and dephosphorylation of WASP at Y291 are both contingent on binding to activated Cdc42. Y291 phosphorylation increases the basal activity of WASP toward Arp2/3 complex and enables WASP activation by new stimuli, namely, SH2 domains of Src-family kinases. The requirement for contingency in both phosphorylation and dephosphorylation enables long-term storage of information by WASP following decay of GTPase signals. This biochemical circuitry allows WASP to respond to the levels and timing of GTPase and kinase signals. It provides mechanisms to specifically achieve transient or persistent actin remodeling, as well as long-lasting potentiation of actin-based responses to kinases (Torres, 2003).
Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Two phosphorylation sites in the VCA domain of WASP have been identified at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases 7-fold the affinity of the VCA domain for the Arp2/3 complex and is required for efficient in vitro actin polymerization by the full-length WASP molecule. It is proposed that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP (Cory, 2003).
The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeleton organization. Immunodeficiency is mainly due to T-cell progressive malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with atypical morphology. The possibility has been investigated that the CD3-restricted defect and some of the cytoskeletal defects of WAS T cells are linked. For this purpose, a number of previously described allospecific WAS T-cell lines has been immortalized by means of infection with Herpesvirus Saimiri. The resulting cells preserve the surface, molecular, and functional phenotypes of their parental lines, including a negligible WASP mRNA expression as well as the CD3-restricted defect and cytoskeleton abnormalities. Results show that, in CD3-stimulated WAS T cells, the pattern of temporal changes in cell shape and F-actin distribution is substantially different from that of control cells. Furthermore, polymerization of actin, a critical step in the CD3-mediated cytoskeleton reorganization, does not occur in WAS T-cell lines in response to OKT3 stimulation. In conclusion, the data link both CD3 and cytoskeletal defects in WAS T cells, strongly suggesting that cytoskeleton abnormalities are an underlying cause for WAS immunodeficiency (Gallego, 1997).
The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, the WASP gene was disrupted in mice by gene-targeted mutation. WASP-deficient mice show apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice have decreased peripheral blood lymphocyte and platelet numbers and develop chronic colitis. Moreover, purified WASP-deficient T cells show markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells show normal responses to anti-Ig stimulation (Snapper, 1998).
The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, WASp roles in lymphocyte development and activation have been examined using mice carrying a WAS null allele. Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality is associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibit impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferate normally in response to phorbol ester/ionomycin. This defect in TCR signaling is associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggers apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appears normal in TCR-stimulated WAS minus cells, TCR-evoked increases in intracellular calcium concentration are decreased in WASp-deficient cells relative to wild-type cells. WAS minus lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS minus neutrophils exhibit reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors (Zhang, 1999).
Cortactin (see Drosophila Cortactin) is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. Cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. Cortactin is shown to activate Arp2/3 complex, based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibits the debranching of filament networks. When cortactin is added in combination with the active VCA fragment of N-WASp, they synergistically enhance Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin are both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells (Weaver, 2001).
Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. How cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex has been investigated. Structural studies show that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrate selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit are mutually exclusive; however, cortactin NTA does not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor does it inhibit activation of Arp2/3 complex by VCA. A saturating concentration of cortactin NTA modestly lowers the binding affinity of VCA for Arp2/3; the results of this experiment provide further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolishes binding of cortactin to Arp2/3 complex. It is concluded that under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40 (Weaver, 2002).
The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrate that N-WASP is located at the tip of the bacterium, whereas cortactin accumulates in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquires 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex is reduced in the presence of both cortactin and actin. Moreover, VCA reduces its affinity for Arp2/3 complex at branching sites that are stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex (Uruno, 2003).
