Inhibition of N-WASP affects actin-mediated cytokinesis during porcine oocyte maturation.

N-WASP is the mammalian ortholog of WASP which is an actin nucleation promoting factor and has been reported to regulate actin nucleation and polymerization for multiple cell activities. However, the expression and functions of N-WASP in porcine oocytes are still unclear. In this study, we showed that N-WASP expressed at all stages during porcine oocyte maturation, and immunofluorescence staining indicated that N-WASP mainly accumulated at the cortex in different stages of meiosis. Inhibition of N-WASP activity by Wiskostatin significantly decreased the rate of first polar body extrusion and disturbed the cell cycle progression of porcine oocytes. Further analysis indicated that cortical actin distribution was interfered by N-WASP inhibition, and this might be through its regulatory roles on the expression and localization of ARP2, a key component of actin nucleator Arp2/3 complex. Moreover, the expression of N-WASP decreased after ROCK activity inhibition, indicating a ROCK-N-WASP-ARP2/3 pathway for actin assembly in porcine oocytes. Taken together, these results suggest that N-WASP is critical for the regulation of actin filaments for cytokinesis during porcine oocyte maturation.

Entry by multiple picornaviruses is dependent on a pathway that includes TNK2, WASL, and NCK1.

Comprehensive knowledge of the host factors required for picornavirus infection would facilitate antiviral development. Here we demonstrate roles for three human genes, TNK2, WASL, and NCK1, in infection by multiple picornaviruses. CRISPR deletion of TNK2, WASL, or NCK1 reduced encephalomyocarditis virus (EMCV), coxsackievirus B3 (CVB3), poliovirus and enterovirus D68 infection, and chemical inhibitors of TNK2 and WASL decreased EMCV infection. Reduced EMCV lethality was observed in mice lacking TNK2. TNK2, WASL, and NCK1 were important in early stages of the viral lifecycle, and genetic epistasis analysis demonstrated that the three genes function in a common pathway. Mechanistically, reduced internalization of EMCV was observed in TNK2 deficient cells demonstrating that TNK2 functions in EMCV entry. Domain analysis of WASL demonstrated that its actin nucleation activity was necessary to facilitate viral infection. Together, these data support a model wherein TNK2, WASL, and NCK1 comprise a pathway important for multiple picornaviruses.

microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements.

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3'-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer.

Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria.

The bacterial pathogen Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42. These three human proteins form a complex that generates tension at apical cell junctions. Listeria relieves this tension and facilitates protrusion formation by secreting a protein called InlC. InlC interacts with a Src Homology 3 (SH3) domain in Tuba, thereby displacing N-WASP from this domain. Interaction of InlC with Tuba is needed for efficient Listeria spread in cultured human cells and infected animals. Recent structural data has elucidated the mechanistic details of InlC/Tuba interaction, revealing that InlC and N-WASP compete for partly overlapping binding surfaces in the Tuba SH3 domain. InlC binds this domain with higher affinity than N-WASP, explaining how InlC is able to disrupt Tuba/N-WASP complexes.

Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation.

Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.

N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods.

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation-promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP's arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.

N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors.

Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.

Impact of the carbazole derivative wiskostatin on mechanical stability and dynamics of motile cells.

Many essential functions in eukaryotic cells like phagocytosis, division, and motility rely on the dynamical properties of the actin cytoskeleton. A central player in the actin system is the Arp2/3 complex. Its activity is controlled by members of the WASP (Wiskott-Aldrich syndrome protein) family. In this work, we investigated the effect of the carbazole derivative wiskostatin, a recently identified N-WASP inhibitor, on actin-driven processes in motile cells of the social ameba Dictyostelium discoideum. Drug-treated cells exhibited an altered morphology and strongly reduced pseudopod formation. However, TIRF microscopy images revealed that the overall cortical network structure remained intact. We probed the mechanical stability of wiskostatin-treated cells using a microfluidic device. While the total amount of F-actin in the cells remained constant, their stiffness was strongly reduced. Furthermore, wiskostatin treatment enhanced the resistance to fluid shear stress, while spontaneous motility as well as chemotactic motion in gradients of cAMP were reduced. Our results suggest that wiskostatin affects the mechanical integrity of the actin cortex so that its rigidity is reduced and actin-driven force generation is impaired.

Claudin-5 participates in the regulation of endothelial cell motility.

