In the absence of chromosome alignment, MN form around lagging chromosomes that travel long distances during anaphase. (A) Stills from time-lapse imaging of KIF18A-depleted cells expressing histone H2B-GFP. Arrows indicate lagging chromosomes that are excluded from the primary DNA mass and form MN. Data were collected from four independent experiments. (B) Representative images of live cells stably expressing GFP-CENP-A and GFP-CENTRIN-1 treated with control, KIF18A, or MAD2 siRNAs. Arrowheads indicate lagging chromosomes. (C–G) Survival plots of poleward anaphase velocity (µm/min; C), poleward anaphase speed (D), starting distance from the pole (E), distance traveled (F), and total anaphase time (G) for kinetochores in each experimental condition indicated. Dashed lines indicate the behavior of lagging chromosomes in KIF18A and MAD2 siRNA-treated cells. Data were compared using a Kruskal–Wallis test with post hoc Dunn’s multiple comparison tests. The starting distance from the pole and distance traveled for lagging chromosomes in KIF18A siRNA cells were significantly different than those in control siRNA cells or total kinetochores in KIF18A KD cells (P < 0.01). The anaphase velocity, speed, and distance traveled for lagging chromosomes in MAD2 KD cells are significantly different than those of kinetochores in control siRNA cells (P < 0.01). Data were collected from three independent experiments.