The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow.

The transition of hematopoiesis from the fetal liver (FL) to the bone marrow (BM) is incompletely characterized. We demonstrate that the Wiskott-Aldrich syndrome verprolin-homologous protein (WAVE) complex 2 is required for this transition, as complex degradation via deletion of its scaffold Hem-1 causes the premature exhaustion of neonatal BM hematopoietic stem cells (HSCs). This exhaustion of BM HSC is due to the failure of BM engraftment of Hem-1-/- FL HSCs, causing early death. The Hem-1-/- FL HSC engraftment defect is not due to the lack of the canonical function of the WAVE2 complex, the regulation of actin polymerization, because FL HSCs from Hem-1-/- mice exhibit no defects in chemotaxis, BM homing, or adhesion. Rather, the failure of Hem-1-/- FL HSC engraftment in the marrow is due to the loss of c-Abl survival signaling from degradation of the WAVE2 complex. However, c-Abl activity is dispensable for the engraftment of adult BM HSCs into the BM. These findings reveal a novel function of the WAVE2 complex and define a mechanism for FL HSC fitness in the embryonic BM niche.

Molecular Control of Actin Dynamics In Vivo: Insights from Drosophila.

The actin cytoskeleton provides mechanical support for cells and generates forces to drive cell shape changes and cell migration in morphogenesis. Molecular understanding of actin dynamics requires a genetically traceable model system that allows interdisciplinary experimental approaches to elucidate the regulatory network of cytoskeletal proteins in vivo. Here, we will discuss some examples of how advances in Drosophila genetics and high-resolution imaging techniques contribute to the discovery of new actin functions, signaling pathways, and mechanisms of actin regulation in vivo.

Neogenin recruitment of the WAVE regulatory complex maintains adherens junction stability and tension.

To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS-WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension.

APP intracellular domain-WAVE1 pathway reduces amyloid-ß production.

An increase in amyloid-ß (Aß) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aß production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aß. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aß levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aß production.

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells.

WASF3 regulates miR-200 inactivation by ZEB1 through suppression of KISS1 leading to increased invasiveness in breast cancer cells.

The WASF3 gene promotes invasion and metastasis in breast cancer cells, which have undergone epithelial-to-mesenchyme transition (EMT). Overexpression of WASF3 in cells that do not show EMT increases their invasion potential as a result of increased ZEB1/2 levels, which specifically suppress the anti-invasion chromosome 1 miR-200a/200b/429 cluster. ZEB1/2 upregulation by WASF3 results from downregulation of KISS1, leading to the release of inhibition of nuclear factor (NF)κB by IκBα. We further show that ZEB1 expression is regulated by the NFκB transcription factor. Knockdown of WASF3 in breast cancer cells leads to reduced ZEB1 levels and increased miR-200 and E-cadherin levels, resulting in loss of invasion potential. The central regulation of this interactive pathway by WASF3 accounts for the increased invasion associated with increased WASF3 expression seen in aggressive breast cancer cells. WASF3, therefore, is a potential target to suppress invasion and metastasis in breast cancer cells.

Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development.

Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.

WAVE1 gene silencing via RNA interference reduces ovarian cancer cell invasion, migration and proliferation.

OBJECTIVE: Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) has been implicated in cancer cell migration and invasion. We have previously shown that the overexpression of WAVE1 in epithelial ovarian cancer (EOC) tissues is associated with a poor prognosis. However, the mechanism of WAVE1 regulating the malignant behaviors in EOC remains unclear. METHODS: In the present study, we knocked down WAVE1 expression in SKOV3 and OVCAR-3 cells through RNA interference to detect the cell biology and molecular biology changes. Moreover, western-blot was used to investigate the underlying mechanism of WAVE1 regulating the proliferative and invasive malignant behaviors in ovarian cancer cells. RESULTS: The down-regulation of WAVE1 had a significant effect on cell morphological changes. WAVE1 silencing decreased cell migration, cell invasion, cell adhesion, colony formation and cell proliferation in vitro. In addition, we found that down-regulation of WAVE1 inhibited malignant behaviors in vivo. Furthermore, our study also indicated that the PI3K/AKT and p38MAPK signaling pathways might contribute to WAVE1 promotion of ovarian cancer cell proliferation, migration, and invasion. CONCLUSIONS: WAVE1 might promote the proliferative and invasive malignant behaviors through the activation of the PI3K/AKT and p38MAPK signaling pathways in EOC.

