Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
Wellcome Trust Centre for Cell Biology, University of Edinburgh King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
Curr Biol. 2017 Jan 9;27(1):137-143. doi: 10.1016/j.cub.2016.11.014. Epub 2016 Dec 22.
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 KNL1 and Mps1 is 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.
纺锤体检查点作为一个有丝分裂监测系统，监测着动粒与纺锤体微管之间的相互作用，并确保染色体的高保真分离[1-3]。检查点通过未连接的动粒被激活，Mps1 激酶磷酸化 KNL1 上保守的 MELT 基序，以生成与 Bub3-Bub1 复合物结合的位点[4-7]。这导致 Mad 蛋白在动粒上的动态募集[8,9]，Mad2 的构象变化[10-12]，以及有丝分裂检查点复合物（MCC：Cdc20-Mad3-Mad2[13-15]）的形成。MCC 的形成抑制了后期促进复合物/周期蛋白（Cdc20-APC/C），从而防止了 securin 和 cyclin 的蛋白水解破坏，并延迟了后期起始。Mps1 依赖性 Bub3-Bub1 募集后在动粒上发生的事情在机制上仍不清楚，也不知道除了 KNL1 之外的动粒蛋白是否在检查点信号转导和 MCC 生成中具有重要作用。在这里，我们采用一种简化的方法，避免了动粒的复杂性，并证明 KNL1 和 Mps1 的共同募集足以产生强大的检查点信号和延长的有丝分裂阻滞。我们证明了 Mad1-Bub1 复合物在合成检查点信号转导过程中形成。对 bub3Δ 突变体的分析表明，Bub3 可抑制过早的检查点信号转导。这个合成系统将使 MCC 生成和检查点沉默的详细机制研究成为可能。在分析了几个影响检查点复合物定位的突变体后，我们得出结论，纺锤体检查点阻滞可以不依赖于它们在动粒、纺锤体极和核膜的定位。
