Dynamic changes in the localization pattern of Plk4 during centriole duplication. (A) Live imaging of Plk4 endogenously tagged with mClover in HCT116 cells, using spinning-disc confocal microscopy. DIC, differential interference contrast. The graph shows the mean ± SD fluorescence of Plk4–mClover (n = 5 cells). The arrows above the graph indicate approximate time points of metaphase. Scale bars, 10 µm. (B) Confocal immunofluorescence images of endogenous Plk4, with deconvolution. Synchronized cells were fixed at the indicated time after thymidine release and stained with antibodies against Plk4 and CEP152 (centriolar marker). Two representative images are shown for each time point. Scale bar, 0.5 µm. (C and D) Quantification of the Plk4 patterns. Representative oval profiles of Plk4 and the associated ring-filling indices (C) and a plot of all ring-filling indices (D) are shown. Representative images in C are from B. Schematics in C show how ring-filling indices are calculated. AU, arbitrary units; deg, degrees.