Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Sci Rep. 2017 Jan 23;7:41072. doi: 10.1038/srep41072.
The mitotic checkpoint complex (MCC) is formed from two sub-complexes of CDC20-MAD2 and BUBR1-BUB3, and current models suggest that it is generated exclusively by the kinetochores after nuclear envelope breakdown (NEBD). However, neither sub-complex has been visualised in vivo, and when and where they are formed during the cell cycle and their response to different SAC conditions remains elusive. Using single cell analysis in HeLa cells, we show that the CDC20-MAD2 complex is cell cycle regulated with a "Bell" shaped profile and peaks at prometaphase. Its formation begins in early prophase before NEBD when the SAC has not been activated. The complex prevents the premature degradation of cyclin B1. Tpr, a component of the NPCs (nuclear pore complexes), facilitates the formation of this prophase form of the CDC20-MAD2 complex but is inactive later in mitosis. Thus, we demonstrate that the CDC20-MAD2 complex could also be formed independently of the SAC. Moreover, in prolonged arrest caused by nocodazole treatment, the overall levels of the CDC20-MAD2 complex are gradually, but significantly, reduced and this is associated with lower levels of cyclin B1, which brings a new insight into the mechanism of mitotic "slippage" of the arrested cells.
有丝分裂检查点复合物（MCC）由 CDC20-MAD2 和 BUBR1-BUB3 两个亚复合物组成，目前的模型表明，它仅在核膜破裂（NEBD）后由动粒产生。然而，这两个亚复合物都没有在体内被可视化，它们在细胞周期中何时以及何地形成，以及它们对不同 SAC 条件的反应仍然难以捉摸。使用 HeLa 细胞的单细胞分析，我们表明 CDC20-MAD2 复合物是细胞周期调控的，呈“钟形”曲线，在前期达到峰值。其形成始于早期前中期，此时 SAC 尚未被激活。该复合物可防止 cyclin B1 的过早降解。Tpr 是 NPC（核孔复合物）的一个组成部分，可促进 CDC20-MAD2 复合物的这种前期形式的形成，但在后期有丝分裂中失活。因此，我们证明 CDC20-MAD2 复合物也可以独立于 SAC 形成。此外，在用 nocodazole 处理引起的长时间阻滞中，CDC20-MAD2 复合物的总体水平逐渐但显著降低，这与 cyclin B1 的水平降低有关，这为有丝分裂“滑溜”阻滞细胞的机制提供了新的见解。
