Activating phosphorylation of Polo is required for cytokinesis. (A) Expression of Polo^WT-GFP, Polo^T182A-GFP, and Polo^T182D-GFP was induced with CuSO₄ (300 µM), and cells were transfected with Polo 3′ UTR dsRNA (or control KAN dsRNA). The next day, protein extracts were analyzed by Western blots. (B) Time-lapse imaging of cells depleted of endogenous Polo and expressing Polo^WT-GFP, Polo^T182A-GFP, or Polo^T182D-GFP. Note the early or late cytokinesis failures in Polo^T182A-GFP–expressing cells. Asterisks indicate nuclei in binucleate cells. Insets show enlarged views of the midbody (taken from the boxed regions). Bars: (main panels) 5 µm; (insets) 1 µm. (C and D) Quantification of cells showing a cytokinesis failure from the experiment in B. (E) Depletion of Map205 rescues cytokinesis in Polo^T182A-GFP expressing cells. Cells were transfected with the indicated dsRNA and analyzed by time-lapse imaging. At least 180 cell divisions were scored for each condition in four independent experiments. Error bars indicate SD (**, P < 0.01; ***, P < 0.001; Student’s t test; ns, nonsignificant).