| 1996 |
N-WASP was identified as a 65 kDa brain protein that binds the SH3 domains of Ash/Grb2 and contains a pleckstrin homology (PH) domain and cofilin-homologous region through which it depolymerizes actin filaments. PH domain mutation (C38W) that reduces PIP2 binding and deletion of the VCA actin-binding domain both abolish cortical actin rearrangements and cause predominantly nuclear localization, establishing that PIP2 binding and actin interaction are required for membrane retention and function. EGF treatment induces complex formation of EGF receptors with N-WASP and produces microspikes. |
Mutagenesis (C38W PH domain mutation, deltaVCA deletion), overexpression in COS7 cells, co-immunoprecipitation with EGF receptor, subcellular localization by immunofluorescence |
The EMBO journal |
High |
8895577
|
| 1998 |
N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP does not, despite structural similarities. In a cell-free system, active Cdc42 stimulates the actin-depolymerizing activity of N-WASP by exposing its actin-depolymerizing region. N-WASP directly interacts with Cdc42 and is required downstream of Cdc42 for filopodium formation. |
Co-expression in cells, cell-free actin polymerization assay, Cdc42-binding experiments |
Nature |
High |
9422512
|
| 1999 |
N-WASP is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP (VCA domain) binds directly to the Arp2/3 complex and dramatically stimulates its actin nucleation activity. Full-length N-WASP activity is greatly enhanced by Cdc42 and PI(4,5)P2, linking signal transduction to actin polymerization through an N-WASP/Arp2/3 core mechanism. |
In vitro actin polymerization assay in Xenopus egg extracts, biochemical binding assays (VCA–Arp2/3 interaction), immunodepletion |
Cell |
High |
10219243
|
| 2000 |
The N-terminal domain of N-WASP physically interacts with its C-terminal effector (VCA) domain in an intramolecular, autoinhibitory interaction that occludes the Arp2/3-binding site. N-WASP is a monomer in solution. PI(4,5)P2 activates N-WASP through a conserved basic sequence element near the Cdc42-binding site (not the WH1 domain), reducing the affinity between N- and C-termini. Cdc42 similarly relieves autoinhibition. In Xenopus extracts, PI(4,5)P2 acts both as a direct N-WASP activator and as an indirect activator of Cdc42. |
In vitro actin polymerization assay, sedimentation/gel filtration (monomer determination), domain-binding assays, mutant N-WASP lacking basic stretch in Xenopus extracts |
The Journal of cell biology |
High |
10995436
|
| 2000 |
N-WASP is recruited to the surface of endosomes and lysosomes that form actin comet tails in Xenopus eggs and in mammalian cell extracts, mediating vesicle propulsion through Arp2/3-complex-dependent actin assembly. |
Live imaging in Xenopus eggs, cell-free reconstitution, immunofluorescence, electron microscopy, acridine orange staining |
The Journal of cell biology |
High |
10662777
|
| 2000 |
The WH1 domain (not the polyproline-rich region) of N-WASP mediates its recruitment to sites of actin polymerization during vaccinia virus motility via direct interaction with WASP-interacting protein (WIP). N-WASP and WIP form a functional complex that integrates signaling cascades leading to actin polymerization. In Shigella motility, WIP is recruited by N-WASP. |
Mutant expression, co-immunoprecipitation, actin comet tail assays in vaccinia/Shigella-infected cells |
Nature cell biology |
High |
10878810
|
| 2001 |
WIP directly interacts with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex and stabilizes actin filaments. WIP and N-WASP act as a functional unit in filopodium formation: anti-N-WASP antibody inhibits WIP-induced filopodia, and anti-WIP antibody blocks N-WASP-induced filopodia. |
