Cep55 S436A is prematurely recruited to the central spindle in anaphase. (A) HeLa cells transiently expressing EGFP-tagged wild-type, TS46, 47AA, and S436A mutant Cep55 were stained for DNA with DAPI and mouse anti–α-tubulin. Bar, 10 µm. (B, left) HeLa cells stably expressing doxycycline-inducible mCherry-Cep55 or the S436A mutant of Cep55 were imaged as they exited mitosis. Insets in phase-contrast images show enlargements of the midbody region (arrows). Times are shown in hours and minutes from the onset of anaphase. Bar, 5 µm. (right) The time of Cep55 recruitment from the start of anaphase was measured in wild-type and S436A-expressing cells and is plotted as a bar graph (n = 10). Error bars indicate SD. (C) The fluorescence intensity of Cep55 was measured for the full 3D central spindle and midbody volume using the quantitation and tracking software and is plotted against time for both wild-type Cep55 and the S436A mutant–expressing cells. Arrows indicate the point of metaphase to anaphase transition obtained by inspection of the chromosomes in phase-contrast images. All timings are set relative to this point.