title: The Toca-1-N-WASP complex links filopodial formation to endocytosis.
abstract: The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Delta95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis.
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138665.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Bu
authors.forename: Ai Mei
authors.initials: AM
authors.lastname: Chou
authors.forename: Kim Buay
authors.initials: KB
authors.lastname: Lim
authors.forename: Thankiah
authors.initials: T
authors.lastname: Sudhaharan
authors.forename: Sohail
authors.initials: S
authors.lastname: Ahmed
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed; D016501:Neurites / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism*; D034622:RNA Interference; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D002352:Carrier Proteins; C482527:FNBP1L protein, human; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein; D020696:rab5 GTP-Binding Proteins