Background:The SH3 domain of IRSp53 interacts with several proteins that control actin dynamics. Results: IRSp53 interacts with mDia1 and WAVE2 within filopodia, and knocking down either protein reduces IRSp53-driven filopodium formation. Conclusion: IRSp53-mediated filopodium formation involves the actin regulators mDia1 and WAVE2. Significance: Identifying proteins involved in filopodium formation enables an understanding of how these structures arise in mammalian cells.

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.Filopodia are dynamic, actin-rich cellular protrusions that are important for processes such as cell migration, neuritogenesis, axon guidance, wound healing, angiogenesis, embryonic development, and phagocytosis (1, 2). Elucidating the exact mechanism(s) by which filopodia form will give a greater understanding of these cellular processes and how such structures play a role in pathological conditions such as metastasis (3) and pathogen invasion (4, 5). The Rho GTPase Cdc42 is a key regulator of cell signaling events that lead to filopodium formation in mammalian cells. It binds to and activates IRSp53 (insulin receptor substrate protein 53 kDa) (6 -8). The Cdc42-IRSp53 complex induces filopodia by coupling membrane protrusion with actin dynamics (8). Interacting partners of IRSp53 include N-WASP (neural Wiskott-Aldrich syndrome protein) (8), Mena (mammalian enabled) (7, 9), Eps8 (EGF receptor kinase substrate 8) (10), and the Rac effectors WAVE (WASP family verprolin homology) isoforms WAVE1 (11-14) and WAVE2 (15). Yeast two-hybrid experiments have shown that IRSp53 binds mDia1 (mammalian Diaphanous 1) (16), but little else is known about this interaction. mDia1 and mDia2 (mammalian Diaphanous 2) belong to the formin family of multidomain eukaryotic proteins that are involved in a wide range of cellular processes that require actin polymerization (17). Formins contain an actin-nucleating formin homology 2 domain, and a proline-rich, formin homology 1 domain that binds Src homology 3 and WW domaincontaining proteins and profilin (18). Diaphanous-r...

We have characterized Drosophila melanogaster ACK (DACK), one of two members of the ACK family of nonreceptor tyrosine kinases in Drosophila. The ACKs are likely effectors for the small GTPase Cdc42, but signaling by these proteins remains poorly defined. ACK family tyrosine kinase activity functions downstream of Drosophila Cdc42 during dorsal closure of the embryo, as overexpression of DACK can rescue the dorsal closure defects caused by dominant-negative Dcdc42. Similar to known participants in dorsal closure, DACK is enriched in the leading edge cells of the advancing epidermis, but it does not signal through activation of the Jun amino-terminal kinase cascade operating in these cells. Transcription of DACK is responsive to changes in Dcdc42 signaling specifically at the leading edge and in the amnioserosa, two tissues involved in dorsal closure. Unlike other members of the ACK family, DACK does not contain a conserved Cdc42-binding motif, and transcriptional regulation may be one route by which Dcdc42 can affect DACK function. Expression of wild-type and kinase-dead DACK transgenes in embryos, and in the developing wing and eye, reveals that ACK family tyrosine kinase activity is involved in a range of developmental events similar to that of Dcdc42.Cdc42 is a member of the Rho family of Ras-related small GTPases originally identified through a mutation in Saccharomyces cerevisiae that affects formation of the bud site. The Cdc42 protein is required for the assembly of a ring of F-actin filaments in the neck of the bud (1). Subsequent work in mammalian fibroblasts demonstrated that Cdc42 drives the formation of F-actin-rich filopodia (40, 50), and numerous later studies have confirmed that Cdc42 regulates the actin cytoskeleton and, as a consequence, cell shape (65). Cdc42 participates in a diverse range of cellular processes including membrane trafficking, transcription, cell growth, and Ras-mediated transformation (65). The various effects of Cdc42 are presumed to be mediated through the interaction of the activated, GTP-bound form of the protein with downstream effectors.Given the important events controlled by Cdc42, intensive efforts have been made to elucidate the signaling pathways activated by this GTPase. This work has largely focused on identifying proteins that interact with GTP-bound Cdc42. Two such proteins are ACK-1 and ACK-2, closely related mammalian nonreceptor tyrosine kinases that bind GTP-bound Cdc42 and not its inactive GDP-bound form (44, 67). ACK-1 and ACK-2 cannot bind either version of the closely related Rho family GTPases Rac1 and RhoA, and these kinases represent likely effectors in Cdc42-specific signaling.To date, much of what is known about Rho family signaling has come from biochemical and cell biological work, but it is now being studied with genetic approaches in a number of model organisms, including Drosophila melanogaster. The Drosophila homolog of Cdc42, Dcdc42, has been studied by using dominantly acting mutant transgenes and loss-of-function mutations. This work has in...

Rif induces dorsal filopodia but the signaling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS (also known as BAIAP2L1) through its I-BAR domain. Rif also interacts with Pinkbar (also known as BAIAP2L2) in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant-negative Rif inhibits the formation of IRTKS-induced morphological structures, and Rif activity is blocked in IRTKS-knockout (KO) cells. To further define the Rif-IRTKS signaling pathway, we identify Eps8 and WAVE2 (also known as WASF2) as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS signaling, whereas WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic Escherichia coli infection, might compete for Rif for interaction with the I-BAR domain of IRTKS. Based on this evidence, we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53 (also known as BAIAP2), IRTKS and Pinkbar, as a central mechanism to modulate cell morphology.

Background: IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through SH3 domain binding partners. Results: Dynamin1 interacts with IRSp53, and its GTPase and actin binding domains are required for filopodial formation. Conclusion: Dynamin1 plays a role in filopodial initiation and assembly. Significance: A novel role for dynamin1 in filopodial dynamics has implications for cell migration and wound healing.