In vitro actin polymerization assay (pyrene-actin), direct binding assay, microinjection of antibodies into NIH 3T3 cells |
Nature cell biology |
High |
11331876
|
| 2001 |
N-WASP knockout mice die before embryonic day 12 with developmental delay. N-WASP is not required for Listeria actin-based motility but is absolutely required for Shigella and vaccinia virus actin-based motility. N-WASP-deficient fibroblasts can still form filopodia and spread via lamellipodia. |
Gene targeting (homologous recombination), genetic knockout, pathogen actin motility assays, cell spreading/morphology analysis |
Nature cell biology |
High |
11584271
|
| 2001 |
Intersectin-l (neuronal variant) functions via its DH domain as a GEF for Cdc42. N-WASP binds directly to intersectin-l and upregulates its GEF activity, generating GTP-bound Cdc42 which in turn activates N-WASP, creating a feed-forward activation loop that drives actin assembly via the Arp2/3 complex. |
GEF activity assay (GDP/GTP exchange), direct binding assay, co-immunoprecipitation, actin polymerization assay, cell-based actin rearrangement analysis |
Nature cell biology |
High |
11584276
|
| 2001 |
A novel adaptor protein WISH binds N-WASP through its SH3 domain and strongly enhances N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro. WISH coexpression with N-WASP induces marked microspike formation even without stimuli; an N-WASP mutant (H208D) that cannot bind Cdc42 still induces microspikes with WISH. |
In vitro actin polymerization assay, co-immunoprecipitation, overexpression in COS7 cells, N-WASP depletion from brain extracts |
The Journal of cell biology |
High |
11157975
|
| 2001 |
N-WASP is involved in insulin-stimulated GLUT4 recycling. Insulin causes PI3K-independent cortical localization of N-WASP and Arp3 plus cortical F-actin polymerization in adipocytes. Dominant-inhibitory N-WASP-DeltaWA attenuates cortical F-actin rearrangements and inhibits insulin-stimulated GLUT4 translocation. TC10 (a Cdc42-related GTPase) acts upstream of N-WASP in this pathway; inhibitory TC10 (T31N) blocks cortical N-WASP localization. |
Dominant-negative expression, immunofluorescence localization, GLUT4 translocation assay in adipocytes, PI3K inhibitor (wortmannin) treatment |
The Journal of biological chemistry |
High |
11694514
|
| 2002 |
Cdc42 regulates Golgi-to-ER protein transport through N-WASP. Cdc42V12 recruits GFP-N-WASP to the Golgi complex. Coexpression of Cdc42 and N-WASP inhibits retrograde Golgi-to-ER transport; this inhibition requires the Arp2/3-binding WA domain of N-WASP, as the N-WASP(ΔWA) mutant does not inhibit transport. |
Overexpression, GFP-N-WASP localization imaging, Shiga toxin retrograde transport assay, KDEL receptor redistribution assay, dominant-active Sar1 assay |
Molecular biology of the cell |
Medium |
11907268
|
| 2002 |
N-WASP is essential for actin assembly at the surface of endomembranes induced by elevated PIP2 levels, leading to actin comet-driven vesicle motility. This process requires WH1 and polyproline domains of N-WASP for vesicle surface recruitment/activation, and Nck, Grb2, and WIP are also recruited. Direct interaction of N-WASP with Cdc42 is not required for reconstitution of vesicle motility. |
N-WASP-deficient cell reconstitution, N-WASP mutant expression, vesicle motility assay, co-immunoprecipitation |
The Journal of biological chemistry |
High |
12147689
|
| 2002 |
Syndapins interact with N-WASP through its proline-rich domain and integrate N-WASP functions in receptor-mediated endocytosis. Co-overexpression of syndapins rescues the endocytosis block caused by N-WASP dominant-negative. Depletion of endogenous N-WASP by sequestration to mitochondria or anti-N-WASP antibodies impairs endocytosis. In vivo reconstitution of the syndapin-N-WASP interaction at cellular membranes triggered local actin polymerization. |
