Aurora B localization and activity at the equatorial cortex. (A) IF for MKLP2 and Aurora B in HeLa cells transfected with siLuc or siPRC1. Dotted line indicates z plane for z-axis view. (B) IF for Anillin and Aurora B in anaphase cells treated with 83 nM nocodazole to depolymerize microtubules that are not part of the spindle midzone. Dotted line indicates z plane for line plot analysis of Anillin (green) and Aurora B (red) intensity (far right). (C) Left: Scheme of the FRET-based Aurora B biosensor fused to Tubby protein (green line). Right: HeLa Flp-In T-Rex cells stably expressing Tubby-Aurora B FRET sensor (green) and H2B-mCherry (red). White boxes indicate the areas where FRET was measured. (D) Measurement of distance between the separating sister chromatids and the width of the ingressing furrow (Δ cortex). (E and F) HeLa cells stably expressing the Tubby-Aurora B FRET sensor and H2B-mCherry were synchronized in G2 by treatment with the CDK1 inhibitor RO3306 and imaged live after release from the G2 block. The emission ratio at the equatorial cortex was calculated for each time point (interval, 25 s; mean ± SD of 10 cells) and plotted with the distance between the separating sister chromatids (E) or with the width of the ingressing furrow (F). (G) Color-coded images of the YPet/mTFP1 emission ratios. Bars: 5 µm (A and B); 10 µm (C and G).