Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

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. Here we examined the possible involvement of N-WASP in the neurite extension process. 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, we prepared a mutant (Deltacof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Deltacof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Deltacof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to 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. Finally, the expression of the Deltacof mutant also severely inhibited 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.

The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.

Cofilin1 (CFL1) is an actin-modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott-Aldrich syndrome protein (N-WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott-Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N-WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N-WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N-WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N-WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over-expressed in ESCC tissue (P < 0.05), while N-WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N-WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC.

N-WASP plays a critical role in fibroblast adhesion and spreading

Crucial relationship of Neural Wiskott Aldrich syndrome protein(N-WASP) and Lysyl oxidase-like 2(LOXL2) in the promotion of pancreatic cancer metastasis

Author SummaryMechanisms to shut down B-cell activation are necessary to ensure termination of an immune response when an infection has been cleared. When this negative regulation goes wrong, it can also lead to autoimmunity. To understand how this inhibitory process is regulated, here we utilized knockout mice containing B cells that are deficient for proteins potentially involved in their negative regulation. We focus on Wiskott–Aldrich syndrome protein (WASP), a key cytoskeletal regulator of hematopoietic cells, and neural WASP (N-WASP), which shares 50% homology with WASP and is ubiquitously expressed. Our study shows that mouse B cells that lack N-WASP protein are activated to a greater level and for longer periods than B cells that express this protein. Furthermore, in mice where B cells do not make N-WASP, the numbers of self-reactive B cells are elevated. We went on to identify molecules that promote or inhibit N-WASP activation and to examine the cellular mechanisms by which N-WASP inhibits B-cell activation. Based on these findings we propose that N-WASP is a critical inhibitor of B-cell activation and serves to suppress self-reactive B cells.

N-WASP is highly expressed in hepatocellular carcinoma and associated with poor prognosis

Coordinated functions of the actin cytoskeleton and microtubules, which require careful control in time and space, are indispensable for the drastic alterations of neuronal morphology during neuromorphogenesis and neuronal network formation. Actin filament formation driven by the Arp2/3 complex and its activator neural Wiskott-Aldrich syndrome protein (N-WASP) is important for proper axon development. The underlying molecular mechanisms for targeting to and specific activation of N-WASP at the neuronal plasma membrane, however, have thus far remained elusive. We show that syndapin I is critical for proper neuromorphogenesis and hereby uses N-WASP as a cytoskeletal effector. Upon N-WASP binding, syndapins release N-WASP autoinhibition. Syndapins hereby cooperate with Cdc42 and phosphatidyl-inositol-(4,5)-bisphosphate. Syndapins furthermore specifically bind to phosphatidylserine-containing membranes via their extended F-BAR domain. Dissecting the syndapin functions actin nucleation and direct membrane binding in vivo, we demonstrate that both functions are physiologically relevant and required. Constitutive plasma membrane-targeting experiments in vivo indicate that specifically actin nucleation at the cell cortex is triggered by syndapins. Consistent with syndapins steering N-WASP as downstream effector for cortical actin nucleation, syndapin-induced neuronal arborization is N-WASP and Cdc42 dependent. The functions of syndapin-N-WASP complexes in neuromorphogenesis were revealed by loss-of-function studies. Knockdown of syndapin I leads to impaired axon development and especially phenocopies the aberrant axon branching observed upon N-WASP and Arp2/3 complex deficiency. In contrast, proper length control involves another N-WASP-binding protein, Abp1. Our data thus reveal that syndapin I is crucial for neuromorphogenesis and that different N-WASP activators ensure fine control of N-WASP activity and have distinct functions during neuronal network formation.

Cortical neuron migration in the developing mouse forebrain is a complex process, which contains several steps related to cytoskeleton dynamics and remodeling. Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP-WAVE family, regulates actin cytoskeleton reorganization through the binding of its VCA domain to the Arp2/3 complex. Here we report expression patterns of N-WASP gene in the mouse developing embryonic cortex (E12.5~ E18.5) and find its expression levels are decreased during embryonic development. By using in utero electroporation (IUE) method, we find that either N-WASP overexpression or knockdown impairs cortical neuron migration, and the defects of cortical neuron migration caused by N-WASP overexpression are much more severe than that by its knockdown. N-WASP protein contains four domains: WH1, GBD, polyPro, and VCA. We generated a series of dominant negative N-WASP mutants by modifying these domains. Overexpression of N-WASP mutant lacking domain polyPro, VCA, or WH1, impairs cortical neuron migration. However, overexpression of N-WASP with the H208D point mutation, which abolishes the Cdc42 binding to N-WASP, causes only a marginal defect of cortical neuron migration. Finally, overexpression of the individual domain polyPro or VCA, but not WH1, can recapitulate the defects by N-WASP overexpression. However, overexpression of WH1-GBD fragment has no apparent effect on cortical neuron migration. In conclusion, our data demonstrate that N-WASP regulates cortical neuron migration mainly through its polyPro and VCA domains.

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis

Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter

WIP is necessary for matrix invasion by breast cancer cells

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. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that 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 its active 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.