Co-overexpression rescue assay, N-WASP depletion via mitochondrial targeting and antibody microinjection, endocytosis assay, in vivo reconstitution at membranes |
The EMBO journal |
High |
12426380
|
| 2003 |
FAK directly interacts with N-WASP and phosphorylates it at Tyr256. Phosphorylation of Tyr256 reduces N-WASP interaction with nuclear importin NPI-1, shifting N-WASP from nuclear to cytoplasmic localization. Nuclear localization of N-WASP also depends on being in the open conformation (Cdc42 activation or VCA truncation). Tyr256 phosphorylation promotes cell migration. |
In vitro kinase assay (FAK phosphorylation of N-WASP), co-immunoprecipitation, subcellular fractionation/localization, co-immunoprecipitation with importin NPI-1, cell migration assay |
The Journal of biological chemistry |
High |
14676198
|
| 2003 |
N-WASP localizes to the nucleus and its nuclear/cytoplasmic shuttling is controlled by phosphorylation by Src family kinases. Phosphorylated N-WASP is exported from the nucleus via a nuclear export signal (NES) in a leptomycin B-sensitive manner; N-WASP also has a nuclear localization signal (NLS) in its basic region. Unphosphorylated nuclear N-WASP suppresses HSP90 expression by binding heat shock transcription factor (HSTF) and enhancing HSTF association with heat shock element (HSE). Reduced HSP90 in turn decreases Src kinase activity. |
Subcellular fractionation, leptomycin B treatment (NES validation), NLS/NES identification, co-immunoprecipitation with HSTF, luciferase/transcription reporter assays, chromatin immunoprecipitation |
The Journal of biological chemistry |
Medium |
12871950
|
| 2004 |
Toca-1 (transducer of Cdc42-dependent actin assembly) was biochemically purified as an essential component of the Cdc42/N-WASP pathway. Toca-1 binds both N-WASP and Cdc42. Toca-1 promotes actin nucleation by activating the N-WASP-WIP complex (the predominant cellular form of N-WASP), and cooperative actions of both N-WASP-WIP and Toca-1 are required for Cdc42-induced actin assembly. |
Biochemical purification, in vitro actin polymerization assay, binding assays (Toca-1 with N-WASP and Cdc42), Xenopus egg extract reconstitution |
Cell |
High |
15260990
|
| 2004 |
mDab1 directly binds N-WASP via the PTB domain of mDab1 interacting with the NRFY sequence near the CRIB motif of N-WASP, and directly activates N-WASP to induce Arp2/3-mediated actin polymerization and filopodium formation in cells. This filopodium formation depends on N-WASP activity. Fyn kinase-mediated phosphorylation of mDab1 leads to its Cbl-dependent ubiquitination and loss of filopodium induction, acting as a negative regulatory switch. |
Direct binding assay (in vitro), in vitro actin polymerization assay, overexpression in COS-7 cells, dominant-negative N-WASP rescue, phosphorylation and ubiquitination assays |
The Biochemical journal |
Medium |
15361067
|
| 2004 |
N-WASP and the Arp2/3 complex are required for invadopodium formation in metastatic carcinoma cells. N-WASP is activated at the base of invadopodia. Upstream regulators Nck1, Cdc42, and WIP are also necessary. Cofilin is required for stabilization and maturation of long-lived invadopodia. EGF receptor signaling drives invadopodium formation through the N-WASP-Arp2/3 pathway. |
RNAi, dominant-negative mutant expression, time-lapse microscopy, EGFR kinase inhibitors, matrix degradation assay |
The Journal of cell biology |
High |
15684033
|
| 2004 |
N-WASP activity is spatially regulated in living cells: N-WASP is activated at the leading edge of lamellipodia and at the base of invadopodia in invasive carcinoma cells, as demonstrated by a FRET biosensor distinguishing active (open) vs. inactive (closed) N-WASP conformations. |
FRET biosensor (N-WASP conformational sensor) in live cells |
