title: Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies.
abstract: The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation [1-3]. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex [4-7]. This leads to dynamic kinetochore recruitment of Mad proteins [8, 9], a conformational change in Mad2 [10-12], and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2 [13-15]). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. Here, we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7and Mps1Mph1is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Ivan
authors.initials: I
authors.lastname: Yuan
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Leontiou
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Priya
authors.initials: P
authors.lastname: Amin
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Karen M
authors.initials: KM
authors.lastname: May
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Sadhbh
authors.initials: S
authors.lastname: Soper Ní Chafraidh
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Eliška
authors.initials: E
authors.lastname: Zlámalová
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK. Electronic address: kevin.hardwick@ed.ac.uk.
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Hardwick
mesh_terms: D059447:Cell Cycle Checkpoints; D020090:Chromosome Segregation; D018386:Kinetochores; D008870:Microtubules; D008938:Mitosis; D010766:Phosphorylation; D012568:Schizosaccharomyces / Q000166:cytology* / Q000502:physiology; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism
chemical_list: D029702:Schizosaccharomyces pombe Proteins
keywords: Bub; Mad; Mps1; cell cycle; checkpoint; kinetochore; mitosis; spindle; synthetic