CRISPR-tagged endogenous GFP::HCP-1 and GFP::HCP-2 localization. (A) Representative pseudokymographs showing midzone localization of mCherry::H2B (magenta) and endogenously tagged GFP::HCP-1 (green, left) or GFP::HCP-2 (green, right) during early central spindle assembly; see also Videos 1 and 2. Time is indicated on the left (imaging every 10 s), where t = 0 s is the metaphase just before anaphase onset. (B) Schematic of the method used for line scan analysis (left) and quantification (right) of GFP::HCP-1 (top) and GFP::HCP-2 (bottom) levels at the kinetochore (metaphase, left) and at the spindle midzone (30 s after chromosome segregation, right). Error bars represent the SEM. (C) Total levels of GFP::HCP-1 and GFP::HCP-2 at the spindle midzone (between chromosomes) 30 s after chromosome segregation in anaphase, normalized to levels in control(RNAi) embryos. Significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD. (D) Schematic of the method used for analysis (left) and quantification (right) of central spindle levels of GFP::HCP-1 and GFP::HCP-2, where t = 0 s represents kinetochore levels at metaphase, and all other time points represent protein levels between chromosomes at the spindle midzone; white dashed line outlines the embryo; error bars represent the SD. Key indicates n for all data shown [GFP::HCP-1: n = 10 control(RNAi), n = 12 hcp-1(RNAi); n = 12 hcp-2(RNAi); and n = 8 hcp-1/2(RNAi); GFP::HCP-2: n = 11 control(RNAi), n = 10 hcp-1(RNAi); n = 12 hcp-2(RNAi); and n = 9 hcp-1/2(RNAi)]. Scale bars, 10 µm. n.s., P ≥ 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.