title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.
abstract: Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.
authors.affiliation: The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Johanna
authors.initials: J
authors.lastname: Roostalu
authors.affiliation: The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Cade
authors.affiliation: The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Surrey
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging / Q000379:methods; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
chemical_list: C454180:CKAP5 protein, human; D018797:Cell Cycle Proteins; D028884:Karyopherins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; C122167:TPX2 protein, human; D028901:alpha Karyopherins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins