Figure 1. Cep295 was identified as an evolutionarily conserved protein required for centriole formation.

(a) Schematic diagrams for Human Cep295 (HsCep295) and Drosophila Ana-1 (DmAna-1). The DDC8-like domain is shown in grey box, the ALMS domain in black box. The positions of two evolutionarily conserved domains are indicated in orange and blue lines, respectively. (b) Alignments of the evolutionarily conserved domains within human, mouse, chicken, Xenopus and Orizias Cep295 and Drosophila Ana1. The orange and blue boxes indicate the conserved region within the DDC8-like domain and PICA (Present in C-terminal of Ana-1) motif, respectively. Identical residues determined by Cluster W2 are shown in red. Asterisks indicate the residues identical in all aligned sequences; colons: conserved substitutions; periods: semi-conserved substitutions. (c–e) Cep295 ensures proper mitotic spindle and centriole formation. Cep295-depleted HeLa cells were stained with the indicated antibodies. Nuclei are shown in blue. Insets show approximately two-fold magnified views around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with the indicated phenotype (d) or with ⩾4 centrin foci (e). Values are mean percentages±standard error of the mean (s.e.m) from three independent experiments (N=30 for each condition). ***P<0.001 (two-tailed t-test). (f,g) Schematic of full-length Cep295 and the deletion constructs used for immunofluorescence assay in HeLa cells in (f). All constructs encode siRNA-resistant forms of Cep295. The table shows presence (+) or absence (−) of centriolar localization of the deletion mutant proteins examined in the cells depleted of endogenous Cep295. In (g), DDC8-like domain (aa 1–564) is required for centriolar localization of Cep295. The cells were stained with the indicated antibodies. Nuclei are shown in blue. Arrows point to the centrioles. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. (h,i) For rescue experiments, Cep295-depleted HeLa cells were transfected with full-length Cep295 and the indicated mutants. The cells were stained with the indicated antibodies. Nuclei are shown in blue. Scale bar, 1 μm. Histograms represent frequency of interphase cells with the indicated number of γ-tubulin foci in each condition. Values are mean percentages±s.e.m from three independent experiments (N>50 for each condition). **P<0.01; *P<0.05; NS, not significant (two-tailed t-test).