LIN7 Mediates the Recruitment of IRSp53 to Tight Junctions

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein–protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin–Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (ΔL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Δ5) or fused to the L27 domain of LIN7 (L27-IRSp53Δ5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7–IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.     

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).     

The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell-ECM signaling

The serine/threonine kinase Par1b promotes cell-cell adhesion and determines the polarity of the luminal domain in epithelial cells. In this study, we demonstrate that Par1b also regulates cell-extracellular matrix (ECM) signaling in kidney-derived Madin-Darby canine kidney (MDCK) cells and identified the rho-guanosine triphosphatase adaptor and scaffolding protein IRSp53 as a Par1b substrate involved in this pathway. Par1b overexpression inhibits basal lamina formation, cell spreading, focal adhesion, stress fiber formation, and compaction, whereas Par1b depletion has the opposite effect. IRSp53 depletion mimics Par1b overexpression on cell-ECM signaling and lumen polarity but had no effect on adherens junction formation. Par1b directly phosphorylates IRSp53 on S366 in cell lysates and stimulates phosphorylation on S453/3/5 via an indirect mechanism. A Par1b phosphorylation-deficient IRSp53 mutant but not the wild-type protein efficiently rescues both the cell spreading and the lumen polarity defects in Par1b MDCK cells. Our data suggest a model in which Par1b phosphorylation prevents recruitment of IRSp53 effector proteins to its Src homology domain 3 by promoting 14-3-3 binding in the vicinity of that domain.     

Contrasting localizations of MALS/LIN-7 PDZ proteins in brain and molecular compensation in knockout mice

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.     

IRSp53 is a novel interactor of SHIP2: A role of the actin binding protein Mena in their cellular localization in breast cancer cells

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P₃, PI(4,5)P₂ and PI(3,4)P₂ content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e., CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.     

Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.     

Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin-1 at epithelial adherens junctions. Nectin-1 serves as a mediator of cell-to-cell spread for Herpes simplex virus 1 (HSV-1). We analyzed the role of AF6 protein in the viral spread and nectin-1 clustering at cell-cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV-1 spreading, however, the clustering of nectin-1 at cell-cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV-1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E-cadherin-dependent cell adhesion.     

Differential expression of PDZ domain-containing proteins in human diseases - challenging topics and novel issues

The general features of the PDZ domain structure and functions have been extensively studied during the last decade. PDZ domains are generally present in proteins that are involved in multiple interactions to assemble functional protein complexes that control key cellular processes. One of the best characterized functions of PDZ domain-containing proteins is control of epithelial cell polarity and cell-cell contacts. In the present review, we summarize the current knowledge on regulation of expression of certain PDZ polarity proteins localized at the intercellular junctions. In addition, we provide a critical overview of recent findings regarding the role of these proteins during development of human diseases. Complete understanding of these issues is valuable for the design of novel therapeutic intervention for common pathologies, such as cancer.     

Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2

mDia1 is a downstream effector of Rho small G protein that is implicated in stress fiber formation and cytokinesis. We isolated an mDia1-binding protein and identified it to be IRSp53/BAIAP2. IRSp53 and BAIAP2 have independently been isolated as a 58/53-kDa protein tyrosine phosphorylated in response to insulin and a BAI1-binding protein, respectively. BAI1 is a brain-specific seven-span transmembrane protein capable of inhibiting angiogenesis. The proline-rich formin homology 1 domain of mDia1 bound the Src homology 3 domain of IRSp53/BAIAP2 in a GTP-Rho-dependent manner. The results suggest that IRSp53/BAIAP2 is a downstream effector of mDia1.     

A Highlights from MBoC Selection: The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics

The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins’ tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.     

Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.

BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.     

Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP(3), and Rac

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).     

Role of Abl Kinase and the Wave2 Signaling Complex in HIV-1 Entry at a Post-Hemifusion Step

Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Gαq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.     

