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.     

I-BAR domains,IRSp53 and filopodium formation

Filopodia and lamellipodia are dynamic actin-based structures that determine cell shape and migration. Filopodia are thought to sense the environment and direct processes such as axon guidance and neurite outgrowth. Cdc42 is a small GTP-binding protein and member of the RhoGTPase family. Cdc42 and its effector IRSp53 (insulin receptor phosphotyrosine 53 kDa substrate) have been shown to be strong inducers of filopodium formation. IRSp53 consists of an I-BAR (inverse-Bin-Amphiphysin-Rvs) domain, a Cdc42-binding domain and an SH3 domain. The I-BAR domain of IRSp53 induces membrane tubulation of vesicles and dynamic membrane protrusions lacking actin in cells. The IRSp53 SH3 domain interacts with proteins that regulate actin filament formation e.g. Mena, N-WASP, mDia1 and Eps8. In this review we suggest that the mechanism for Cdc42-driven filopodium formation involves coupling I-BAR domain-induced membrane protrusion with SH3 domain-mediated actin dynamics through IRSp53.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.     

WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac

We previously reported that IRSp53 binds both Rac and WAVE2, inducing formation of Rac/IRSp53/WAVE2 complex that is important for membrane ruffling. However, recent reports noted a specific interaction between IRSp53 and Cdc42 but not Rac, which led us to re-examine the binding of IRSp53 to Rac. Immunoprecipitation analysis and pull-down assay reveal that full-length IRSp53 binds Rac much less efficiently than the N-terminal fragment, which may be caused by intramolecular interaction. Interestingly, the intramolecular interaction is interrupted by the binding of WAVE2 and full-length IRSp53 associates with Rac in the presence of WAVE2. We also report that IRSp53 induces spreading and neurite formation of N1E-115 cells, which presumably reflect functional cooperation with Rac.     

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.     

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.     

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.     

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.     

Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin beta3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the beta3 subunit participate in the effect of CD47 on neurite formation.     

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 Eps8/IRSp53/VASP network differentially controls actin capping and bundling in filopodia formation

There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia.