Depletion of Megator restores Mad1 KT recruitment and mitotic fidelity in Drosophila mps1-null neuroblasts. (A and B) Immunofluorescence images with ploidy histograms (A) and quantifications (B) of Mad1 levels at unattached KTs of w¹¹¹⁸, InscGal4>UAS-MegatorRNAi, homozygous mps1^G4422, or homozygous mps1^G4422;InscGal4>UAS-MegatorRNAi neuroblasts treated with colchicine (50 µM) for 90 min (n ≥ 91 KTs). (C–E) Mitotic progression (C), mitotic timing (D), and percentage of anaphases with lagging chromosomes (E) in neuroblasts with indicated genotypes coexpressing the microtubule-associated protein Jupiter-GFP and H2B-mRFP. The mitotic timing corresponds to the time from NEB to anaphase onset (AO; n ≥ 14 neuroblasts from at least two independent experiments). The arrowhead in C points to a lagging chromosome. (F) Model for the control of Mad1 subcellular redistribution during the G2/M transition. In interphase, inactive Mps1 (unphosphorylated T-loop) is in the cytoplasm and Mad1-C-Mad2 is docked to Megator at the nucleoplasmic side of NPCs, where it catalyzes the assembly of premitotic MCC. During prophase, active Mps1 (phosphorylated T-loop) becomes detectable in the nucleus and is now able to phosphorylate Megator, disrupting the interaction with Mad1. This ensures timely release of Mad1-C-Mad2 from NPCs, which enables Mad1-C-Mad2 to accumulate at prometaphase KTs and instate robust SAC signaling. In A–C and E, data are presented as mean ± SD. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). Scale bars, 5 µm (inset, 0.5 µm).