Current biology |
Medium |
15084285
|
| 2005 |
HSP90 binds directly to N-WASP. Binding alone does not affect basal actin polymerization rate, but HSP90 enhances v-Src-mediated phosphorylation of N-WASP, leading to increased actin polymerization. HSP90 also protects phosphorylated/activated N-WASP from proteasome-dependent degradation, amplifying N-WASP activity. HSP90-N-WASP association is increased proportional to N-WASP activation. Blocking HSP90 binding to N-WASP inhibits podosome formation and neurite extension. |
Direct binding assay (pull-down), in vitro actin polymerization assay, phosphorylation assay (v-Src), proteasome inhibitor rescue, co-immunoprecipitation at podosomes, wiskostatin/HSP90 inhibitor treatment |
The EMBO journal |
High |
15791211
|
| 2005 |
Abi1, an essential component of the WAVE protein complex, also binds N-WASP with nanomolar affinity and cooperates with Cdc42 to potently stimulate N-WASP activity in vitro. Abi1 and N-WASP (but not WAVE) regulate actin-based vesicular transport, EGFR endocytosis, and EGFR/TfR cell-surface distribution. |
In vitro actin polymerization assay, direct binding affinity determination, RNAi knockdown, EGFR endocytosis assay, receptor surface distribution analysis |
Nature cell biology |
High |
16155590
|
| 2006 |
N-WASP is present in the nucleus within a large protein complex containing PSF-NonO, nuclear actin, and RNA polymerase II. N-WASP interacts with the PSF-NonO complex and couples it to RNA polymerase II to regulate transcription. Nuclear actin polymerization promoted by N-WASP plays an important role in this transcriptional regulation. |
Co-immunoprecipitation of nuclear complex, RNA polymerase II co-IP, transcription reporter assays, nuclear fractionation |
Nature cell biology |
Medium |
16767080
|
| 2006 |
IQGAP1 controls co-localization of N-WASP with the Arp2/3 complex in lamellipodia. The C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner; the N-terminal half of IQGAP1 antagonizes this by associating with the C-terminal region of IQGAP1 (autoinhibition). Signal-induced relief of IQGAP1 autoinhibition allows it to activate N-WASP for Arp2/3-dependent actin assembly. |
Quantitative co-localization, IQGAP1 downregulation, co-immunoprecipitation, pull-down with N-WASP domains, kinetic actin polymerization assay |
The Journal of biological chemistry |
High |
17085436
|
| 2007 |
WASP and N-WASP have combined, partially redundant roles in T cell development. Double knockout (lacking both WASP and N-WASP) in T cells causes thymic hypocellularity, reduced peripheral T cells, impaired DN-to-DP transition with reduced cycling DN3 cells, and decreased SP cell migration. N-WASP single deficiency in T cells is indistinguishable from wild-type. |
Homologous recombination plus conditional Cre-loxP knockout, RAG-2-deficient blastocyst complementation, flow cytometry, migration assays |
Proceedings of the National Academy of Sciences |
High |
17878299
|
| 2007 |
N-WASP and the Arp2/3 complex regulate the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localizes to spines and active synapses. RNAi knockdown or wiskostatin treatment decreases spine and excitatory synapse number. Deletion of the C-terminal VCA domain that binds/activates Arp2/3 dramatically decreases spines and synapses. Cdc42 knockdown phenocopies N-WASP knockdown, placing Cdc42 upstream. |
RNAi knockdown, wiskostatin pharmacological inhibition, VCA deletion mutant, FM4-64 dye loading (functional synapse marking), immunofluorescence co-localization |
The Journal of biological chemistry |
High |
18430734
|
| 2007 |
Abp1 (F-actin-binding protein) directly interacts with N-WASP and releases N-WASP autoinhibition in cooperation with Cdc42, promoting N-WASP-triggered Arp2/3-mediated actin polymerization. Abp1 knockdown in neurons increases axon length, phenocopying Arp2/3 complex inhibition. Abp1, N-WASP and Arp2/3 colocalize at actin polymerization sites in neurons. |
