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Spindle biology5/8/2023 ![]() ![]() BubR1/Mad3 appears to be the other crucial player. Consistent with this idea is the finding that Mad1 and a proportion of Mad2 are stably localized to the kinetochore whereas the remaining Mad2 and a pool of Cdc20 rapidly cycle on and off the kinetochore with similar dynamics ( Howell et al., 2004 Shah et al., 2004 Vink et al., 2006).Īlthough Mad2 is a good in vitro APC/C inhibitor, formation of Mad2-Cdc20 is unlikely to be sufficient to inhibit the APC in vivo. However, this has recently been challenged by two new models in which kinetochore-bound Mad1–C-Mad2 is a stable complex that acts as a template recruiting O-Mad2, which is then able to bind Cdc20 ( De Antoni et al., 2005 Yu, 2006). Because Mad1 and Cdc20 compete for the same binding site on Mad2, it was initially thought that kinetochore recruitment stimulated exchange of inactive Mad2 from Mad1 to an active form that binds Cdc20. ![]() In solution, free Mad2 adopts an open conformation (O-Mad2) but, on binding to Mad1 or Cdc20, this changes to a stable closed conformation (C-Mad2) ( De Antoni et al., 2005). Mad2 binds to Cdc20 this interaction is essential for checkpoint-dependent inhibition of the APC/C ( Hwang et al., 1998 Kim et al., 1998). However, interpreting such experiments is complicated because microtubule attachment is stabilised by tension ( Nicklas et al., 2001).įRAP experiments have shown that Bub1 and Mad1 are stably associated with unattached kinetochores, suggesting that they function as scaffolds recruiting the dynamic BubR1/Mad3 and Mad2 proteins, which are candidates for the inhibitory signal ( Howell et al., 2004 Shah et al., 2004). Chemical inhibition of spindle dynamics, which relieves tension but does not destroy kinetochore-microtubule attachments, also activates the checkpoint ( Clute and Pines, 1999 Skoufias et al., 2001). Conversely, kinetochores lacking tension because both sisters are attached to microtubules from the same pole (syntelic attachment) activate the checkpoint even though kinetochore-microtubule attachments are made, indicating that lack of tension can be sufficient for checkpoint activation. This indicates that lack of microtubule attachment elicits the checkpoint response. Laser ablation of the last unattached kinetochore relieves the checkpoint-dependent arrest and the cell enters anaphase even though the remaining sister kinetochore is not under tension ( Rieder et al., 1995). When a chromosome is attached to microtubules from opposite poles, tension is generated across the sister kinetochores by the pulling forces of the spindle. ![]()
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