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.

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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.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissuespecific protein ablation. We show that this approach works in multiple tissues -the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons -where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.