Generation of a spindle checkpoint arrest from synthetic signaling assemblies

Ivan Yuan, Ioanna Leontiou, Priya Amin, Karen May, Sadhbh Soper ní chafraidh, Eliška Zlámalová, Kevin Hardwick

Research output: Contribution to journalArticlepeer-review

Abstract

The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules, 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 signalling and MCC generation. Here we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1- Bub1 complex is formed during synthetic checkpoint signalling. Analysis of bub3- mutants demonstrates that Bub3 acts to suppress premature checkpoint signalling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analysing several mutants that affect localisation of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole and nuclear envelope localisation.
Original languageEnglish
Pages (from-to)137-143
Number of pages7
JournalCurrent Biology
Volume27
Issue number1
Early online date22 Dec 2016
DOIs
Publication statusPublished - 9 Jan 2017

Keywords

  • bub
  • mad
  • Mps1
  • cell cycle
  • checkpoint
  • kinetochore
  • mitosis
  • spindle
  • synthetic

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