Direct interaction assay (in vitro pull-down), in vitro actin polymerization assay, Abp1 RNAi knockdown, N-WASP mutants lacking Abp1 or Cdc42 binding, immunofluorescence |
PLoS ONE |
Medium |
17476322
|
| 2008 |
EFC/F-BAR domain proteins (Toca-1 and FBP17) activate the N-WASP-WIP complex-mediated actin polymerization depending on membrane curvature in the presence of phosphatidylserine-containing membranes, even in the absence of Cdc42 and PIP2. Toca-1/FBP17 recruit N-WASP-WIP to the membrane and position it spatially via conserved acidic residues near their SH3 domain. |
In vitro actin polymerization assay with defined lipid vesicles of varying curvature, mutant analysis of acidic residues, N-WASP-WIP recruitment assay |
The EMBO journal |
High |
18923421
|
| 2008 |
Toca-1 is required in intact mammalian cells for the conversion of N-WASP from a closed (inactive) to an open (active) conformation during Shigella actin tail initiation. N-WASP recruitment to Shigella is dependent on the bacterial IcsA, whereas Toca-1 recruitment is mediated by type III secretion effectors, showing two independently hijacked nodes of the N-WASP actin assembly pathway. |
Toca-1 RNAi knockdown, conformation-sensitive N-WASP antibody assay, cell infection assays, fluorescence microscopy |
Cell host & microbe |
Medium |
18191793
|
| 2009 |
N-WASP exchange rate limits the extent of Arp2/3-dependent actin-based motility of vaccinia virus. N-WASP rapidly turns over at the virus surface (FRAP), and its turnover depends on its ability to stimulate Arp2/3 actin polymerization. Disrupting N-WASP interaction with Grb2 or barbed ends increases N-WASP exchange rate and results in faster virus movement. N-WASP thus controls the rate of actin-based motility by regulating actin polymerization extent. |
FRAP (fluorescence recovery after photobleaching), N-WASP mutant analysis, vaccinia actin motility assay |
Nature |
High |
19262673
|
| 2009 |
Amphiphysin 1 directly interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the SH3 and N-BAR domains of amphiphysin 1 are required for stimulation. Acidic liposome-triggered N-WASP-dependent actin polymerization is strongly impaired in amphiphysin 1 knockout mouse brain cytosol. FRET-FLIM confirmed the association in vivo in Sertoli cells; association is enhanced by phosphatidylserine receptor stimulation. |
Direct binding assay, in vitro actin polymerization assay, amphiphysin 1 knockout mouse brain cytosol, FRET-FLIM in cells |
The Journal of biological chemistry |
High |
19759398
|
| 2009 |
Nck and PI(4,5)P2 show reciprocal interdependence in promoting localized N-WASP-mediated actin polymerization. Nck knockdown/knockout suppresses PIP5K-induced actin comets. PI(4,5)P2 is necessary for localized Nck-induced actin polymerization. PI(4,5)P2 and PIP5K are enriched at Nck-induced actin comets. The extent of N-WASP-mediated actin polymerization is modulated by PI(4,5)P2-sensitive N-WASP mutants. |
Nck RNAi knockdown, Nck knockout cells, PIP5K overexpression, inositol 5-phosphatase coclustering, N-WASP mutant analysis, live-cell imaging of actin comets |
Molecular cell |
High |
19917259
|
| 2010 |
Nebulin and N-WASP form a complex at Z bands of myofibrils upon IGF-1 stimulation, downstream of PI3K-Akt signaling through inhibition of GSK-3β. Importantly, the nebulin-N-WASP complex promotes unbranched actin filament nucleation from Z bands without Arp2/3 complex, representing a non-canonical Arp2/3-independent function of N-WASP. N-WASP is required for IGF-1-induced muscle hypertrophy. |
Co-immunoprecipitation, in vitro actin polymerization assay (with and without Arp2/3), IGF-1 stimulation, GSK-3β inhibitor treatment, N-WASP conditional knockout in muscle |
