Jcb_201311113 1..8

The mitotic checkpoint inhibits the anaphase promoting complex/cyclosome (APC/C) in the presence of unattached kinetochores and silences this inhibition once all kinetochores are stably attached to spindle microtubules. Checkpoint activity (i.e., APC/C inhibition) and silencing are correlated with changes in the kinetochore localization of checkpoint proteins, including Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, and Cdc20 (Kops and Shah, 2012). These proteins are enriched at kinetochores until stable MT attachment and are required for checkpoint function. A central challenge in understanding the mitotic checkpoint is to dissect how local changes in checkpoint protein occupancy at kinetochores drive global changes in checkpoint activity. Preventing the removal of Mad1 or Mps1 from kinetochores via genetic fusion to the stable kinetochore component Mis12 blocks anaphase, which demonstrates that the removal of these proteins is required for checkpoint silencing (Jelluma et al., 2010; Maldonado and Kapoor, 2011). To probe checkpoint activation (i.e., switching the checkpoint from an “off” state to an “on” state), experimental intervention when the checkpoint is silenced, such as at metaphase, is an attractive approach. The checkpoint can be reactivated at metaphase by disrupting kinetochore–microtubule attachments, using either spindle poisons or laser microsurgery (Clute and Pines, 1999; Dick and Gerlich, 2013). It is unknown if the checkpoint can be reactivated after metaphase without compromising kinetochore– microtubule attachment. Metaphase kinetochores are stably attached and depleted of checkpoint proteins, so they provide a context in which to test the effect of increasing the kinetochore concentration of an individual protein in the absence of the full set of signals associated with the unattached state. We tested whether increasing kinetochore localization of Mad1 at metaphase is sufficient to reactivate the checkpoint. Mad1 and its partner Mad2 are essential checkpoint proteins (Li and Murray, 1991). Mad1 constitutively binds a single copy of Mad2 in the closed conformation, and this bound population of Mad2 serves as the kinetochore receptor for cytosolic, open-conformation Mad2 (Luo et al., 2004; De Antoni et al., 2005; Lara-Gonzalez et al., 2012). Continual recruitment of open Mad2 and its conversion to the closed conformation, concomitant with binding to Cdc20, constitutes the catalytic engine of the spindle assembly checkpoint at kinetochores (Han et al., The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/ cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores. Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint