Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

SUMMARY: Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

SUMMARY: Protein phosphatase 1 (PP1) is essential for spindle assembly checkpoint (SAC) silencing and mitotic exit, but its regulation during mitosis remains ill-defined. Here, we demonstrate in vitro and in Drosophila cells that the mitotic kinase POLO phosphorylates PP1α87B at a conserved residue (T286) within a pocket implicated in the recognition of RVxF-containing target proteins. Phosphorylation of T286 inhibits PP1α87B binding to the RVxF motif of the SAC kinase MPS1, dampening the dephosphorylation of MPS1 T-loop. Phosphorylation of T286 is dynamically regulated during mitosis. It occurs at unattached/tensionless kinetochores and decreases as chromosomes congress. Expression of phosphomimetic PP1α87B ^T286D prevents MPS1 inactivation in metaphase and causes a SAC-dependent delay of anaphase onset. Conversely, an unphosphorylatable PP1α87B ^T286A mutant impairs MPS1 activation at unattached kinetochores and weakens the SAC. In vivo , larval neuroblasts expressing PP1α87B ^T286 phosphomutants exhibit frequent chromosome mis-segregation and aneuploidy. Thus, our findings identify POLO-mediated phosphorylation of PP1α87B as a critical regulatory strategy that fine-tunes phosphatase activity to ensure a robust and timely SAC and prevent genome instability.

Accurate transition into mitosis driven by cyclin B1-CDK1 activity is essential to avoid chromosome segregation errors and preserve genome integrity. How this activity is spatially controlled to trigger mitotic onset remains unclear. Here, we show that chromosome condensation triggers an increase in nuclear envelope (NE) tension. This increased tension is required for translocation of cyclin B1 into the nucleus and dynein loading on nuclear pore complexes (NPCs), ensuring timely mitotic entry. Micromanipulation experiments further indicate this tension-dependent mechanism requires SUN proteins on the NE. Impairment of chromosome condensation leads to the nuclear accumulation of the G2 checkpoint kinase Wee1 and an inhibition of CDK1 activity, which result in a temporary delay in mitotic entry. This delay can be overridden by increasing tension on the NE, which accelerates the nuclear translocation of cyclin B1 and dynein loading. We propose that mitotic onset is controlled by a chromosome-dependent NE tension mechanism that enables robust spatiotemporal coupling between chromosome condensation and the NE structural changes required for an efficient mitosis.