title: Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].
abstract: Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.
authors.affiliation: Dipartimento di Biologia e Biotecnologie, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Ple. A. Moro 5, 00185 Rome, Italy; Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Valeria
authors.initials: V
authors.lastname: Palumbo
authors.affiliation: Dipartimento di Biologia e Biotecnologie, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Ple. A. Moro 5, 00185 Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Pellacani
authors.affiliation: Proteomics Facility, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Heesom
authors.affiliation: Department of Statistics, University of Oxford, South Parks Road, Oxford OX1 3TG, UK.
authors.forename: Kacper B
authors.initials: KB
authors.lastname: Rogala
authors.affiliation: Department of Statistics, University of Oxford, South Parks Road, Oxford OX1 3TG, UK.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Deane
authors.affiliation: Dipartimento di Biologia e Biotecnologie, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Ple. A. Moro 5, 00185 Rome, Italy.
authors.forename: Violaine
authors.initials: V
authors.lastname: Mottier-Pavie
authors.affiliation: Dipartimento di Biologia e Biotecnologie, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Ple. A. Moro 5, 00185 Rome, Italy; Istituto di Biologia e Patologia Molecolari del CNR c/o Sapienza Università di Roma, 00185 Rome, Italy; Institute of Molecular and Cellular Biology SD RAS, Novosibirsk 630090, Russia.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Gatti
authors.affiliation: Dipartimento di Biologia e Biotecnologie, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Ple. A. Moro 5, 00185 Rome, Italy. Electronic address: silvia.bonaccorsi@uniroma1.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Bonaccorsi
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Electronic address: j.g.wakefield@exeter.ac.uk.
authors.forename: James G
authors.initials: JG
authors.lastname: Wakefield
mesh_terms: D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D056404:Chaperonin Containing TCP-1 / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D018832:Molecular Chaperones / Q000378:metabolism; D058105:Polymerization; D034622:RNA Interference; D008941:Spindle Apparatus / Q000378:metabolism / Q000502:physiology*; D059008:Time-Lapse Imaging; D014404:Tubulin / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; C572493:Mgr protein, Drosophila; D018832:Molecular Chaperones; D014404:Tubulin; C105947:mst protein, Drosophila; D056404:Chaperonin Containing TCP-1