A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein

Insulin receptor tyrosine kinase substrate p53 (IRSp53) has been identified as an SH3 domain-containing adaptor that links Rac1 with a Wiskott-Aldrich syndrome family verprolin-homologous protein 2 (WAVE2) to induce lamellipodia or Cdc42 with Mena to induce filopodia. The recruitment of these SH3-binding partners by IRSp53 is thought to be crucial for F-actin rearrangements. Here, we show that the N-terminal predicted helical stretch of 250 amino acids of IRSp53 is an evolutionarily conserved F-actin bundling domain involved in filopodium formation. Five proteins including IRSp53 and missing in metastasis (MIM) protein share this unique domain and are highly conserved in vertebrates. We named the conserved domain IRSp53/MIM homology domain (IMD). The IMD has domain relatives in invertebrates but does not show obvious homology to any known actin interacting proteins. The IMD alone, derived from either IRSp53 or MIM, induced filopodia in HeLa cells and the formation of tightly packed parallel F-actin bundles in vitro. These results suggest that IRSp53 and MIM belong to a novel actin bundling protein family. Furthermore, we found that filopodium-inducing IMD activity in the full-length IRSp53 was regulated by active Cdc42 and Rac1. The SH3 domain was not necessary for IMD-induced filopodium formation. Our results indicate that IRSp53, when activated by small GTPases, participates in F-actin reorganization not only in an SH3-dependent manner but also in a manner dependent on the activity of the IMD.     

CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high‐density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53‐dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.     

SH2B1 increases the numbers of IRSp53-induced filopodia

BackgroundFilopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53 kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1β is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1β may act together to regulate filopodium formation.MethodsTo determine the contribution of IRSp53 and SH2B1β in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1β to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1β.ResultsIn this study, we show that SH2B1β interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1β to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation.ConclusionsTaken together, this study provides a novel finding that SH2B1β interacts with IRSp53-containing complexes to increase the number of filopodia.General significanceA better understanding of how SH2B1β and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.     

Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.     

SPIN90-IRSp53 complex participates in Rac-induced membrane ruffling

SPIN90 is a key regulator of actin cytoskeletal organization. Using the BioGRID^beta database (General Repository for Interaction Datasets), we identified IRSp53 as a binding partner of SPIN90, and confirmed the in vivo formation of a SPIN90-IRSp53 complex mediated through direct association of the proline-rich domain (PRD) of SPIN90 with the SH3 domain of IRSp53. SPIN90 and IRSp53 positively cooperated to mediate Rac activation, and co-expression of SPIN90 and IRSp53 in COS-7 cells led to the complex formation of SPIN90-IRSp53 in the leading edge of cells. PDGF treatment induced strong colocalization of SPIN90 and IRSp53 at membrane protrusions. Within such PDGF-induced protrusions, knockdown of SPIN90 protein using siRNA significantly reduced lamellipodia-like protrusions as well as localization of IRSp53 at those sites. Finally, competitive inhibition of SPIN90-IRSp53 binding by SPIN90 PRD dramatically reduced ruffle formation, further suggesting that SPIN90 plays a key role in the formation of the membrane protrusions associated with cell motility.     

Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. Here we show that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. We provide a molecular mechanism for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion.     

The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics

The Cdc42 effector IRSp53 is a strong inducer of filopodia formation and consists of an Src homology domain 3 (SH3), a potential WW-binding motif, a partial-Cdc42/Rac interacting binding region motif, and an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain.We show that IRSp53 interacts directly with neuronal Wiskott-Aldrich syndrome protein (N-WASP) via its SH3 domain and furthermore that N-WASP is required for filopodia formation as IRSp53 failed to induce filopodia formation in N-WASP knock-out (KO) fibroblasts. IRSp53-induced filopodia formation can be reconstituted in N-WASP KO fibroblasts by full-length N-WASP, by N-WASPDeltaWA (a mutant unable to activate the Arp2/3 complex), and by N-WASPH208D (a mutant unable to bind Cdc42). IRSp53 failed to induce filopodia in mammalian enabled (Mena)/VASP KO cells, and N-WASP failed to induce filopodia when IRSp53 was knocked down with RNA interference. The IRSp53 I-BAR domain alone induces dynamic membrane protrusions that lack actin and are smaller than normal filopodia ("partial-filopodia") in both wild-type N-WASP and N-WASP KO cells. We propose that IRSp53 generates filopodia by coupling membrane protrusion through its I-BAR domain with actin dynamics through SH3 domain binding partners, including N-WASP and Mena.