The signal cascade of the spindle checkpoint

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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.