Measuring the parameters of daughter centriole growth. (A) Schematic summary of the Sas-6-GFP image acquisition and processing procedure used to monitor Sas-6-GFP dynamics over time (see also Video 3). (B) Graph shows the Sas-6-GFP fluorescence intensity over time measured from four different centriole-pair tracks during nuclear cycle 12 (A.U., arbitrary units; Cent. Sep., time centriole pair separation first detected at the start of S-phase; NEB, nuclear envelope breakdown). Note that when Sas-6-GFP is imaged using spinning-disk confocal microscopy, the mother and growing daughter centrioles cannot be resolved, and so they appear as a single fluorescent focus. Therefore, to measure the growth of the daughter centriole, all centriolar Sas-6-GFP intensities were normalized by subtracting the mean initial intensity of all the mother centrioles in that embryo at the start of S-phase (so that the mean Sas-6-GFP fluorescence at the start of S-phase is 0 in every embryo). (C) Same as in B, but the mean has been taken of the fluorescence intensity of >100 centriole pairs from the same embryo. (D) The S-phase (green line) and M-phase (dark blue line) data from C were fitted by regression analysis (see Fig. S2 for a summary of the models tested). R2 is used as a measure of goodness-of-fit. From this model, several parameters of centriole growth were measured for each individual embryo (as indicated in the figure). Data are represented as mean ± SD.
