Accurate genome inheritance by daughter cells requires that sister chromatids in the mother attach to microtubules emanating from opposite poles of the mitotic spindle (bi-orientation). A surveillance mechanism named the spindle assembly checkpoint (SAC) monitors the microtubule attachment process, temporarily halting sister chromatid separation and mitotic exit until completion of bi-orientation1. SAC failure results in abnormal chromosome numbers (aneuploidy), a hallmark of many tumours. The HORMA domain protein MAD2 is a subunit of the SAC effector mitotic checkpoint complex (MCC). Structural conversion from open to closed MAD2 is required for MAD2 incorporation in MCC1. In vitro, MAD2 conversion and MCC assembly requires several hours2–4, while the SAC response in cells is established in a few minutes5–7. To address this discrepancy, we reconstituted with purified components a near-complete SAC signalling system and monitored MCC assembly with real-time sensors. Dramatic acceleration of MAD2 conversion and MCC assembly was observed when MPS1 phosphorylated the MAD1:MAD2 complex, triggering its template function in the MAD2 conversion and contributing to the establishment of a physical platform for MCC assembly. Thus, catalytic activation of the SAC depends on regulated protein-protein interactions that accelerate the spontaneous but rate-limiting conversion of MAD2 required for MCC assembly.

The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures.

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.DOI: http://dx.doi.org/10.7554/eLife.21007.001

The Rod–Zw10–Zwilch (RZZ) complex assembles as a fibrous corona on kinetochores before microtubule attachment during mitotic spindle formation. Mosalaganti et al. provide new structural insight into the Spindly–RZZ complex that suggests that it resembles a dynein adaptor–cargo pair in the kinetochore corona.