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The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C.

To satisfy the mitotic checkpoint and drive chromosome congression, the Mps1 kinase lets go of kinetochores by phosphorylating itself in trans (see also related papers by Maciejowski et al. and Santaguida et al. in this issue)., Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.

The NSL1 subunit structures interactions between the MIS12, NDC80, and KNL1 kinetochore complexes (see also a related paper by Maskell et al. in this issue)., Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of ∼22 nm. Through biochemical analysis, cross-linking–based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms.

Addition of reversine to dividing cells ejects Mad1 and the RZZ complex from unattached kinetochores and prevents resolution of incorrect chromosome–microtubule attachments (see also related papers by Hewitt et al. and Maciejowski et al. in this issue)., The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD–ZWILCH–ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome–microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery.

A chemical biology study characterizes the role of Haspin kinase in centromere recruitment of the chromosome passenger complex and in spindle assembly checkpoint function., By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.