Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. (B–D) Growth curves of DT40 wild-type (WT) and INCENP^ON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENP^WT (B), TrAP-INCENP^WT plus TrAP-INCENP^W766G (C), and TrAP-INCENP^WT plus TrAP-INCENP^F802A (D). Growth of cultures expressing TrAP-INCENP^WT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENP^ON/OFF cells (E) or INCENP^ON/OFF cells expressing TrAP-INCENP^W766G (F) or TrAP-INCENP^F802A (G). INCENP^OFF cells expressing TrAP-INCENP^WT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.