In support of Fig. 2: Temporal analysis of de novo centriole biogenesis at extended spatial resolution. (A) Visualization of centrosome biogenesis in a Drosophila egg extract by 3D-Structured Illumination Microscopy. Maximum-intensity z projections from a time-lapse acquisition of an unfertilized egg explant overexpressing Plk4. Centrioles (insets) are detected as barrel-shaped structures surrounded by the PCM component Spd2 (green) associated with a MT array (magenta), reported by the MT-associated protein Jupiter. Insets are single-plane images of three different centrosomes. Scale bar, 0.5 µm. Centrioles formed de novo can duplicate. Time is reported as minutes:seconds. (B) Time-lapse (top row) of an egg explant overexpressing GFP–Plk4, in which centriole form de novo over time (arrows) as shown in magnified views (bottom). Numbers indicate sequence of formation. Inter-event times are shown in Fig. S4 A. (C) Time-lapse (top row) of an egg explant overexpressing Asl–mCherry, in which centrioles form de novo over time (arrows) as shown in magnified views (bottom). Numbers indicate sequence of formation; after de novo event #1, two centrioles formed concomitantly within the temporal resolution (#2 and #2′′) followed by another event (#3). Inter-event times are shown in Fig. S4 B. (D) Histogram of frame-to-frame (instantaneous) displacements of first event centrosomes. Most of the centrosomes performed random movement and only in rare cases they moved away in a directed fashion from the explant boundaries. (E) Comparison of centrosome movement versus distance between registered biogenesis. Cumulative distribution functions of frame-to-frame displacement (black) in comparison with all subsequent inter-event distances as presented in Fig. 3. This comparison shows that the probability for a biogenesis event to quickly displace a distance typically seen between biogenesis events is extremely low. Any subsequent event after the first biogenesis is unlikely a duplication-and-run event.