A FRET-based biosensor for Plk1 activity at kinetochores is dephosphorylated as chromosomes align at metaphase. (A) The YFP/CFP emission ratio was averaged over multiple mitotic cells (n ≥ 9) expressing an untargeted Plk1 phosphorylation sensor and treated with an inhibitor for Aurora B (ZM) or Plk1 (BI2536) or with Plk1 siRNA, as indicated. (B) The YFP/TFP emission ratio was analyzed in cells expressing a kinetochore-targeted Plk1 sensor at metaphase or treated with nocodazole or BI2536, as indicated (n ≥ 10 cells, n ≥ 15 kinetochores per cell). (C and D) Cells expressing a kinetochore-targeted Plk1 sensor (C and D) or a kinetochore-targeted Aurora B sensor (D) were imaged live after nocodazole washout. Images (C) of the Plk1 sensor (YFP emission) show kinetochore alignment after washout. To compare FRET changes for the two sensors (D), the ratios for each were normalized by dividing by the maximum value for that sensor (n ≥ 10 cells, n ≥ 15 kinetochores per cell).