The spindle assembly checkpoint (SAC) relies on the recruitment of Mad1-C-Mad2 to unattached kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that activation of Mps1 during prophase triggers Mad1 release from NPCs and that this is required for kinetochore localization of Mad1-C-Mad2 and robust SAC signaling. We find that Mps1 phosphorylates Megator/Tpr to reduce its interaction with Mad1 in vitro and in Drosophila cells. Importantly, preventing Mad1 from binding to Megator/Tpr restores Mad1 accumulation at kinetochores, the fidelity of chromosome segregation, and genome stability in larval neuroblasts of mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is tightly coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.

35The strength of the Spindle Assembly Checkpoint (SAC) depends on the amount of the 36 Mad1-C-Mad2 heterotetramer at kinetochores but also on its binding to Megator/Tpr at 37 nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings 38 controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis 39 remain elusive. Here, we show that Mps1-mediated phosphorylation of Megator/Tpr 40 abolishes its interaction with Mad1 in vitro and in Drosophila cells. Timely activation of 41 Mps1 during prophase triggers Mad1 release from NPCs, which we find to be required for 42 competent kinetochore recruitment of Mad1-C-Mad2 and robust checkpoint response. 43 Importantly, preventing Mad1 binding to Megator/Tpr rescues the fidelity of chromosome 44 segregation and aneuploidy in larval neuroblasts of Drosophila mps1-null mutants. Our 45 findings demonstrate that the subcellular localization of Mad1 is stringently coordinated with 46 cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs 47 in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently 48 preserve genomic stability.