Science |
High |
21148390
|
| 2011 |
N-WASP regulates the epithelial junctional actin cytoskeleton through a nucleation-independent pathway at the zonula adherens. N-WASP depletion decreases junctional F-actin but does not affect junctional actin nucleation (dominantly mediated by Arp2/3). An N-WASP mutant unable to directly activate Arp2/3 rescues the junctional defect. N-WASP stabilizes newly formed actin filaments via the WIP-family protein WIRE, which binds the N-WASP WH1 domain. |
RNAi knockdown, rescue with Arp2/3-activation-deficient N-WASP mutant, WIRE binding assay, junctional F-actin quantification |
Nature cell biology |
High |
21785420
|
| 2011 |
N-WASP is required for membrane wrapping and myelination by Schwann cells. Schwann cell-specific N-WASP knockout mice fail to myelinate (cells arrest at promyelinating stage); a limited number form unusually short internodes with thin myelin and occasional myelin misfoldings. Schwann cells can sort and ensheath axons without N-WASP. |
Conditional knockout (Schwann cell-specific Cre-loxP), electron microscopy, histological analysis, nerve morphology assessment |
The Journal of cell biology |
High |
21263026
|
| 2012 |
N-WASP is an essential negative regulator of B cell receptor (BCR) signaling. B-cell-specific N-WASP deletion causes enhanced and prolonged BCR signaling, elevated autoantibodies, increased F-actin at the B-cell surface, enhanced spreading, delayed contraction, inhibition of BCR microcluster merging into central clusters, and blockage of BCR internalization. WASP is activated first upon BCR activation, followed by N-WASP; N-WASP activation is suppressed by Bruton's tyrosine kinase-induced WASP activation and restored by SHIP-mediated WASP inactivation. |
B-cell-specific conditional N-WASP knockout, TIRF microscopy, actin imaging, BCR cluster analysis, serum autoantibody measurement, signaling assays |
PLoS biology |
High |
24223520
|
| 2012 |
N-WASP-mediated invadopodium formation is essential for breast cancer invasion, intravasation and lung metastasis in vivo. Both N-WASP shRNA and dominant-negative N-WASP constructs decrease invadopodium formation, extracellular matrix degradation, tumor intravasation, and lung metastasis in a rat mammary tumor model. |
Stable shRNA knockdown, dominant-negative expression, intravital imaging, lung metastasis counting, in vivo tumor intravasation assay |
Journal of cell science |
High |
22389406
|
| 2012 |
N-WASP coordinates delivery of MT1-MMP to invasive pseudopods from late endosomes and stabilizes MT1-MMP at the plasma membrane via direct tethering of its cytoplasmic tail to F-actin. N-WASP promotes assembly of elongated pseudopodia required for matrix degradation in 3D. |
Co-immunoprecipitation (N-WASP with MT1-MMP), live-cell trafficking assays, dominant-negative N-WASP, 3D matrix invasion assay, immunofluorescence |
The Journal of cell biology |
High |
23091069
|
| 2012 |
N-WASP is required for structural integrity of the blood-testis barrier (BTB). Sertoli cell-specific N-WASP knockout leads to mislocalization of junctional/cytoskeletal elements, disruption of BTB function, and complete spermatogenic arrest. N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling, mediating restructuring through endocytic recycling of BTB components. |
Sertoli cell-specific conditional knockout, electron microscopy, junction protein localization, BTB permeability assay |
PLoS genetics |
High |
24967734
|
| 2012 |
N-WASP binds p120-catenin through its VCA domain and links p120-catenin to the Arp2-cortical actin polymerization machinery to stabilize endothelial adherens junctions. This interaction requires Tyr256 phosphorylation of N-WASP by FAK. Phosphomimicking Y256D-N-WASP or VCA expression stabilizes junctions and facilitates barrier recovery after thrombin. |
