Local concentration of Aurora B is required for kinase activity. (A) HeLa cells were transfected with either INCENP-mCherry or CB^DBD–INCENP-mCherry with or without Borealin siRNA as indicated and fixed and stained for DNA, Borealin, and Aurora B. (B) A schematic showing targeting of phosphorylation sensors by fusion to Hec1 (kinetochores), CB (centromeres), or histone H2B (bulk chromatin). (C) Cells were transfected as described in A, together with an Aurora B phosphorylation sensor targeted to chromatin, and imaged live. The YFP/CFP emission ratio was analyzed to measure phosphorylation changes and averaged over multiple cells (n = 12 cells for each bar). 2 µM ZM was used to dephosphorylate the sensor, which is indicated by an increased emission ratio. The letters to the left of the vertical axis indicate how normalized phosphorylation was calculated: (c − b)/(a − b) for wt-INCENP or (d − b)/(a − b) for CB^DBD–INCENP. (D) The experiment described in C was repeated with sensors targeted either to centromeres or to chromatin. Normalized values were calculated for each experiment as in C and averaged over three independent experiments. (E) U2OS-LacO cells were transfected with mCherry-LacI or with LacI-INCENP–mCherry with or without Borealin siRNA as indicated and then were fixed and stained for DNA, Borealin, and Aurora B. (F) Cells were transfected as described in E, together with the chromatin-targeted Aurora B phosphorylation sensor, and imaged live. The normalized YFP/CFP emission ratio was averaged over three independent experiments. Bars, 5 µm.