title: Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis.
abstract: Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation 'switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Weronika E
authors.initials: WE
authors.lastname: Borek
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Lynda M
authors.initials: LM
authors.lastname: Groocock
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Itaru
authors.initials: I
authors.lastname: Samejima
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Juan
authors.initials: J
authors.lastname: Zou
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Flavia
authors.initials: F
authors.lastname: de Lima Alves
authors.affiliation: 1] Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK [2] Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin 13355, Germany.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Sawin
mesh_terms: D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D010766:Phosphorylation; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
chemical_list: D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; C505279:mto2p protein, S pombe