Co-immunoprecipitation, N-WASP depletion, VCA domain expression, phosphomimetic mutant (Y256D), endothelial permeability assay, actin imaging |
The Journal of biological chemistry |
Medium |
23212915
|
| 2012 |
N-WASP is required for muscle-cell fusion during mouse skeletal myogenesis. N-WASP-deficient myoblasts fail to fuse but otherwise differentiate normally, maintain motility, and attach normally. N-WASP activity is required in both partners of a fusing myoblast pair. |
Conditional N-WASP knockout in muscle, primary satellite cell cultures, cell fusion quantification, motility and morphology assays |
Proceedings of the National Academy of Sciences |
High |
22736793
|
| 2013 |
WIP acts as an essential link between Nck and N-WASP. WIP (or its homolog WIRE) is required for N-WASP recruitment and actin-based motility of vaccinia virus. WIP contains two Nck-binding sites; it is recruited to the virus by the second SH3 domain of Nck. N-WASP's recruitment depends on its interaction with WIP rather than directly with Nck. The first and third SH3 domains of Nck stimulate actin assembly but are not required for WIP-N-WASP recruitment. |
MEF knockouts (Nck, WIP, N-WASP), vaccinia actin motility assay, co-immunoprecipitation, domain-specific mutant analysis |
Current biology |
High |
23707428
|
| 2013 |
Cdc42 cooperates with Nck to promote actin tail formation by stabilizing N-WASP beneath vaccinia virus. Cdc42 activation is mediated by the Rho-GEF intersectin-1 (ITSN1), which is recruited to the virus before actin-based motility. Cdc42, ITSN1, and N-WASP function in a feed-forward loop to promote actin polymerization. This pathway also operates in FcγR-mediated phagocytosis. |
RNAi knockdown, co-immunoprecipitation, vaccinia actin tail assay, phagocytosis assay, genetic epistasis |
Journal of cell science |
Medium |
24284073
|
| 2013 |
N-WASP is required for stabilization of podocyte foot processes. Podocyte-specific N-WASP knockout mice develop proteinuria and kidney failure. N-WASP-deficient podocytes show impaired dynamic actin reorganization (dorsal ruffle formation). N-WASP-mediated Arp2/3 actin nucleation of branched microfilament networks is specifically required for foot process maintenance. |
Podocyte-specific conditional N-WASP knockout, electron microscopy of foot processes, proteinuria measurement, primary culture actin dynamics assay |
Journal of the American Society of Nephrology |
High |
23471198
|
| 2014 |
Cdc42/N-WASP signaling controls β cell delamination and differentiation during pancreatic development. Expression of constitutively active Cdc42 inhibits β cell delamination and differentiation associated with junctional actin and cell-cell junction disassembly. Genetic ablation of N-WASP in constitutively active Cdc42-expressing β cells partially restores both delamination and β cell differentiation, placing N-WASP downstream of Cdc42 in this process. |
Conditional mouse genetics (Cre-loxP), constitutively active Cdc42 expression, N-WASP conditional knockout, immunofluorescence of junction proteins and differentiation markers |
Development |
High |
24449844
|
| 2014 |
PC1 (polycystin-1), Pacsin 2, and N-WASP are in the same protein complex. Both PC1 and Pacsin 2 are required for N-WASP/Arp2/3-dependent actin remodeling and directional cell migration in kidney epithelial cells. |
Yeast two-hybrid, co-immunoprecipitation, PC1/Pacsin2 siRNA knockdown, directional migration assay, actin remodeling assay |
Human molecular genetics |
Medium |
24385601
|
| 2019 |
N-WASP drives pancreatic cancer metastasis through chemotaxis and matrix remodeling. N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid (LPA)-induced RhoA-mediated contractility and force generation by controlling LPA receptor (LPAR1) recycling and preventing its degradation. N-WASP-depleted cells do not recognize LPA gradients, showing altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. |
