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.     

IRSp53 Mediates Podosome Formation via VASP in NIH-Src Cells

Podosomes are cellular “feet,” characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.     

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.     

mDia1 and WAVE2 proteins interact directly with IRSp53 in filopodia and are involved in filopodium formation

Filopodia are dynamic actin-rich cell surface protrusions involved in cell migration, axon guidance, and wound healing. The RhoGTPase Cdc42 generates filopodia via IRSp53, a multidomain protein that links the processes of plasma membrane deformation and actin dynamics required for their formation in mammalian cells. The Src homology 3 domain of IRSp53 binds to the actin regulators Mena, Eps8, WAVE1, WAVE2, mDia1, and mDia2. We show that mDia1 and WAVE2 synergize with IRSp53 to form filopodia. IRSp53 also interacts directly with these two proteins within filopodia, as observed in acceptor photobleaching FRET studies. Measurement of filopodium formation by time-lapse imaging of live cells also revealed that depleting neuronal cells of either mDia1 or WAVE2 protein decreases the ability of IRSp53 to induce filopodia. In contrast, IRSp53 does not appear to partner WAVE1 or mDia2 to give rise to these structures. In addition, although all three isoforms of mDia are capable of inducing filopodia, IRSp53 requires only mDia1 to do so. These findings suggest that mDia1 and WAVE2 are important Src homology 3 domain partners of IRSp53 in forming filopodia.     

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.     

Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics.     

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.     

Rif-mDia1 interaction is involved in filopodium formation independent of Cdc42 and Rac effectors

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.     

The mammalian Verprolin, WIRE induces filopodia independent of N-WASP through IRSp53

The mammalian verprolin family of proteins, WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) regulate the actin cytoskeleton through WASP/N-WASP (Wiskott Aldrich Syndrome Protein and Neural-WASP). In order to characterize the WASP/N-WASP-independent function of WIRE, we screened and identified IRSp53 (Insulin Receptor Substrate) as a WIRE interacting protein. Expression of IRSp53 with WIRE in N-WASP^−/− mouse fibroblast cells induced filopodia while co-expression of IRSp53 with WIP did not. The induction of filopodia is dependent on WIRE-IRSp53 interaction as mutation in the SH3 domain of IRSp53 abolished WIRE-IRSp53 interaction as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of WIRE is critical for IRSp53-WIRE interaction and for filopodia formation. The interaction between WIRE and IRSp53 is regulated by Cdc42 as mutations which abolish Cdc42-IRSp53 interaction lead to loss of IRSp53-WIRE interaction as shown by pull down assay. The plasma membrane localization of IRSp53 is dependent on Cdc42 and WIRE. Expression of Cdc42^G12V (active mutant) with WIRE-IRSp53 caused significant increase in the number of filopodia per cell. Thus our results show that Cdc42 regulates the activity of IRSp53 by regulating the IRSp53-WIRE interaction as well as localization of the complex to plasma membrane to generate filopodia.     

Functional analysis of Dictyostelium IBARa reveals a conserved role of the I-BAR domain in endocytosis

I-BAR (inverse-Bin/amphiphysin/Rvs)-domain-containing proteins such as IRSp53 (insulin receptor substrate of 53 kDa) associate with outwardly curved membranes and connect them to proteins involved in actin dynamics. Research on I-BAR proteins has focussed on possible roles in filopod and lamellipod formation, but their full physiological function remains unclear. The social amoeba Dictyostelium encodes a single I-BAR/SH3 (where SH3 is Src homology 3) protein, called IBARa, along with homologues of proteins that interact with IRSp53 family proteins in mammalian cells, providing an excellent model to study its cellular function. Disruption of the gene encoding IBARa leads to a mild defect in development, but filopod and pseudopod dynamics are unaffected. Furthermore, ectopically expressed IBARa does not induce filopod formation and does not localize to filopods. Instead, IBARa associates with clathrin puncta immediately before they are endocytosed. This role is conserved: human BAIAP2L2 (brain-specific angiogenesis inhibitor 1-associated protein 2-like 2) also tightly co-localizes with clathrin plaques, although its homologues IRSp53 and IRTKS (insulin receptor tyrosine kinase substrate) associate with other punctate structures. The results from the present study suggest that I-BAR-containing proteins help generate the membrane curvature required for endocytosis and implies an unexpected role for IRSp53 family proteins in vesicle trafficking.     

SH2B1 and IRSp53 Proteins Promote the Formation of Dendrites and Dendritic Branches

SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the β isoform (SH2B1β) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1β and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching.     

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.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.     

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.     

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers?a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in?vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.     

I-BAR domain proteins: linking actin and plasma membrane dynamics

Dynamic plasma membrane rearrangements occur during many cellular processes including endocytosis, morphogenesis, and migration. Actin polymerization together with proteins that directly deform membranes, such as the BAR superfamily proteins, is essential for generation of membrane invaginations during endocytosis. Importantly, recent studies revealed that direct membrane deformation contributes also to the formation of plasma membrane protrusions such as filopodia and lamellipodia. Inverse BAR (I-BAR) domain proteins bind phosphoinositide-rich membrane with high affinity and generate negative membrane curvature to induce plasma membrane protrusions. I-BAR domain proteins, such as IRSp53, MIM, ABBA, and IRTKS also harbor many protein-protein interaction modules that link them to actin dynamics. Thus, I-BAR domain proteins may connect direct membrane deformation to actin polymerization in cell morphogenesis and migration.     

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.     

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).     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

The Rho family GTPase Rif induces filopodia through mDia2

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.