Phosphorylation of EB3 by spatially regulated Aurora B. (A) Frames from time-lapse movies of cells expressing α-tubulin–mRFP and either EB3-FL or EB3-S176D tagged with GFP in the presence or absence of the Aurora B inhibitor ZM447439 upon EB3 RNAi. Higher magnification images highlight the midbodies before and after dissolution. Time is given in hours:minutes. Bars: (main panels) 10 µm; (insets) 3 µm. (B) Quantification of the percentage of cells that fail cytokinesis after inhibition of Aurora A (MLN8054) or Aurora B (ZM447439), and the respective dependence on EB3 phosphorylation. Error bars indicate mean ± SEM. (C) Correlation between the midbody dissolution time and cytokinesis outcome in cells expressing either EB3-FL or EB3-S176D and treated with the Aurora B inhibitor upon EB3 RNAi. Note the positive correlation between the midbody dissolution time and cytokinesis success. (D) Immunolocalization of endogenous pEB3-S176. Cells expressing the EB3-MT-GFP construct were used to facilitate detection of MT plus ends. The ratio between pEB3-S176 and EB3-MT-GFP is shown on the right. Bars, 10 µm. (E) Quantification of the subcellular colocalization of endogenous pEB3-S176. Note that phosphorylation of EB3 at S176 occurs throughout the spindle in early mitosis but is concentrated in the midbody in telophase. Error bars indicate mean ± SEM. (F) Inhibition of Aurora B leads to a significant decrease in the levels of pEB3-S176 in the midbody when normalized relative to EB3-MT-GFP and tubulin. (*, P < 0.001 using nonparametric ANOVA followed by a post-hoc Dunn’s test). Experiments were done in triplicate and n represents the number of cells quantified in each condition.