Figure 4.

Low amounts of Cyclin B1 are sufficient to trigger mitosis and the threshold is reached by mid S phase. (A) To establish the threshold of Cyclin B1–Venus that can drive null cells into mitosis, fluorescence intensity was used as a read-out for the amount of protein and measured in individual blastomeres of −/− embryos; y axis arbitrary intensity units. The threshold concentration to trigger mitosis lies between 100 and 200 units above background intensity, corresponding to ∼1/10 of the sampled intensity range (n = 21 blastomeres from five experiments). (B) Wild-type Cyclin B1–Venus was injected into one blastomere of −/− embryos at the two-cell stage to rescue the arrest phenotype. Signal intensity was measured at the 8- to 16-cell transition after addition of Wee1 inhibitor. Only injected cells with a Cyclin B1 level above a threshold of ∼150 arbitrary units can be forced into mitosis by Wee1 inhibition; n = 17, 1 experiment. Error bars are the SD of the mean. (C–F) To test whether there is a time dependency of the inhibitory phosphorylation of Cdk1 by Wee1 the inhibitor MK1775 was added at different phases of the cell cycle to +/+ and +/− embryos. (C) Inhibitor added during mitosis or G1. Only 30% of cells enter to mitosis. 70% of cells arrest after a variable S phase (n = 35, six experiments). (D) Inhibitor added during early S phase leads to mitotic entry during mid-S phase in most cases; 80% (n = 36; six experiments). (E) Inhibitor added during mid-S phase forces cells into mitosis mostly in mid- and late S (together 85%; n = 34, six experiments). (F) Inhibitor added during late S phase drives cells into mitosis mostly with a much shortened G2 (see text for details; n = 24, five experiments). ES, early S phase; LS, late S phase; MS, mid-S phase.