Plk1 destruction controls the timing of Cep55 recruitment to the midbody. (A) HeLa cells were mock treated or treated with 100 µM MG132 for 120 min and stained for DNA with DAPI and mouse anti–α-tubulin, rabbit anti-Cep55 or MKlp1 (green), and goat anti-Plk1 (red). Bars, 10 µm. The number of Plk1-positive and Cep55-negative midbody structures in the presence and absence of 100 µM MG132 was counted for 120 cells in each of three independent experiments. (B and C) HeLa cells stably expressing mCherry-Cep55 and either doxycycline-inducible EGFP-tagged wild-type Plk1 or the D-box mutant of Plk1 were imaged every minute with a spinning-disk confocal microscope. At the times indicated (arrows), 100 µM MG132 or 1 µM BI2536 was added. Fluorescence intensity of Plk1 and Cep55 was measured for the full central spindle and midbody volume and is plotted against time. A bar graph shows the times spent in the different stages of mitosis for 15 cells in three independent experiments. (D and E) Still images of Cep55 behavior in control, MG132, and BI2536 (D) cells and wild-type and D-box mutant Plk1 (E) cells are shown. Times are shown in hours:minutes. Error bars indicate SD. Bar, 5 µm.