WAVE2 regulates epithelial morphology and cadherin isoform switching through regulation of Twist and Abl.

BACKGROUND: Epithelial morphogenesis is a dynamic process that involves coordination of signaling and actin cytoskeletal rearrangements. PRINCIPAL FINDINGS: We analyzed the contribution of the branched actin regulator WAVE2 in the development of 3-dimensional (3D) epithelial structures. WAVE2-knockdown (WAVE2-KD) cells formed large multi-lobular acini that continued to proliferate at an abnormally late stage compared to control acini. Immunostaining of the cell-cell junctions of WAVE2-KD acini revealed weak and heterogeneous E-cadherin staining despite little change in actin filament localization to the same junctions. Analysis of cadherin expression demonstrated a decrease in E-cadherin and an increase in N-cadherin protein and mRNA abundance in total cell lysates. In addition, WAVE2-KD cells exhibited an increase in the mRNA levels of the epithelial-mesenchymal transition (EMT)-associated transcription factor Twist1. KD of Twist1 expression in WAVE2-KD cells reversed the cadherin switching and completely rescued the aberrant 3D morphological phenotype. Activity of the WAVE2 complex binding partner Abl kinase was also increased in WAVE2-KD cells, as assessed by tyrosine phosphorylation of the Abl substrate CrkL. Inhibition of Abl with STI571 rescued the multi-lobular WAVE2-KD 3D phenotype whereas overexpression of Abl kinase phenocopied the WAVE2-KD phenotype. CONCLUSIONS: The WAVE2 complex regulates breast epithelial morphology by a complex mechanism involving repression of Twist1 expression and Abl kinase activity. These data reveal a critical role for WAVE2 complex in regulation of cellular signaling and epithelial morphogenesis.

WASPs and WAVEs: from molecular function to physiology in hematopoietic cells.

The actin cytoskeleton is critically involved in a variety of cell functions. The Arp2/3 complex mediates branching of filamentous actin. The members of the Wiskott-Aldrich syndrome protein (WASP) family are major regulators of the complex. As such, the family proteins are also involved in numerous aspects of cell biology. In this short review, we first define the expanding WASP family. Next, we compare the domain structure of the members, and explain the known or proposed functions of each domain or region. Finally, we demonstrate the well-characterized roles of the proteins in specific cellular functions.

Scar/WAVE3 contributes to motility and plasticity of lamellipodial dynamics but not invasion in three dimensions.

The Scar (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex plays a major role in the motility of cells by activating the Arp2/3 complex, which initiates actin branching and drives protrusions. Mammals have three Scar/WAVE isoforms, which show some tissue-specific expression, but their functions have not been differentiated. In the present study we show that depletion of Scar/WAVE3 in the mammalian breast cancer cells MDA-MB-231 results in larger and less dynamic lamellipodia. Scar/WAVE3-depleted cells move more slowly but more persistently on a two-dimensional matrix and they typically only show one lamellipod. However, Scar/WAVE3 appears to have no role in driving invasiveness in a three-dimensional Matrigel™ invasion assay or a three-dimensional collagen invasion assay, suggesting that lamellipodial persistence as seen in two-dimensions is not crucial in three-dimensional environments.

HIF1A induces expression of the WASF3 metastasis-associated gene under hypoxic conditions.

The WASF3 (WAVE3) gene is an important mediator of cell motility, invasion and metastasis and is expressed at high levels in some advanced stage tumors. In our survey of breast cancer cells, we now demonstrate that exposure to hypoxic conditions increases WASF3 expression levels in MDA231, SKBR3 and MCF7 cells. The WASF3 promoter region contains HIF1A response elements (HRE). ChIP assays demonstrate that HIF1A binds to these HRE elements in the promoter region, and luciferase reporter assays using the WASF3 gene minimal promoter shows that hypoxia results in its upregulation. Phosphorylation of WASF3 is required for its ability to affect invasion and increased phosphoactivation of WASF3 is also seen in cells challenged with hypoxia. These cells also show increased motility in the scratch wound assay. Cells in which WASF3 has been knocked down show no response to hypoxia as expected, implicating the specificity of the hypoxic response to WASF3. Overall, these experiments demonstrate WASF3 is a HIF1A-regulated gene and suggests a mechanism to explain the observation of elevated expression of WASF3 in advanced stage tumors.

