Figure 1.

Plk4 Levels Oscillate at the Centriole in a Process Entrained by the CCO

(A) Top panel: micrograph shows an image from a time-lapse movie of an embryo expressing Plk4-NG. Middle panels: micrographs illustrate the centriolar Plk4-NG oscillation during nuclear cycle 12—obtained by superimposing all the Plk4-NG foci (n = 60) at each time point (see STAR Methods). Bottom panel: quantification of centriolar Plk4-NG levels during nuclear cycles 11–13 in a single embryo (red arrows highlight equivalent time points in the middle panels).

(B) Graphs show the mathematical regression of centriolar Plk4-NG dynamics during S-phase of cycles 11–13 (regression mean ± SEM). R² values indicate goodness-of-fit. N ≥ 15 embryos; n = 24, 37, and 53 centrioles (mean) per embryo over cycles 11–13, respectively.

(C) The bar charts quantify the oscillation parameters—derived from the data shown in (B). Data are presented as mean ± SD. Statistical significance was assessed using an ordinary one-way ANOVA test (for Gaussian-distributed data) or a Kruskal-Wallis test (^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001; ns, not significant).

(D) Micrographs show, and pie charts quantify, the distribution of Plk4-NG at centrioles assessed by 3D-SIM at the indicated phases of the nuclear cycle (see STAR Methods). N = 6 embryos per cell-cycle stage; n = 20 centrioles per embryo; all images were scored blindly by 3 assessors and the mean score is shown (scale bar, 0.5 μm).

(E) Graph shows the mean regression of Plk4-NG oscillations in nuclear cycle 12 of WT embryos (green), or in embryos where the genetic dose of either cyclin B (CycB^1/2; blue) or grapes (Drosophila Chk1) (grp^1/2; red) has been halved to slow or speed-up the nuclear cycles, respectively. Dashed lines mark the center (peak) of the Plk4-NG oscillations (denoted with C), and dotted lines indicate the time of NEB (denoted with N) for each genotype. N ≥ 14 embryos for each condition; n = 55, 43, and 44 centrioles (mean) per embryo in WT, CycB^1/2, and grp^1/2 embryos, respectively. To clearly illustrate the phase shift in the oscillations, the highest mean fluorescence signal for each group was normalized to 1.

(F) Bar charts quantify the time at which the Plk4-NG oscillations peaked, the length of S-phase, and the ratio between them (C/N)—derived from the data shown in (E). Data are presented as mean ± SD. Statistical significance was assessed using an ordinary one-way ANOVA test (for Gaussian-distributed data) or a Kruskal-Wallis test (^∗∗p < 0.01; ^∗∗∗p < 0.001; ns, not significant).

See also Figures 6, S1, and S2.