Posttranslational control of Cdc25 degradation terminates Drosophila's early cell-cycle program

Abstract

In most metazoans, early embryonic development is characterized by rapid mitotic divisions that are controlled by maternal mRNAs and proteins that accumulate during oogenesis. These rapid divisions pause at the midblastula transition (MBT), coinciding with a dramatic increase in gene transcription and the degradation of a subset of maternal mRNAs. In Drosophila, the cell-cycle pause is controlled by inhibitory phosphorylation of Cdk1, which in turn is driven by downregulation of the activating Cdc25 phosphatases. Here, we show that the two Drosophila Cdc25 homologs, String and Twine, differ in their dynamics and that, contrary to current models, their downregulations are not controlled by mRNA degradation but through different posttranslational mechanisms. The degradation rate of String protein gradually increases during the late syncytial cycles in a manner dependent on the nuclear-to-cytoplasmic ratio and on the DNA replication checkpoints. Twine, on the other hand, is targeted for degradation at the onset of the MBT through a switch-like mechanism controlled, like String, by the nuclear-to-cytoplasmic ratio, but not requiring the DNA replication checkpoints. We demonstrate that posttranslational control of Twine degradation ensures that the proper number of mitoses precede the MBT.

Figure 1. String and Twine undergo different down-regulations, which are not controlled by mRNA levels

Representative His2Av-RFP, String-GFP (A) and His2Av-RFP, Twine-GFP (B) images from time-lapse confocal microscopy. Time from the beginning of the movie as well as interphase number are reported in every panel. C) Images of an embryo used for immunofluorescence analysis. DNA was visualized by Hoechst staining, String using an anti-String rabbit antibody, Twine using an anti-Twine rat antibody. Comparison between the dynamics measured by live imaging, the dynamics inferred by immunofluorescence and the mRNA dynamics measured using q-PCR for String (D) and Twine (E). For both live imaging and immunofluorescence, we quantified the nuclear fluorescence intensity and normalized the data to the maximum value. F) Comparison between the protein dynamics of String and Twine. See also Figure S1 and Movies S1-S2.

Figure 2. String and Twine levels are controlled by the N/C ratio but respond differently to the DNA replication checkpoints

String-GFP fluorescence intensity, endogenous String intensity measured with immunofluorescence and mRNA levels measured by q-PCR for diploid and haploid embryos (embryos laid by mothers mutant for the gene sesame (ssm) [24]) as a function of time (A) and the N/C ratio (B). Twine-GFP fluorescence intensity, endogenous Twine intensity measured with immunofluorescence and mRNA levels measured by q-PCR for diploid and haploid embryos as a function of time (C) and the N/C ratio (D). Comparison of the String-GFP (E) and Twine-GFP (F) dynamics in wt and grp lok embryos. The inset in (F) shows the dynamics of Twine-GFP in the last maternal cycle in wt (cycle 14) and grp lok (cycle 15). Fluorescence intensities were normalized to c_MBT defined as the maximum concentration in the last maternal cycle. See also Figure S2 and Movie S3.

Figure 3. A post-translational switch controls Twine degradation at the onset of the MBT

A) Representative Twine-Dronpa and String-Dronpa images for the measurement of protein stability. Time from the beginning of the movie as well as interphase number are reported in every panel. B) Conceptual scheme for the measurement of protein degradation rate. Degradation rate of Twine (C) and String (D) as a function of inferred developmental time. E) The mass action reactions and equations describing Twine and String dynamics. String-Dronpa and Twine-Dronpa can exist in two states (an immature state p and a mature fluorescent state p*) with first order transition from the immature to the mature state (with rate k_mat). m indicates the amount of mRNA, k_tr the protein translation rate and k_deg(t) the time dependent degradation rate measured experimentally. Comparison between the measured fluorescence intensity and the numerical solution of the mass action kinetic equations for Twine (F) and String (G). See also Figure S3.

Figure 4. The signals targeting Twine and String for degradation are transient

A) Quantification of Twine dynamics in embryos expressing Twine-GFP maternally and zygotically. B) Quantification of Twine dynamics in embryos expressing String-GFP maternally and zygotically. Estimate of the degradation rate (and half-life) of Twine (C) and String (D) following their reaccumulation in interphase 14 and interphase 15. See also Figure S4 and Movie S4.