Actin polymerization is required for many types of cell motility, such as chemotaxis, nerve growth cone movement, cell spreading, and platelet activation. In the lamellipodia that push forward the leading edge of motile cells, polymerizing filaments form a meshwork consisting of 'Y branches' with the pointed end of one filament attached to the side of another filament. This meshwork presumably may provide a rigid body against which polymerization can drive membrane protrusion. A major unanswered question is how cells integrate signals coming through a variety of pathways to control when and where actin polymerizes. The filaments grow from a huge pool of unpolymerized actin maintained by monomer-binding proteins at a concentration approximately 1000-fold higher than required for spontaneous polymerization of actin. The monomer-binding protein profilin biases the direction of filament elongation, allowing growth at the fast growing barbed end but not the slow growing pointed end. In cells capping proteins block the barbed end of most filaments, so some mechanism is required to start new filaments (Higgs, 1999a and references therein: Full text).
Cells might trigger actin polymerization in three ways: (1) de novo nucleation of filaments from monomeric actin; (2) severing existing filaments to create uncapped barbed ends, and (3) uncapping existing barbed ends. There is evidence for each of these mechanisms in various cellular processes, but new filaments are often created during cell motility, placing emphasis on mechanisms 1 and 2. Although activation of de novo nucleation by cell stimulation has long been an attractive model, no barbed end nucleating factors were known until it was discovered that Arp2/3 complex promotes actin nucleation, creating filaments that grow at their barbed ends. Because nucleation is rate-limiting in actin polymerization and strongly suppressed by monomer-binding proteins, Arp2/3 complex may be a key mediator of actin polymerization in cells. Arp2/3 complex also cross-links actin filaments end-to-side, indistinguishable from the Y branches at the leading edge (Higgs, 1999a and references therein).
Based on these biochemical activities, the dendritic nucleation model, whereby Arp2/3 complex both creates new filaments and cross-links them into a branching meshwork, was proposed (Mullins, 1998). Cellular observations support this model. Arp2/3 complex is concentrated at the leading edge of motile cells, specifically at the junctions of the Y branches. It exists in all eukaryotes examined, and ablation of Arp2/3 complex subunits in Saccharomyces cerevisiae and Schizosaccharomyces pombe is lethal or severely debilitating (Higgs, 1999a and references therein).
The next breakthrough was the discovery that ActA, a cell surface protein from the pathogenic bacterium, Listeria monocytogenes, stimulates Arp2/3 complex to nucleate actin in vitro. Listeria uses force generated by actin polymerization to propel itself around the cytoplasm of eukaryotic cells. ActA is the only bacterial protein required to induce polymerization, but ActA cannot stimulate actin filament formation by itself. This work suggested that cellular factors might activate Arp2/3 complex to nucleate actin. WASp/Scar proteins have been identified as the first example of such factors. These proteins also interact with a variety of cell signaling molecules known to influence cytoskeletal dynamics, thus connecting surface receptor stimulation and actin polymerization. Analysis of Wiskott-Aldrich syndrome protein (WASp) and its neural homolog N-WASP reveal a binding site for Rho family GTPases and other domains that affect actin assembly in cells. Study of GTPS1-stimulated actin polymerization in extracts of vertebrate cells, Dictyostelium, and Acanthamoeba, demonstrate that the Rho family GTPase Cdc42 mediates the effect of GTP and that Arp2/3 complex is required. Similar experiments with extracted yeast suggested that Bee1p (a WASp homolog) and Arp2/3 complex are required for actin patch assembly (Higgs, 1999a and references therein).
The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. The effect of WASp WA on actin polymerization was characterized in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. It is proposed that filament side binding by the Arp2/3 complex enhances its activation by WASp WA (Higgs, 1999b).
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).
The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits, has been shown to physically interact with WASP. Attempts have been made to determine whether WASP is a cellular activator of the Arp2/3 complex and it has been found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerize actin, form actin tails, and exhibit actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes are non-motile and exhibit only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization (Yarar, 1999).
Using fluorescence anisotropy assays it has been shown that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol-1 drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex (Marchand, 2001).