A key step in metastasis is the interaction and penetration of the vascular endothelium by cancer cells. Tight Junctions (TJ) are located between the cancer epithelial cells and between the endothelial cells functioning in an adhesive manner. They represent a critical barrier which the cancer cells must overcome in order to penetrate and initiate metastasis. Claudin-5 is a protein member of the Claudin family, a group of TJ proteins expressed in both endothelial and epithelial cells. This study examined in vitro the effect of altering levels of expression of Claudin-5 in HECV cells. Insertion of Claudin-5 gene in HECV cells resulted in cells that were significantly less motile and less adhesive to matrix (P < 0.001). These cells also exhibited a significant decreased in the angiogenic potential (P < 0.001). Results also revealed a link between Claudin-5 and cell motility. Furthermore, a possible link between Claudin-5 and N-WASP, and Claudin-5 and ROCK was demonstrated when interactions between these proteins were seen in the cell line. Moreover, followed by treatment of N-WASP inhibitor (Wiskostatin) and ROCK inhibitor (Y-27632), cell motility and angiogenic potential were assessed in response to the inhibitors. Results showed that the knockdown of Claudin-5 in HECV cells masked their response to both N-WASP and ROCK inhibitors. In conclusion, this study portrays a new and interesting role for Claudin-5 in cell motility involving the N-WASP and ROCK signalling cascade which is beyond the primarily role of Claudin-5 in keeping the cell barrier tight as it was originally reported.

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway.

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization.

BACKGROUND INFORMATION: Cholesterol/sphingolipid-rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (muscle-specific kinase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. RESULTS: In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the -MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid-ordered phase by methyl-ß-cyclodextrin abolished this association. We further show that actin and the actin-nucleation factors, N-WASP (neuronal Wiscott-Aldrich syndrome protein) and Arp2/3 (actin-related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N-WASP activity perturbed agrin-elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR-enriched rafts. CONCLUSIONS: The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.

Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway.

The integrity of actin cytoskeletal organization in aqueous humor outflow pathway is thought to play a critical role in modulation of aqueous humor outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in aqueous humor outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on aqueous humor outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous humor outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of aqueous plexi and deformation of aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases aqueous humor outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of aqueous humor outflow.

Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility.

Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.

Epithelial ICAM-1 and ICAM-2 regulate the egression of human T cells across the bronchial epithelium.

Egression of inflammatory cells from the lung interstitium into the airway lumen is critical for the resolution of inflammation, but the underlying mechanisms of this egression are unclear. Here, we use an in vitro system, in which human T cells migrate across a bronchial epithelial monolayer, to investigate the molecules involved. We show that although inhibition of T-cell LFA-1 blocks egression by 75 +/- 5.6% (P<0.0001), inhibition of the LFA-1-ligand ICAM-1 on the epithelium only inhibits by 52.7 +/- 0.06% (P=0.0001). We, therefore, looked for other epithelial ligands for LFA-1 and demonstrate that ICAM-2, but not ICAM-3, is expressed on the bronchial epithelium. Blocking ICAM-2 inhibits egression by 50.95 +/- 10.79% (P=0.04), and blocking both ICAM-1 and ICAM-2 inhibits egression by 69.6 +/- 5.2% (P< 0.0001). Inhibition of LFA-1/ICAM-1 and ICAM-2 interactions on the basolateral epithelium does not prevent egressing T cells from adhering, polarizing, or moving over the basal epithelium, but it does prevent their recognition of the interepithelial junctions. In conclusion, we show that egression of T cells involves three distinct sequential steps: adhesion, junctional recognition, and diapedesis; we further demonstrate that ICAM-2 is expressed on the bronchial epithelium and, together with ICAM-1, has an essential function in the clearance of T cells from the lung.

Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition.

Actin polymerization in the mammalian cytosol can be locally activated by mechanisms that relieve the autoinhibited state of N-WASP, an initiator of actin assembly, a process that also requires the protein Toca-1. Several pathogenic bacteria, including Shigella, exploit this host feature to infect and disseminate efficiently. The Shigella outer membrane protein IcsA recruits N-WASP, which upon activation at the bacterial surface mediates localized actin polymerization. The molecular role of Toca-1 in N-WASP activation during physiological or pathological actin assembly processes in intact mammalian cells remains unclear. We show that actin tail initiation by S. flexneri requires Toca-1 for the conversion of N-WASP from a closed inactive conformation to an open active one. While N-WASP recruitment is dependent on IcsA, Toca-1 recruitment is instead mediated by S. flexneri type III secretion effectors. Thus, S. flexneri independently hijacks two nodes of the N-WASP actin assembly pathway to initiate localized actin tail assembly.

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination.

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, alpha-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.

CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization.

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl(-) currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels.

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts.

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.

Biochemical properties and inhibitors of (N-)WASP.

The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho GTPase Cdc42 and a key component of signaling pathways that regulate the actin cytoskeleton. WASP is regulated by a number of ligands, and the mechanisms by which these act are beginning to be understood through detailed biochemical analyses. Here we describe the protocols we use to study WASP proteins, including the methods we use to purify signaling components and the assays we use to quantitatively characterize the biochemical and biophysical properties of WASP, its activation by Cdc42, and its inhibition by the small molecule wiskostatin. These methods have broad use within the WASP-related cytoskeletal-signaling pathway but are also applicable to investigations of other intramolecular and intermolecular interactions.