Figure 2.

Centrioles assemble de novo, mature, and duplicate within the same explants, in the absence of cell-cycle progression. Images show maximum-intensity z projections from time-lapse videos of cytoplasmic explants extracted from noncycling unfertilized eggs overexpressing Plk4. Newly assembled centrosomes load Plk4 (A and B), Ana1 (C and D), Asl (E and F), and Spd2 (G, H, and J) shown in green, and nucleate MTs as reported by the MT-associated protein Jupiter (magenta). The larger green blobs result from yolk autofluorescence, highly noticeable in the Plk4 and Spd2 panels. (B, D, and F) Centrioles formed de novo also duplicate, which was inferred from changes in the intensity profile along the axis $\overline{AB}$ across the centrosomal signal (bottom plots); from a symmetrical Gaussian curve to a Gaussian mixture, suggesting the presence of more than one diffraction-limited structure (centriole). A uni- or bimodal Gaussian distribution was fitted to each de novo and canonical intensity profile, respectively (dashed lines represent modes from fit). The coefficient of determination (R²) is presented for each fit. Scale bars, 0.5 µm. (G) Centrioles form de novo and canonically over time; therefore, both biogenesis pathways co-occur. Centriole duplication was inferred from the change in the intensity profile across the Spd2 signal (H, bottom plots). Uni- or bimodal Gaussian fitting as in B–F. Colors represent one centrosome that first assembled de novo and later duplicated. (I) The duplication time depicted in the graph is the time elapsed between the documentation of the first centriole formed de novo (unimodal density) and the detection of a centriole pair (bimodal density). The horizontal line and error bars represent the median and interquartile range (n = 66 explants from different eggs). (K) Insets of the first three centrosomes formed de novo in time-lapse (J) and their corresponding normalized and bleach-corrected intensity of Spd2 (L) and Jupiter reporting MTs (M), plotted over time. Time is reported in minutes:seconds. Scale bar, 0.5 µm. Norm., normal.