HCP-1 cooperates with HCP-2 to promote robust central spindle assembly. (A) Representative pseudokymographs of AIR-2::GFP (green) and mCherry::H2B (magenta) localization throughout cell division; see Video 5. Time on the left; t = 0 s is the metaphase before anaphase onset [or chromosome segregation in hcp-1(RNAi); hcp-2Δ embryos that did not form a metaphase plate]. Images were acquired every 15 s and are shown every 30 s; scale bar, 10 µm. (B) Method used for line scan analysis of AIR-2::GFP distribution (left) and quantification (right) of average fluorescence intensity at the central spindle 30 s, 60 s, 90 s, and 120 s after chromosome segregation on sum projections of all Z-planes. Error bars represent the SEM; n is listed in the key [n = 10 control(RNAi), n = 10 hcp-1(RNAi); hcp-2Δ, n = 12 hcp-1Δ, n = 10 hcp-2Δ, n = 12 spd-1(oj5ts), and n = 12 zen-4(or153ts)]. (C) Top: Representative images of SPD-1::sfGFP (inverted contrast, top; green, bottom) and mCherry::H2B (magenta, bottom) at the central spindle 60 s after anaphase onset [or chromosome segregation in hcp-1/2(RNAi) embryos that did not form a metaphase plate]; scale bar, 5 µm. Bottom: Method used for SPD-1::sfGFP midzone analysis (left; black dashed line outlines embryo; scale bar, 10 µm) and quantification (right) at 60 s after chromosome segregation. Genotype is indicated below graphs; n is listed in the key [right; n = 12 control(RNAi), n = 11 hcp-1/2(RNAi), n = 13 hcp-1(RNAi), n = 13 hcp-2(RNAi), and n = 9 spd-1(RNAi)]. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD. n.s., P ≥ 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.