Phosphatidylinositol 4,5-bisphosphate [PIP(2)] has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains (called 'rafts'), in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promotes actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induces comets even in the absence of PIP5KI overexpression. PV increases PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhances PV-induced comet formation, and these stimuli together potentiate the PIP5KI effect. The vesicles at the heads of comets are enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduces comets, establishing that rafts promote comet formation. It is concluded that sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking (Rozelle, 2000).
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).
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 have been sought and the LAS17/BEE1 gene (encoding the yeast homolog of the human Wiskott-Aldrich Syndrome protein, WASp) has been 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). However, LAS17 is unable to suppress a full deletion of END5 (end5 delta), 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).
The spatial and temporal control of actin assembly was examined in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appear on a subset of cytoplasmic vesicles that are enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo is stimulated by the PKC activator phorbol myristate acetate (PMA), and this process can be reconstituted in a cell-free system. This system was used to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. N-WASP is recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulate the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails have the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleate actin assembly and move in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton (Taunton, 2000).
Syndapins are potential links between the cortical actin cytoskeleton and endocytosis because this family of dynamin-associated proteins can also interact with the Arp2/3 complex activator N-WASP. Evidence is provided for involvement of N-WASP interactions in receptor-mediated endocytosis. The observed dominant-negative effects of N-WASP are dependent exclusively on the proline-rich domain, the binding interface of syndapins. These results therefore suggest that syndapins integrate N-WASP functions in endocytosis. Both proteins co-localize in neuronal cells. Consistent with a crucial role for syndapins in endocytic uptake, co-overexpression of syndapins rescue the endocytosis block caused by N-WASP. An in vivo reconstitution of the syndapin-N-WASP interaction at cellular membranes triggers local actin polymerization. Depletion of endogenous N-WASP by sequestering it to mitochondria or by introducing anti-N-WASP antibodies impairs endocytosis. These data suggest that syndapins may act as important coordinators of N-WASP and dynamin functions during the different steps of receptor-mediated endocytosis and that local actin polymerization induced by syndapin-N-WASP interactions may be a mechanism supporting clathrin-coated vesicle detachment and movement away from the plasma membrane (Kessels, 2002).
Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, the propulsive motility of lipid-coated glass beads were reconstituted using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation (Co, 2007).
This model for actin/membrane attachment extends the dendritic nucleation model by considering the fate of barbed ends at the membrane surface, which cycle through elongation, attachment, and release steps until filament growth and membrane attachment are ultimately terminated by capping protein. Reversible capture of a membrane-proximal barbed end by WH2 or WH2/actin establishes the molecular link between the dendritic network and the membrane. It is not clear whether barbed end capture occurs primarily via free WH2 domains or WH2/actin monomers. Based on the preference of WASP WH2 for ATP-actin (5-fold higher binding affinity relative to ADP-actin), it is possible that barbed end binding by free WH2 domains is sensitive to the nucleotide state of the terminal actin subunit. This effect may be exacerbated by the R438A and K444Q mutations, leading to catastrophic detachment of the actin network as the concentration of ATP-actin declines in the in vitro motility assay (Co, 2007).
A barbed end attachment model may seem counterintuitive given the high dissociation rate of WH2 from barbed ends in solution (2500 s−1 if similar to the profilin-barbed end dissociation rate. This dissociation rate has not been measured, but it must be fast because the rate of barbed end polymerization is only slightly decreased by saturating concentrations of WH2-containing peptides. Nevertheless, in the context of a dendritic actin network attached to a membrane surface, rebinding of a free barbed end to a WH2 domain or WH2/actin complex is likely to be rapid. First, the surface density (or 'local concentration') of N-WASP on moving lipid-coated beads is high, estimated from fluorescence measurements to be ~50,000 molecules per μm2, with a maximum distance between N-WASP molecules of ~5 nm. Second, actin filaments in the attachment zone are crosslinked in a dendritic network, and thus their movement is highly constrained. Ligand rebinding effects between membrane-associated N-WASP and membrane-proximal barbed ends most likely drive the attachment interaction, despite the low affinity of WH2 domains for filament barbed ends in solution (Co, 2007).
