Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

Abstract

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly.

Figure 1.

Cyclin-type effect on interphase timing. (A) As illustrated for RNAi knockdown of CycA (A) and CycB3 (B3), after pairwise cyclin knockdown, the dose of the remaining cyclin (CycB, B) did not significantly affect interphase length (McCleland et al., 2009b). Halving the gene dose of the remaining cyclin (performed for all combinations) had a minimal effect on interphase length (comparison on the left), arguing that cyclin level is not a major determinant of interphase length in these conditions. In contrast, the type of cyclin remaining (comparison on the right) influences interphase duration (this study). (B and C) Real-time records of histone (RFP-H2AvD) and centrosomes (GFP-TACC) show that RNAi against CycB+B3 (C), which created a situation in which the cell cycle was running mainly on CycA, greatly extended the interphase length in cycle 13 compared with the control (B; 28:12 vs. 13:46 [minutes and seconds]). Bar, 10 µm. (D) Interphase durations in control and each of the pairwise cyclin knockdown embryos. Mean interphase 13 length was 12.02 ± 0.92 min in control embryos, whereas knockdown of CycA+B, CycA+B3, or CycB+B3 extended interphase to 19.19 ± 1.57, 16.76 ± 1.67, or 24.81 ± 1.75 min, respectively (±SD). Horizontal lines show means.

Figure 2.

Diversity of cyclin types influences interphase length, whereas amount of a single cyclin has little effect. (A) Schematic of the experiment. dsRNA to two of the three cyclins was introduced throughout the embryo in cycle 10. Rhodamine-tagged GST-CycB protein was then injected at one pole during interphase 13. Embryos were imaged in regions 1 and 2, and the timing of mitosis in the two regions was determined. (B and C) Video frames of cycle 13 in which the remaining cyclin is CycB (B) or CycA (C). After injection of the GST-CycB protein, red fluorescence is seen in the injected pole (rhodamine). The absence of red at the other pole shows that significant GST-CycB does not reach the other pole in this time frame. Noting time stamps (minutes and seconds) on the images, it can be seen that GST-CycB does not advance mitosis when introduced into the CycB alone embryo but does advance mitosis when introduced into the CycA alone embryo. Bar, 5 µm. (D) A compilation of timing results from these experiments. Error bars represent SDs. n = 3.

Figure 3.

Interphase progression in cyclin RNAi-treated embryos. (A–D) Video frames of GFP-PCNA (top; white) and histone-RFP (bottom; red) during the thirteenth syncytial cycle. Time is given in minutes and seconds. (A) Control embryos had a short gap phase before mitotic entry (<2 min, between 11:12 and 13:37; Video 1). (B–D) Pairwise knockdown of mitotic cyclins prolonged interphase, mainly by extending the G2 (e.g., ∼6 min between 15:57 and 21:59 in B; Video 2). (E) S-phase duration. Mild prolongations of S phase were observed in CycA+B and CycA+B3 RNAi-treated embryos as compared with the control, whereas treatment with CycB+B3 RNAi had no significant effect. (F) Gap-phase duration. All the combinations of cyclin RNAi treatment extended the gap phase. CycB+B3 RNAi was most dramatic. Error bars represent SDs. n > 3. (G and H) Alexa Fluor 546–dUTP (red) was incorporated into DNA when injected before but not after the dispersal of PCNA foci (green) in CycB+B3 RNAi-treated embryos. Bars, 5 µm.

Figure 4.

The G2 and prolonged interphase after pairwise cyclin knockdown requires the replication checkpoint. (A) Inactivation of Chk1 (grp⁻) or deletion of S phase (by Geminin injection) shortened interphase in control and cyclin RNAi-treated embryos and reduced the differences among cyclin types. Error bars represent SDs. Detailed measurements are listed in Table S1. Data for cyclin RNAi experiments in wild-type embryos are reproduced from Fig. 1 D for purpose of comparison. (B–E) Video frames of grp⁻ mutant embryos in cycle 13 (GFP-PCNA is shown in white, and histone-RFP is shown in red) aligned at the start of DNA condensation (t = 00:00). Time is given in minutes and seconds. Bars, 5 µm. (F) A schematic model in which checkpoint inhibition of cyclin–Cdk1 (gray) is reversed by compartment-specific action of cyclins plus a slow communication between compartments (arrows).