WAVE regulatory complex activation by cooperating GTPases Arf and Rac1.

The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant WRC and activate it, but when both GTPases were bound at the membrane, recruitment and concomitant activation of WRC were dramatically enhanced. This cooperativity between the two GTPases was sufficient to induce WAVE-dependent bead motility in cell extracts. Our findings suggest that Arf GTPases may be central components in WAVE signalling, acting directly, alongside Rac1.

WAVE2, N-WASP, and Mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments.

Cell migration is accomplished by the formation of cellular protrusions such as lamellipodia and filopodia. These protrusions result from actin filament (F-actin) rearrangement at the cell cortex by WASP/WAVE family proteins and Drosophila enabled (Ena)/vasodilator-stimulated factor proteins. However, the role of each of these actin cytoskeletal regulatory proteins in the regulation of three-dimensional cell invasion remains to be clarified. We found that platelet-derived growth factor (PDGF) induces invasion of MDA-MB-231 human breast cancer cells through invasion chamber membrane pores. This invasion was accompanied by intensive F-actin accumulation at the sites of cell infiltration. After PDGF stimulation, WAVE2, N-WASP, and a mammalian Ena (Mena) colocalized with F-actin at the sites of cell infiltration in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Depletion of WAVE2, N-WASP, or Mena by RNA interference (RNAi) abrogated both cell invasion and intensive F-actin accumulation at the invasion site. These results indicate that by mediating intensive F-actin accumulation at the sites of cell infiltration, WAVE2, N-WASP, and Mena are crucial for PI3K-dependent cell invasion induced by PDGF.

WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes.

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.

WAVE1 regulates P-glycoprotein expression via Ezrin in leukemia cells.

For children with acute myeloblastic leukemia (AML), multidrug resistance (MDR) reduces treatment effectiveness, and often leads to poor patient survival. While a number of factors have been described that affect MDR, the mechanisms underlying this effect remain unclear. In this study, the role of WAVE1 in MDR was investigated. Among 62 children with AML, high levels of WAVE1 were associated with poor patient outcomes. Proteomic techniques were used to identify novel WAVE1-interacting proteins from leukemia cells, one of which was the cytoskeleton regulator Ezrin. In leukemia cells, WAVE1 co-localized with both Ezrin and P-glycoprotein (P-gp), a critical regulator of the MDR phenotype. Overexpression of WAVE1 in K562 leukemia cells up-regulated P-gp and Ezrin, and reduced K562 cells' sensitivity to the chemotherapy drug adriamycin. The opposite effect was seen when WAVE1 expression was reduced via RNA interference. Critically, overexpression of WAVE1 in the absence of Ezrin did not affect P-gp levels or MDR. These data suggest that WAVE1 affects P-gp and MDR of leukemia cells through Ezrin.

WASP and WAVE family proteins: friends or foes in cancer invasion?

Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) family proteins activate cells' major actin nucleating machinery, the actin-related protein 2/3 (Arp2/3) complex, leading to the formation and remodeling of cortical actin filament networks. Cortical actin regulation is critical in many aspects of cell physiology including cell-cell adhesion and cell motility, whose dysregulation is directly associated with cancer invasion and metastasis. In line with this association, the WASP and WAVE family proteins have been reported to be involved in cancer malignancies. What is puzzling, however, is that they can act as either enhancers or suppressors of cancer malignancies depending on the type of cancer and its pathological stage. We are still far from understanding the roles of the WASP and WAVE family proteins in cancer progression. Here, we summarize the recent advances of studies of the WASP and WAVE family proteins with respect to cancer invasion and we offer a model that can account for the diverse outcomes originating from dysregulated WASP and WAVE family proteins in cancer development.