SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the γ-tubulin–mediated addition of centriolar microtubules

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Figure S2. The centriolar GFP:SAS-6 and GFP:SAS-4 signals are not subject to detectable photobleaching under the imaging conditions used. Because our recruitment profiles are generated using fluorescence intensity measurements from multiple z series per embryo, we needed to exclude the possibility that photobleaching was affecting our results. To determine whether our imaging conditions resulted in detectable photobleaching, we took advantage of the fact that the sequences from different embryos begin at different time points after fertilization (depending on their stage when we dissect them out of the mother) and end at cytokinesis onset during the first mitotic division, which we use to time-align the sequences. Therefore, we can determine if photobleaching is occurring by plotting the individual fluorescence measurements made during a small time window with respect to cytokinesis onset as a function of the number of previous exposures for that embryo. If photobleaching were occurring, one would expect to see less signal in embryos that had been filmed from meiosis forward and had therefore received more exposures before the test interval than in embryos that had just been mounted on the microscope stage when the interval began. For this analysis, we chose a small time interval during which there was no overall change in the mean centriolar signal for each GFP fusion (intervals are indicated by the boxes on the recruitment curves in A and C; −800 to −400 s for SAS-6 and −800 to −500 s for SAS-4). The individual fluorescence intensity measurements made during these intervals from embryos imaged during one experiment were then plotted versus the number of previous exposures that that embryo had received (B and D; the number of prior exposures varied from 1 to just over 200 because the maximum number of 11-frame z series taken per embryo was 20). Because there was no negative correlation between fluorescence signal and the number of previous exposures for either marker, we conclude that our conditions did not result in detectable photobleaching. Error bars indicate the 90% confluence level.