title: Filament formation of MSF-A, a mammalian septin, in human mammary epithelial cells depends on interactions with microtubules.
abstract: Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.
authors.affiliation: Divisions of Biochemistry and Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.
authors.forename: Koh-Ichi
authors.initials: K
authors.lastname: Nagata
authors.forename: Aie
authors.initials: A
authors.lastname: Kawajiri
authors.forename: Seiya
authors.initials: S
authors.lastname: Matsui
authors.forename: Mihoko
authors.initials: M
authors.lastname: Takagishi
authors.forename: Takashi
authors.initials: T
authors.lastname: Shiromizu
authors.forename: Noriko
authors.initials: N
authors.lastname: Saitoh
authors.forename: Ichiro
authors.initials: I
authors.lastname: Izawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
authors.forename: Tomohiko J
authors.initials: TJ
authors.lastname: Itoh
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Hotani
authors.forename: Masaki
authors.initials: M
authors.lastname: Inagaki
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001940:Breast / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D003703:Demecolcine / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014404:Tubulin / Q000737:chemistry
chemical_list: D003598:Cytoskeletal Proteins; D011994:Recombinant Proteins; D014404:Tubulin; D003571:Cytochalasin B; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D003703:Demecolcine