title: Dynamin1 is a novel target for IRSp53 protein and works with mammalian enabled (Mena) protein and Eps8 to regulate filopodial dynamics.
abstract: 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.
authors.affiliation: From the Institute of Medical Biology, Immunos, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Ai Mei
authors.initials: AM
authors.lastname: Chou
authors.affiliation: From the Institute of Medical Biology, Immunos, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Kai Ping
authors.initials: KP
authors.lastname: Sem
authors.affiliation: From the Institute of Medical Biology, Immunos, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Graham Daniel
authors.initials: GD
authors.lastname: Wright
authors.affiliation: From the Institute of Medical Biology, Immunos, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Thankiah
authors.initials: T
authors.lastname: Sudhaharan
authors.affiliation: From the Institute of Medical Biology, Immunos, 8A Biomedical Grove, Singapore 138648, Singapore sohail.ahmed@imb.a-star.edu.sg.
authors.forename: Sohail
authors.initials: S
authors.lastname: Ahmed
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D034284:Dynamin I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011554:Pseudopodia / Q000502:physiology*; D034741:RNA, Small Interfering; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C119539:BAIAP2 protein, human; C489989:EPS8 protein, human; C511939:Enah protein, human; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D034284:Dynamin I
keywords: Actin; Cell Biology; Cell Migration; Dynamin; Filopodia; Fluorescence Resonance Energy Transfer (FRET); IRSp53; Imaging; TIRF