Sas-6-GFP is incorporated irreversibly into the growing daughter centriole. (A and B) Micrographs show a 3D-SIM-FRAP analysis of Sas-6-GFP (A) or Ana2-GFP (B) dynamics. A 3D-SIM image of a centriole pair was acquired in early S-phase (Pre-bleach); the centrioles were then photobleached (Bleach), and a post-bleach image was acquired 5–10 min later. (C and D) The same image acquisition protocol was followed in embryos that simultaneously express the mother centriole marker Asl-mCherry along with either Sas-6-GFP (C) or Ana2-GFP (D; note that bleaching the Sas-6- or Ana2-GFP, as an unintended consequence, also bleaches the Asl-mCherry fluorescence). Asl-mCherry fluorescence rapidly recovers at the mother centriole, as reported previously (Novak et al., 2014, 2016), as does Ana2-GFP fluorescence, indicating that both proteins turn over at the fully grown mother centrioles. In contrast, Sas-6-GFP does not detectably recover at the mother centriole, but does recover at the daughter. This suggests that once Sas-6-GFP molecules are incorporated into the cartwheel structure they do not turn over, at least not over the time course of these experiments. The underlying schematics illustrate our interpretation of the behavior of the GFP and mCherry fusions. Bars, 0.2 µm. n = 4 embryos; n = 15 centriole pairs for each protein in each experiment.