The S-phase checkpoint delays nuclear CycB accumulation and CDK1 activation by a Grp and Wee1-dependent mechanism. (A–E) Wild-type (A and C), grp (B and D), and wee1 (E) embryos expressing RLC-GFP (cyan) were injected with a mix of Aph and CHX (CHX + Aph) at metaphase of cycle 12. 10 min after the onset of the next interphase, they were injected with CycB-R (red) at the specified concentrations. The schematic describes the injection and imaging sequence. Time is given in minutes/seconds (see Videos 6, 7, and 8 for A, B, and E, respectively). The right column of each panel is an enlargement of the area outlined with the white box shown on the first image of the left column. However, for A and B, the areas are at a focal plane 2 μm above the focal plane shown in the left columns. Blue outlines mark the areas with cytoplasmic CDK1 activity, and orange outlines mark the areas undergoing NEB. (C and D) Red arrows point to the nuclear localization of CycB-R. n, number of embryos injected. Bars: (left columns) 20 μm; (right columns) 5 μm. (F–H) 100 (F), 56 (G), and 13 μM (H) CycB-R was injected 10 min after the onset of interphase in CHX + Aph–treated embryos. The timing of cytoplasmic and nuclear CDK1 activation was determined as described in Fig. 2. (I) The timing of CycB-R accumulation in nuclei was determined when CycB-R appeared at kinetochores in at least three nuclei. (F–I) Data are represented as mean ± AVD. n, number of embryos injected.