The model resembles filament end-tracking models for Listeria motility in that barbed ends, rather than filament sides or nascent branches, mediate attachment to the membrane. A key difference, however, is that WH2 domains need not act as processive end-tracking proteins dependent on ATP hydrolysis by actin (although WH2/barbed end affinity may be modulated by ATP hydrolysis). Individual barbed ends can dissociate from the membrane because attachment is mediated by a multivalent array of hundreds or thousands of crosslinked filaments. Disruption of WH2/barbed end attachment interactions may be rate-limiting for vesicle movement. The increased speed of lipid-coated beads driven by R438A N-WASP before they detach is consistent with this idea (Co, 2007).
In the context of a rocketing vesicle, WH2 domains are necessary and sufficient to localize N-WASP fragments to membrane sites enriched in nascent barbed ends. Although this study focused primarily on attachment of the actin network to moving vesicles, the results imply that barbed ends can reciprocally capture a pool of diffusing N-WASP molecules at membrane sites of dendritic actin nucleation. This suggests a mechanism for localized signal amplification, which is likely defective in WH2 mutants. Reversible capture of diffusing N-WASP molecules by nascent barbed ends increases the frequency of local nucleation and branching events, resulting in the expansion of the dendritic network. The reduced number of large podosome arrays assembled by RA/RA N-WASP is consistent with this proposal. Although the mechanism of podosome assembly is largely unknown, it is speculated that N-WASP capture by nascent barbed ends is a critical determinant of podosome expansion, fusion and/or fragmentation. Similarly, WH2-mediated capture of WAVE/SCAR proteins by nascent barbed ends may locally amplify actin nucleation signals at the leading edge of moving cells (Co, 2007).
Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. The possible involvement of N-WASP in the neurite extension process has been examined. Since verprolin, cofilin homology and acidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, a mutant (Deltacof) was prepared lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP are mutated in some Wiskott-Aldrich syndrome patients. Expression of Deltacof N-WASP suppresses neurite extension of PC12 cells. In support of this, the VCA region of Deltacof cannot activate Arp2/3 complex enough, when compared with wild-type VCA. Furthermore, H208D mutant, which is unable to bind Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Deltacof double mutant has no significant dominant negative effect. The expression of the Deltacof mutant also severely inhibits the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization (Banzai, 2000).
Neurite extension is a key process for constructing neuronal circuits during development and remodeling of the nervous system. Src family tyrosine kinases and proteasome degradation signals synergistically regulate N-WASP in neurite extension. Src family kinases activate N-WASP through tyrosine phosphorylation, which induces Arp2/3 complex-mediated actin polymerization. Tyrosine phosphorylation of N-WASP also initiates its degradation through ubiquitination. When neurite growth is stimulated in culture, degradation of N-WASP is markedly inhibited, leading to accumulation of the phosphorylated N-WASP. However, under culture conditions that inhibit neurite extension, but favor proliferation, the phosphorylated N-WASP is degraded rapidly. Collectively, neurite extension is regulated by the balance of N-WASP phosphorylation (activation) and degradation (inactivation), which are induced by tyrosine phosphorylation (Suetsugu, 2002).
Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. This study shows that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. These observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation (Jin, 2011).
Cell migration is crucial for many biological and pathological processes such as chemotaxis of immune cells, fibroblast migration during wound healing, and tumor cell invasion and metastasis. Cells migrate forward by extending membrane protrusions. The formation of these protrusions is driven by assembly of actin filaments at the leading edge. Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitous member of the WASP family, induces actin polymerization by activating Arp2/3 complex and is thought to regulate the formation of membrane protrusions. However, it is totally unclear how N-WASP activity is spatially and temporally regulated inside migrating cells. To detect and image sites of N-WASP activity during cell motility and invasion in carcinoma cells, an N-WASP fluorescence resonance energy transfer (FRET) biosensor was designed that distinguishes between the active and inactive conformations and mimics the function of endogenous N-WASP. The data show that N-WASP is involved in lamellipodia extension, where it is activated at the leading edge, as well as in invadopodia formation of invasive carcinoma cells, where it is activated at the base. This is the first time that the activity of full-length N-WASP has been visualized in vivo, and this has lead to new insights for N-WASP function (Lorenz, 2004).
