Different requirements for the chromosomal passenger complex to maintain a mitotic checkpoint arrest induced by different spindle poisons. Example of an experiment in which osteosarcoma cells (U2OS) were transfected with mock shRNA, Mad2 shRNA, or shRNA for INCENP (which results in knock-down of all chromosomal passenger complex components). Transfected cells were released from a 24 h thymidine block into the indicated drugs. Eighteen hours after the release cells were fixed and prepared for FACS analysis. The MPM2 antibody was used to determine the mitotic index. This type of experiment shows that knock-down of a classical checkpoint protein (Mad2) does not allow cells to accumulate in mitosis with any of the drugs, while knock-down of the chromosomal passenger complex affects the response to paclitaxel and monastrol more dramatically than the response to nocodazole.

The chromosomal passenger complex generates an alternative APC/C inhibitory signal that amplifies the 'wait anaphase' signal from unattached kinetochores. Based on different sets of data (see text for details), we propose that in response to inappropriately attached kinetochores, the chromosomal passenger complex not only destabilises these attachments but also elicits an additional signal that inhibits the APC/C [35]. This could be via direct phosphorylation of APC/C subunits, or via direct control of the spindle assembly checkpoint. Ways be which the chromosomal passenger complex could exert direct control over the spindle checkpoint are through regulation of BubR1/Bub1 kinetochore levels, modulation of the mitotic checkpoint complex or via an as yet unknown pathway. Regardless the mechanism, this additional signal is thought to amplify the unattached kinetochore-derived signal, resulting in a robust checkpoint response when the number of unattached kinetochores is low.

The chromosomal passenger complex influences spindle checkpoint activity indirectly, through the creation of unattached kinetochores. Upon entry into mitosis chromosomes start to make random connections with microtubules from the mitotic spindle. To finally obtain bipolar spindle attachments, improper attachments (syntelic – depicted here-, and merotelic attachments) need to be corrected to prevent chromosome segregation errors. Aurora B in complex with its fellow passenger proteins is necessary for this correction process. Through the phosphorylation of kinetochore proteins that bind microtubules it modifies the stability or affinity of the kinetochore-microtubule interaction. As a consequence microtubules detach from the kinetochore allowing new rounds of attachments until bipolarity is obtained. However, during this correction process unattached are created capable of inhibiting the APC/C [33]. As such Aurora B's role in checkpoint function can be considered an indirect consequence of its microtubule destabilising activity.

Model explaining the role of the chromosomal passenger complex/Aurora B in spindle checkpoint function. (A) Treatment with the microtubule destabilising drug nocodazole results in a long checkpoint-dependent mitotic arrest. However, even when all kinetochores are unattached the spindle assembly checkpoint is not capable of inhibiting all APC/C's which might explain why these cells do eventually exit from mitosis (mitotic slippage) [58]. If Aurora B is inactivated in these cells, less APC/C's will be inhibited. Still this is sufficient to allow a mitotic delay, but this delay is significantly shorter than when Aurora B is active [14, 15]. (B) Treatment with the microtubule stabilising agent paclitaxel induces a mitotic arrest with a few unattached kinetochores [35] most likely inhibiting less APC/C's than when all kinetochores are unattached. Yet, this number of inhibited APC/C's is sufficient to sustain a robust checkpoint-dependent arrest. Since the unattached kinetochores are generated under the influence of the chromosomal passenger complex/Aurora B [35], inhibition of Aurora B will now silence both the unattached kinetochore-derived checkpoint signal and the additional amplification signal, resulting in an override of the spindle assembly checkpoint. (C) Expression of a chromosomal passenger complex that lacks the coiled-coil domain of INCENP does not affect the microtubule destabilising activity of Aurora B. In the presence of paclitaxel unattached kinetochores are therefore generated but this does not result in a checkpoint-dependent arrest [35]. We propose that due to the low number of unattached kinetochores that are now inhibiting the APC/C, the spindle checkpoint becomes more dependent on this additional chromosomal passenger complex-generated amplification signal to inhibit a sufficient number of APC/C's that allow a robust mitotic arrest.