N-WASP depletion (RNAi), LPAR1 trafficking assay (receptor recycling vs. degradation), RhoA activation assay, traction force microscopy, in vivo metastasis model, co-immunoprecipitation with SNX18 |
Developmental cell |
High |
31668663
|
| 2010 |
CIP4 (Cdc42 interacting protein-4), an F-BAR protein, interacts with N-WASp in an EGF-dependent manner. CIP4 silencing causes decreased tyrosine phosphorylation of N-WASp at the Src-dependent site Y256, impairs invadopodium formation and gelatin degradation, and reduces migration and invasion. |
Co-immunoprecipitation, siRNA knockdown of CIP4, invadopodium assay, phospho-Y256 N-WASP Western blot, invasion/migration assays |
Cancer research |
Medium |
20940394
|
| 2010 |
CIP4 promotes GLUT4 endocytosis by interacting with both N-WASp and Dynamin-2 in an insulin-dependent manner. Knockdown of CIP4 increases surface GLUT4 by decreasing endocytosis. FRET confirmed insulin-dependent subcellular coordination of CIP4-N-WASp and CIP4-Dynamin-2 interactions at the plasma membrane and in cytosol. |
Co-immunoprecipitation, FRET, siRNA knockdown, GLUT4 surface quantification by flow cytometry, glucose uptake assay |
Journal of cell science |
Medium |
19509061
|
| 2011 |
N-WASP and CK2 (casein kinase 2) form a complex and co-localize at clathrin-coated vesicles. N-WASP binds to and is phosphorylated by CK2, thereby reducing CK2 kinase activity. Conversely, N-WASP-promoted actin polymerization is decreased by CK2 phosphorylation. Both CK2 and N-WASP knockdown inhibit the initial rate of EGFR clathrin-mediated endocytosis (CME). Full rescue requires reconstitution of the N-WASP-CK2 complex; N-WASP controls F-actin presence at clathrin-coated structures. |
Co-immunoprecipitation, in vitro kinase assay, CK2/N-WASP knockdown, EGFR endocytosis rate measurement, TIRF microscopy of clathrin-coated structures, F-actin quantification |
Journal of cell science |
High |
21610097
|
| 2007 |
The VCA domain of N-WASP binds the Arp2/3 complex in a 1:1 stoichiometry even with excess VCA. VCA-Arp2/3 binds one actin in a 1:1:1 complex (latrunculin A-sensitive), with binding of the second actin to VCA weakened in the ternary complex. Each of the two WH2 (V) domains independently binds G-actin in 1:2 complexes. V, VC, and VCA enhance barbed end depolymerization but do not nucleate or sever filaments. |
Protein crystallography (partial VC-actin crystal structure), hydrodynamic methods, spectrofluorimetry, in vitro actin polymerization/depolymerization assays |
The Journal of biological chemistry |
High |
22847007
|
| 2007 |
Multiple WIP-binding epitopes (three distinct regions in WIP residues 451-485) are required for functional interaction with the N-WASP EVH1 (WH1) domain. A central polyproline motif occupies the canonical EVH1 binding site in a reversed orientation; flanking hydrophobic contacts (WIP residues 454-459 and 475-478) augment binding. Disruption of any of the three WIP epitopes reduces N-WASP binding in cells. |
NMR structure determination of WIP-EVH1 complex, binding affinity measurements, site-directed mutagenesis, co-immunoprecipitation in cells |
The Journal of biological chemistry |
High |
17229736
|
| 2021 |
The Chlamydia trachomatis type III secretion effector TmeA directly activates N-WASP to promote Arp2/3-dependent actin polymerization during chlamydial invasion. TmeA and TarP influence separate but synergistic pathways for chlamydial entry. |
Chlamydial gene deletion (FRAEM), proximity labeling, direct binding assay, actin polymerization assay, infection assays with TmeA deletion mutants |
mBio |
Medium |
33468693
|