Figure 1.

Two distinct sizes of Plk4 ring structures in the absence or presence of the Cep152 ring around Cep192-decorated centrioles. (a) 3D -SIM images showing asynchronously growing U2OS cells co-immunostained with anti-Plk4 (red), anti-Sas6 (blue; pseudo-colored in gray), Alexa 647 (magenta)-conjugated anti-Cep192 N-terminal (N), and Alexa 488 (green)-conjugated anti-Cep152 middle region (M) antibodies. Arrows in the 1^st panel indicate the outer diameters of two Plk4 rings—one from a daughter (D) and the other from a mother (M) centriole before and after Cep152 recruitment, respectively. A bracket on the 2^ndpanel indicates Plk4 signals colocalized with a nascent Cep152 toroid assembling at a daughter centriole. Arrowheads on the 3^rd and 4^th panels indicate dot -like Plk4 (red) signals colocalized with Sas6 (gray) on Cep152 toroids. Scale bars, 0.5 μm. (b) Quantification of the outer diameters of Cep192, Cep152, and Plk4 ring signals for the samples in Figure 1a. G1 centrioles prior to Cep152 recruitment (n=25) or after Cep152 recruitment (n=69), or S centrioles without discernable Plk4 ring signals (n=41) were measured. Error bars, s.d. (c) An immunoblot showing U2OS cells silenced for control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). Asterisk, cross-reacting protein. (d,e) Immunostaining (d) of the cells in c and subsequent quantification (e) from three independent experiments (n=105 for GL siRNA, n=116 for CEP152 siRNA). Arrows in d, the outer diameters of Cep192 and Plk4 rings; scale bars in d, 0.5 μm; error bars in e, s.d. An uncropped blot image for c is shown in Supplementary Figure 8a.