PP1-docking mutants of KNL-1 exhibit kinetic defects in load-bearing attachment formation and are synthetically lethal with checkpoint inhibition. (A) An image of a metaphase plate in living one-cell embryos from strains harboring the indicated KNL-1^RR::mCherry transgenes and depleted of endogenous KNL-1; the control image is reproduced from Fig. 2 A. Bar, 2 µm. (B) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The WT trace is reproduced from Fig. 2 D. (C) Frames from time-lapse sequences of the first embryonic division for the indicated KNL-1 mutants. Bar, 3 µm. (D) Biochemical analysis of KNL-1 interaction with GSP-2 performed as in Fig. 5 E. Molecular mass is indicated in kilodaltons. (E) NEBD–anaphase onset interval in one-cell stage embryos for the indicated conditions. See Table S1 for statistical analysis. (F) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The WT trace is reproduced from Fig. 2 D. (G) Embryonic viability analysis for the indicated conditions. Lethality was measured during two intervals (21–41 and 42–64 h) after endogenous KNL-1 depletion. In the earlier time point, embryos are depleted of maternal load but potentially inherit some dsRNA that affects zygotic KNL-1 expression; at the later time point, the maternal load is depleted, but zygotic expression is likely unaffected (see the legend of Fig. S2). (H) NEBD–chromosome decondensation interval in AB cells with bipolar or monopolar spindles for the indicated conditions. The red dashed line marks the duration of AB cell mitosis induced by monopolar spindles in the same strain. See Table S2 for statistical analysis. (E and H) The gray dashed lines mark the duration of AB cell mitosis for the WT transgene. Error bars represent the SEM with a 95% confidence interval.