Microtubule-binding mutants of KNL-1 do not affect formation of load-bearing attachments or chromosome segregation. (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. Bar, 2 µm. (B) A schematic of analysis performed after crossing in GFP::histone H2b and GFP::γ-tubulin and depleting endogenous KNL-1. (C) Frames from time-lapse sequences of the first embryonic division for the indicated KNL-1 mutants. Bar, 3 µm. (D) Spindle pole separation kinetics for the indicated conditions. Error bars represent the SEM with a 95% confidence interval. The no transgene knl-1(RNAi) trace is reproduced from Fig. 1 K. (E) Timing of anaphase onset in the indicated conditions. The first visible sign of sister chromatid separation (based on the GFP::H2b signal) was scored as anaphase onset. The gray dashed line indicates the NEBD–anaphase interval for the WT transgene (reproduced from the inset in Fig. 1 K). Error bars represent the SEM with a 95% confidence interval. For comprehensive statistical analysis, see Table S1. (F) Embryonic viability analysis of KNL-1 microtubule-binding mutants. L4-stage worms were injected with dsRNA-targeting endogenous knl-1, and the embryos laid by the injected worms were collected 21–41 h after injection and scored for hatching to form larvae.