CLS-2^CLASP::GFP localization at the spindle midzone with and without hcp-1/2 disruption. (A) Representative pseudokymographs of CLS-2^CLASP::GFP (green) and mCherry::H2B (magenta) localization during central spindle assembly; see also Video 3. t = 0 s (imaging every 10 s) represents the time point just before chromosome segregation in anaphase [or sooner in the absence of metaphase plate assembly in hcp1/2(RNAi); see Edwards et al. (2018)]. Time (in s) after chromosome segregation is indicated on the left; scale bar, 10 µm. (B) Schematic of method used for CLS-2^CLASP::GFP analysis (top), and quantification (bottom) of CLS-2^CLASP::GFP fluorescence intensity along a line scan at the kinetochores (0 s, left) and spindle midzone (30 s, right) on sum projections of all Z-planes. (C) Schematic depicting the method used for CLS-2^CLASP::GFP intensity quantification at the kinetochores and central spindle on sum projections of all Z-planes; white dashed line outlines the embryo. (D) Quantification of CLS-2^CLASP::GFP fluorescence intensity at the kinetochores (left) and at the central spindle 10 s and 20 s after chromosome segregation (right). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD. (E) CLS-2^CLASP::GFP levels over time at the kinetochores (t = 0 s) and central spindle (t = 10–90 s). n is listed to the right of the graph in the indicated color for each genotype [n = 12 control(RNAi), n = 13 hcp-1(RNAi); hcp-2Δ, n = 13 hcp-1(RNAi); and n = 12 hcp-2(RNAi)]; error bars represent the SEM; scale bars, 10 µm. n.s., P ≥ 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.