Cdk1 phoshorylation of Cdc20 is required for an efficient mitotic arrest in response to microtubule poisons. (A) Live cell imaging of Hela cells depleted of endogenous Cdc20 and transiently expressing untagged Cdc20 WT, 3DD, or 3AA in the presence of nocadazole (30 ng/ml). Percentage of cells that exit mitosis during the course of imaging from three independent experiments (mean and SD indicated). (B) As in A, time from nuclear envelope breakdown (NEBD) to mitotic exit or end of filming from three independent experiments (each dot represents a single cell, red line indicates mean and SD, n values are shown in the graph and represent single cells analyzed per condition; statistical analysis with Mann-Whitney test; ****P > 0.0001). (C) Live cell imaging of stable cell lines that were depleted of endogenous Cdc20 and expressing indicated YFP-tagged Cdc20 proteins in the presence of nocodazole (30 ng/ml). The R132A mutation prevents Mad2 binding, while the 4A mutations prevent BubR1 binding. Time from NEBD to mitotic exit (each dot represents a single cell, red line indicates mean and standard deviation, n values are shown in the graph and represent single cells analyzed per condition, statistical analysis with Mann-Whitney test; ****P > 0.0001). (D) Model: MCC-bound BubR1 is preferentially dephosphorylated by PP2A-B56, whereas free Cdc20 is phosphorylated by Cdk1.