Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENPOFF cells expressing TrAP-INCENPWT (A), TrAP-INCENPW766G (B), or TrAP-INCENPF802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENPOFF cells expressing TrAP-INCENPW766G or TrAP-INCENPF802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENPW766G, where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.
