The signal cascade of the spindle checkpoint

纺锤体检查点是一种监督机制，确保有丝分裂过程中染色体的平等分离。从前期到中期，它延迟了姐妹染色单体的分离，直到所有的动点都连接到有丝分裂的纺锤体上。在本研究项目中，我们重点研究了在纺锤体检查点的信号级联中起核心作用的蛋白质MAD2，并试图揭示其生化性质以及负调控MAD2的分子机制。我们发现，MAD2在平衡状态下采用两种不同的折叠构象(称为N1-MAD2和N2-MAD2)。N_2-MAD2对APC/C的抑制作用更强。这两个MAD2构象在体外相互转换很慢，但MAD2上游调节因子Mad1的片段加速了相互转换。我们还证明了负调控P31彗星与N_2-MAD2选择性相互作用。通过对p31彗星的生化分析，确定动力蛋白中间链DNCI是p31彗星的结合伙伴。在有丝分裂后期，这两种蛋白质在物理上相互作用。缺乏DNCI的细胞停滞于中期，中期平板上的染色体排列正确。这一表型提示DNCI和p31comet在纺锤体检查点沉默中起着重要作用。

The spindle checkpoint is a surveillance mechanism that ensures equal segregation of chromosomes in mitosis. From prophase to metaphase it delays sister chromatid separation until all the kinetochores are connected to the mitotic spindles. In this research project we focused on Mad2, a protein which plays a central role in the signal cascade of the spindle checkpoint and attempted to reveal its biochemical nature as well as a molecular mechanism to negatively regulate Mad2.We found that Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). N2-Mad2 is more potent in APC/C inhibition. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. We also demonstrated that a negative regulator p31comet selectively interacts with N2-Mad2. Biochemical analysis of p31comet allowed identification of DNCI (intermediate chain of dynein) as a binding partner of p31comet. The two proteins physically interact each other in late mitosis. Cells lacking DNCI were arrested at metaphase with properly aligned chromosomes at the metaphase plate. This phenotype would suggest that DNCI together with p31comet play an important role in silencing the spindle checkpoint.