ne. Numbers above arrows indicate time in hours. Ctrl, control; IF, immunofluorescence; STA, single thymidine arrest. Bars, 5 m. Reversine is an MPS1 inhibitor Santaguida et al. 83 phosphorylate some of its centromeric substrates, suggesting that it is impairing a specific function of the chromosome passenger complex in spindle checkpoint control. In many additional settings, including experiments with yeast temperaturesensitive mutants or small molecule inhibitors, the ATL-962 inhibition of AURORA B has been shown to reduce the strength of the checkpoint arrest to unattached kinetochores but not to lead to complete override. It is possible that these effects result from residual AURORA B activity as a conse quence of incomplete depletion or inactivation. Small residual AURORA B activity may be sufficient to maintain the arrest under the strong checkpointactivating conditions created by spindledepolymerizing agents. However, the requirements on MPS1 may be more stringent, explaining why it is relatively easier to observe a checkpoint override when targeting MPS1. A confusing aspect of the relationship between error correction and the spindle checkpoint is that the inhibition of error correction can influence the pattern of kinetochore local ization of the spindle checkpoint proteins and therefore the strength of the checkpoint response at suboptimal concentra tions of spindledepolymerizing drugs such as nocodazole. Evidence of this can be extrapolated from Fig. 6 B: the same concentration of reversine has significantly different effects on the duration of mitotic arrest at low or high nocodazole doses. Thus, residual microtubules may contribute to checkpoint satisfaction if kinetochores cannot let go of them because error correction is impaired. A pathway that removes the checkpoint proteins from microtubulebound kinetochores is likely responsible for this phenomenon. Future studies will have to refer to the rigorous test PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833188 proposed by Yang et al. for evaluating the participation of MPS1, AURORA B, and other proteins in the checkpoint response. The test consists in evaluating the effects from ablating a putative checkpoint component when spindledepolymerizing drugs are present at concentrations that remove any residual tubulin polymer. By applying this test to AURORA B, Yang et al. demonstrated that at 100 nM hesperadin, the presence or absence of residual micro tubules results in dramatic differences in the localization of the checkpoint protein MAD2 to kinetochores. At high nocodazole concentrations, MAD2 is retained on kinetochores despite the presence of hesperadin. Conversely, at low nocodazole concentrations and at the same concentration of hesperadin, MAD2 is absent from kinetochores. This result predicts that previous studies implicating AURORA B in MAD2 recruitment might have been at least 
in part biased by the relatively low nocodazole concentrations used. However, we find that at higher hesperadin concentrations, MAD1 and the RZZ complex are lost from kinetochores even at high concentrations of nocodazole. Thus, AURORA B may be ultimately required for the recruitment of these checkpoint proteins, but higher levels of inhibition may be required for its 84 JCB VOLUME 190 NUMBER 1 2010 involvement to become explicit. We show that at least in vitro, these higher concentrations of hesperadin do not inhibit BUB1 and MPS1, but it remains formally possible that hesperadin inhibits additional kinases in the MAD1 and RZZ recruitment pathway. We