These data suggest that N-WASP plays a role in initiation of invadopodia where N-WASP activity and the activity of its downstream effector, the Arp2/3 complex, is constrained to the base. This supports findings where N-WASP has been implicated in the formation of filopodia, similarly shaped tubular protrusions. While it is possible that Arp2/3 complex can be activated throughout the invadopod resulting from some other activators, these results support the model proposed for filopodium protrusion by convergent elongation of filaments originating from Arp2/3 complex constrained to the base of filopodia. The biosensor results are the first to localize N-WASP activity in the basal region of tubular protrusions, suggesting that Arp2/3 activity is localized only to the base and that filament elongation of the protrusion does not involve nucleation of filaments within the protrusion itself. Thus, the first step to initiate invadopodia would be to generate localized dendritic nucleation at the ventral surface by N-WASP activation of the Arp2/3 complex. The convergent elongation of newly nucleated filaments supported by Ena/Vasp family proteins or Diaphanous-related formin 3 (Drf3) as suggested for filopodia would push the plasma membrane forward into the ECM. If the force produced by elongation of existing filaments is sufficient to push out the membrane, the N-WASP activity could decrease without affecting further elongation. At this point invadopodium growth would rely upon elongation rather than nucleation. The fact that N-WASP activity is also seen throughout the leading edge of lamellipodia suggests that the decision to convert the geometry of pushing force of the elongating filaments of the dendritic network from a lamellipodial to a filopodial shape occurs at a step independent of the activation of N-WASP alone. Invadopodium formation greatly enhances the invasion of carcinoma cells. N-WASP has been shown to potentiate invasion, and the biosensor results suggest a mechanism for the involvement of N-WASP in invadopodium-mediated invasion. Together, these observations emphasize N-WASP as a target for therapies directed at inhibiting the invasion of carcinomas (Lorenz, 2004).
Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation (LTF) and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). cDNAs encoding Aplysia Rho, Rac, and Cdc42 have been isolated and it has been found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities (Udo, 2005).
Actin is enriched in both the pre- and postsynaptic compartments of most neurons. Although the activity-dependent modulation of actin dynamics at postsynaptic spines has been well documented, the extent and role of actin regulation in presynaptic terminals is not well understood. During development, reorganization of actin in growth cones has been shown to play an important role in axonal pathfinding. However, in mature neurons, it has been suggested that the presynaptic actin network functions more as an intracellular scaffold rather than as a propulsive force, that contributes directly to the type of frank structural remodeling reported for postsynaptic dendritic spines. In Aplysia, repeated applications of 5-HT (which lead to LTF) induce a slower and more persistent alteration in the dynamics of the presynaptic actin network, leading to the growth of new sensory neuron synapses. The data indicate that Cdc42 is one of the key molecular regulators of this learning-related modulation of presynaptic actin organization (Udo, 2005).
The family of Rho GTPases has been shown to play an important role in neuronal development, for example, the establishment of polarity, axon guidance, and the growth and maintenance of dendritic spines. ApCdc42 is involved in long-term synaptic plasticity in Aplysia, suggesting that Cdc42 may also have a role in learning and memory storage in the mature nervous system. It was surprising that Rac, which is functionally related to Cdc42 and known to regulate spine morphology and memory consolidation in mice, does not significantly contribute to LTF and the associated structural changes. By contrast, Rho tends to oppose the effects of Cdc42 on long-term synaptic plasticity, which is consistent with the ways in which these two proteins regulate actin dynamics (Udo, 2005).
