Real-time observation of phosphorylation spreading from centromeres. (A–C) Cells were transfected with the chromatin-targeted Aurora B phosphorylation sensor together with CB-mCherry to label centromeres and then were treated with monastrol, MG132, and ZM. Cells were imaged live during activation of Aurora B by ZM washout. (A) The YFP/CFP emission ratio was averaged over multiple cells (n = 11) to determine the kinetics of phosphorylation during ZM washout. The arrow indicates the time point analyzed in C. (B, top) Centromeres (CB-mCherry) and chromosomes (YFP emission) for a single time point. (bottom) Color-coded YFP/CFP emission ratio at different time points. The timestamp (minutes and seconds) is relative to ZM washout at t = 0. Bar, 5 µm. (C) The spatial phosphorylation gradient was analyzed by averaging the emission ratio over lines extending outward from mCherry-labeled centromeres (B, white lines) at t = 8 min (n = 11 cells, four centromeres per cell).