Sas-6-GFP is incorporated into the proximal end of the growing daughter centriole. (A) Experimental scheme to test the site of centriolar Sas-6-GFP incorporation. Embryos expressing Sas-6-GFP and Asl-mCherry were allowed to proceed through S-phase until daughter centrioles were approximately halfway through their growth period. The distance (d1) between the centroid of the mother (calculated using the Asl-mCherry signal) and daughter (calculated using the Sas-6-GFP signal) was measured (time = T1). The centriole pair was bleached (time = T2; note that bleaching the GFP fluorescence unintentionally also bleached the Asl-mCherry fluorescence, but Asl-mCherry fluorescence at the mother rapidly recovered), and a second image acquired 1 min later (time = T3), when the distance between the centroids was measured again (d2). The difference between d2 and d1 will depend upon the site of Sas-6-GFP incorporation, as illustrated. Micrographs below the schematic illustrate examples of the images acquired at the corresponding time points. Bar, 0.2 μm. (B) Graph quantifies d1 and d2: in unbleached centrioles, which have grown slightly between T1 and T3, d2 is greater than d1; after bleaching d2 is less than d1, indicating that Sas-6-GFP is incorporated proximally. n = 10 embryos; n = 23 centriole pairs for control (unbleached) and 24 pairs for FRAP. (C and D) These panels show similar data to that shown in A and B, but for a control experiment examining where the distal-end-binding protein GFP-Cep97 is incorporated. n = 10 embryos; n = 25 centriole pairs for control (unbleached) and 28 pairs for FRAP. In this control, GFP-Cep97 appears to be incorporated distally, as expected, indicating that these methods are sensitive enough to distinguish between the proximal incorporation of Sas-6-GFP (B) and the distal incorporation of GFP-Cep97 (D). Bar, 0.2 μm. Midlines represent the median, whiskers (error bars) mark the minimum to maximum, and bottom/top of the boxes indicate the first/third quartile of the distribution, respectively. Statistical significance was assessed using a paired t test (**, P < 0.01; ****, P < 0.0001).