In Aplysia, the activation of ApCdc42 in sensory neurons leads to the outgrowth of filopodia from presynaptic varicosities. Interestingly, 5-HT stimulation by itself naturally induces filopodia; this induction is dependent on the activation of ApCdc42. Filopodia have been proposed to be morphological precursors of dendritic spines in the mammalian central nervous system, and this process may be regulated by neuronal activity. The 5-HT-induced activation of Cdc42 in Aplysia triggers not only the formation of filopodia but also the molecular maturation of neurotransmitter release sites. A major synaptic vesicle protein, synaptophysin, accumulates at the tips of 5-HT-induced filopodia, some of which then give rise to new varicosities. These observations support the following ideas: (1) filopodia represent one type of morphological precursor for the growth of new presynaptic varicosities during learning-related synaptic plasticity, and (2) the formation of filopodia and initial assembly of the presynaptic compartment can be induced by the activation of Cdc42 (Udo, 2005).
5-HT is a modulatory neurotransmitter released from facilitating interneurons that make synaptic contacts onto sensory neurons. Most of the 5-HT receptors are known to be G protein coupled, and G proteins such as Gα12 and Gα13 have been shown to link to Rho. However, the signaling pathways for Rac/Cdc42 appear to be different from those for Rho, and the molecular cascade between G protein-coupled receptors and Cdc42 is not well understood. The current results suggest that 5-HT activates ApCdc42 through pathways involving PLC and PI3 kinase. PLC produces two independent second messengers (diacylglycerol and inositol triphosphate) to initiate a variety of cellular functions. It has also been shown that some isoforms of PLC are able to directly bind to Rac/Cdc42 and to enhance its activity. Since an increase in the internal concentration of calcium is known to stimulate actin polymerization, PLC may send multiple signals to regulate actin-related structures. Like PLC, PI3 kinase also plays a key role in regulating cell growth and survival. PI3 kinase is thought to interact directly with Cdc42 and stimulate its activity. Although PI3 kinase is usually activated by receptor tyrosine kinases such as Trk receptors, recent evidence suggests that some PI3 kinase isoforms (such as type 1B) can be upregulated by their interaction with Gβγ. Thus, it is possible that PI3 kinase may be activated by 5-HT receptors as well as Trk-like receptors and that PLC and PI3K may send signals to ApCdc42 via independent pathways (Udo, 2005).
The synapse-specific nature of the 5-HT-induced activation of ApCdc42 suggests the possibility of a coordinated interaction between the presynaptic sensory neuron and the postsynaptic motor neuron. This could be mediated by a variety of different cell adhesion or trans-synaptic signaling molecules. For example, ephexin and IQGAP, which bind to the EphA receptor and cadherin, respectively, are known to modulate the activity of Cdc42. The identification of such molecules in Aplysia should provide additional molecular insights into the upstream signaling pathways that activate ApCdc42 (Udo, 2005).
The differential activation of ApCdc42 at a spatially distinct subset of presynaptic sensory neuron varicosities is consistent with previous studies, which have shown that the initial segment and cell body of the postsynaptic motor neuron is a preferred site for new sensory neuron varicosity formation induced by 5-HT. It is proposed that 5-HT receptors coupled to G proteins (Gαq and Gβγ) activate the PLC and PI3 kinase pathways, which in turn upregulate ApCdc42 at specific presynaptic varicosities. The selective activation of ApCdc42 leads to the formation of actin-based filopodia by activating downstream effector proteins such as N-WASP and to a lesser extent PAK. Presynaptic components, including synaptic vesicles, appear to be recruited to the tips of specific filopodia, possibly through actin-myosin-dependent transport, which then become transformed into new functional sensory neuron varicosities. Thus, 5-HT-induced regulation of the Cdc42 signaling pathways and the consequent reorganization of the presynaptic actin network appear to be a part of the initial molecular cascade required for the growth of new sensory neuron synapses associated with long-term memory (Udo, 2005).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.