During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be guaranteed. Accuracy requires that chromosomes become correctly attached to the microtubule spindle apparatus via their kinetochores. When not correctly attached to the spindle, kinetochores activate the spindle assembly checkpoint network, which in turn blocks cell cycle progression. Once all kinetochores become stably attached to the spindle, the checkpoint is inactivated, which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed. Here we review recent progress in our understanding of how the checkpoint signal is generated, how it blocks cell cycle progression and how it is extinguished.

SummaryAccurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31 comet is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31 comet antagonises the checkpoint is unclear. A prevalent model is that p31 comet acts as a 'cap' by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1-C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31 comet localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31 comet downstream of kinetochores. We show that p31 comet binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31 comet results in more Mad2 bound to BubR1-Cdc20, and conversely, overexpression of p31 comet results in less Mad2 bound to BubR1-Cdc20. Addition of recombinant p31 comet to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31 comet mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31 comet prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31 comet negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.

Summary Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres.

Centromeres play essential roles in equal chromosome segregation by directing the assembly of the microtubule binding kinetochore and serving as the cohesion site between sister chromatids. Here, we review the significant recent progress in our understanding of centromere protein assembly and how centromere proteins form the foundation of the kinetochore.

A fundamental challenge for the survival of all organisms is maintaining the integrity of the genome in all cells. Cells must therefore segregate their replicated genome equally during each cell division. Eukaryotic organisms package their genome into a number of physically distinct chromosomes, which replicate during S phase and condense during prophase of mitosis to form paired sister chromatids. During mitosis, cells form a physical connection between each sister chromatid and microtubules of the mitotic spindle, which segregate one copy of each chromatid to each new daughter cell. The centromere is the DNA locus on each chromosome that creates the site of this connection. In this review, we present a brief history of centromere research and discuss our current knowledge of centromere establishment, maintenance, composition, structure, and function in mitosis.