Figure 4: Cep295 is crucial for the integrity of a newly born daughter centriole at the older mother centriole.
From: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole
(a) 3D-SIM images showing centriolar distribution of Cep192 in control and Cep295-depleted HeLa cells (M: mother centriole; D: daughter centriole). Arrows point to the daughter centriole wall (N=5). Dotted lines indicate the shape of centriole cylinders in this figure. Scale bar, 400 nm. (b) U2OS cells were transfected with Flag-tagged Cep192 plasmid for 24 h. The cells were fixed and stained with antibodies against Flag and Cep295 for STED microscope. The right panels show magnified views. Arrowhead points to the cap-like structure of Cep295 at the procentriole assembly site before recruitment of Cep192 (N=10). Scale bar, 500 nm. The schematic illustrates the centriolar localization of Cep295 and Cep192. (c) STED images showing centriolar distribution of Cep295 in the presence or absence of HsSAS-6. HeLa cells were treated with control siRNA or HsSAS-6 siRNA for 48–60 h and stained with the indicated antibodies. To make sure the efficacy of HsSAS-6 depletion, we chose the Hs-SAS-6-depleted cells having only one mother centriole with a mono-polar spindle. Arrowhead shows centriolar distribution of Cep295 without the cartwheel structure (N=3). Scale bar, 500 nm. (d) Control and Cep295-depleted HeLa cells were visualized by STED microscope using the indicated antibodies. Arrows point to poly-glutamylation of centriolar microtubules stained with GT335 antibody at the daughter centrioles (N=3). Scale bar, 500 nm. (e,f) (e) HeLa cells treated with control siRNA or siRNA targeting endogenous Cep295 for 24 h were stained with antibodies against centrin1 (green) and Cep135 (red; a proximal component of centrioles). Nuclei are shown in blue. The arrow indicates defective recruitment of Cep135 to the disengaged daughter centriole. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. (f) Histograms represent frequency of late-mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m. from three independent experiments (N=30 for each condition). **P<0.01, (two-tailed t-test).
