Fig. 7: Mutations in the conserved CC motif of Cep152 result in misorganizing pericentriolar Cep152.

a–e 3D MINFLUX data for U2OS cells expressing the indicated RNAi-insensitive mGFP-Cep152 constructs (WT, ∆23, and 2D) and depleted of endogenous Cep152 by RNAi. Raw fluorescence signals acquired with an anti-GFP nanobody fused to a single Alexa Fluor 647 (i.e., one fluorophore per nanobody) were filtered and processed as shown in Supplementary Fig. 7b, c (see also Methods). Representative images in (a) are shown with dotted boundaries [the maximum (excluding outliers; white dotted line) and the 5th–95th percentiles of the entire minimum–maximum values (yellow dotted lines) of Cep152 WT (see Supplementary Fig. 7d, e)] for easy comparison. Numbers, the radial width and height of the mGFP-Cep152 WT. Concentrations of mGFP-Cep152 in (b) were calculated from Supplementary Fig. 7e. Rendered 3D images (top, generated by binning localizations into 0.5 × 0.5 × 0.5 nm voxels, also see Methods), and all localizations plotted in 3D (bottom; respective movies in Supplementary Movie 3) are provided. To reveal mislocalized mGFP-Cep152 signals (c–e), the entire min–max range data points shown in Supplementary Fig. 7e were plotted for WT, ∆23, and 2D (c, d), and the population outside of the 5th–95th percentiles of the WT radius was determined (e). Note in (d) that, unlike Cep152 WT (gray dots), both ∆23 an 2D mutants are spread out over a larger area, yielding a greater mislocalized population. All quantifications in (a–e), n = 15 centriole images for each group obtained from three independent experiments. Bars in (b, e), mean of n ± s.d. *P < 0.05, ****P < 0.0001 (unpaired two-tailed t test). Vertical lines in (c, d), median with 5th–95th (thin red lines) and 1st–99th percentiles (thin blue lines). f FRAP analysis for the mGFP-Cep152-mCherry fluorescence localized around a centriole. Images were acquired for 42 minutes at 3-minute intervals. Representative confocal images acquired before and after photobleaching are shown in Supplementary Fig. 7g. Relative signal intensities were quantified from three independent experiments [n = 18 for Cep152/siCep152 (n = 6/experiment); n = 17 for Cep152 (Δ23)/siCep152 (n ≥ 5/experiment); n = 18 for Cep152 (2D)/siCep152 (n = 6/experiment)]. Bars, mean of n ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test). n.s., not significant.