Plk1 phosphorylation negatively regulates the interaction of Cep55 with the midbody component MKlp1. (A, top) HeLa cells stably expressing EGFP-tagged α-tubulin (green) and mCherry-tagged histone H2B (red) were treated with 1 µM BI256. Still images from videos are indicated with arrows to mark the position of the midbody and intracellular bridge. Bar, 10 µm. (bottom) The model depicts the decay in Plk1 levels and activity in anaphase while chromosome segregation by the microtubule spindle is taking place and the outcomes of inhibiting Plk1 in early or late anaphase. Lines at the second time point indicate the extent of chromosome segregation. Dotted lines in the last time point mark the outline of the binucleate cells. (B) HeLa cells in anaphase or undergoing cytokinesis were treated with DMSO (control) or 1 µM BI2536 for 25 min. Cep55 and MKlp1 complexes were isolated with sheep antibodies and analyzed by quantitative nano liquid chromatography MS/MS. The ratio of identified proteins in BI2536 and control conditions was plotted for anaphase versus midbody stages. Green dots indicate midbody proteins, red dots mark proteins increased after Plk1 inhibition in anaphase, and blue dots indicate other proteins. (C) The response of midbody proteins to Plk1 inhibition is summarized in the table. The number of peptides found is listed in the control and BI2536 columns. Fold change in abundance based on the summed intensities of the peptides forming each protein group was calculated using MaxQuant. IP, immunoprecipitation. (D) The same samples were Western blotted. (E) Western blotting of MKlp1 and Cep55 complexes isolated from synchronized HeLa cells at the different stages of mitosis. Western blot markers are given in kilodaltons.