Defective anaphase spindle structure in the FF1154AA PRC1-binding mutant of KIF4A. (A) Superresolution images of WT KIF4A and mutants, showing anaphase localization of KIF4A and PRC1. Arrows mark the spread of PRC1 signal at the central spindle. (B) HeLa Flp-In T-Rex cells depleted of endogenous KIF4A induced to express GFP-PRC1 and cotransfected with either mCherry-KIF4A WT or the FF1154AA mutant were imaged every 2 min while entering anaphase. Brightfield (BF) reference images are also shown. (C–F) Graphs shows the length of the PRC1 overlap at the midzone (C) and chromosome separation as a function of time for the WT and the FF1154AA mutant (D). The start of furrowing is marked. Plots in E and F measure the distribution of PRC1 and the diameter of the cell during mitotic exit for the WT and the FF1154AA mutant. For PRC1 overlap in C, the mean and SD are plotted; in all other cases, the mean and SEM are plotted (WT n = 7, FF1154AA n = 10).