Two conserved FF motifs in the C-terminal tail of KIF4A support interaction with either condensin I or PRC1. (A) Sequence alignment of the KIF4A C-terminal tail from a group of metazoans reveals a high degree of conservation. (B) Endogenous KIF4A was immunoprecipitated from HeLa cells exiting mitosis. A 0-min time point was taken 25 min after nocodazole washout. Synchronous progression of mitotic cells into anaphase was triggered with 2 µM MPS1 inhibitor (AZ3146) to override the spindle checkpoint. The temporal association of KIF4A to condensin I and PRC1 was followed over time by Western blot. (C) Immunoprecipitation (IP) of WT FLAG-KIF4A (WT), deletion mutants, and alanine point mutants from anaphase HeLa cells. (D and E) Superresolution images of FlpIn T-REx eGFP-KIF4A HeLa cells induced with doxycycline to express KIF4A WT and point mutants in metaphase (D) and anaphase (E). Arrows mark the weak metaphase spindle staining for KIF4A FF1220AA and the cortical spread of microtubules in the FF1154AA PRC1-binding mutant and FF1154/1220AA double mutant in anaphase.