Fig. 3
Expression of Mutant CEP164 in Renal Epithelial Cells Abrogates Localization to Centrosomes (A) Immunofluorescence using α-SDCCAG8/NPHP10-CG antibody in MDCK cells, labels both centrioles, whereas α-CEP164-ENR antibody demonstrates CEP164 staining at the mother centriole only. (B) Inducible overexpression of N terminally GFP-tagged human full-length CEP164 isoform 1 (NGFP-CEP164-WT) in IMCD3 cells demonstrates, in addition to a cytoplasmic expression pattern, localization at one of the two centrioles (inset, arrow heads) consistent with selective localization to the mother centriole Graser et al., 2007). Both centrioles are stained with an anti-γ-tubulin antibody (C) In contrast, the centrosomal signal is abrogated upon overexpression of an N terminally GFP-tagged truncated CEP164 construct representing the mutation p.Q525X. (D) The number of centrosomes positive for CEP164 is reduced upon overexpression of C terminally GFP-tagged human full-length CEP164 isoform 1 (CGFP-hCEP164-WT), which mimics the mutation p.X1460WextX57 that causes a read-through of the stop-codon X1460, adding 57 aberrant amino acid residues to the C terminus of CEP164. Similar data were obtained upon CEP164 expression in hTERT-RPE cells (see also Figures S3B–S3D). IMCD3 cells were stably transfected with the respective CEP164 constructs in a retroviral vector for doxycyclin-inducible expression (pRetroX-Tight-Pur). Scale bars, 10 μm (E–H) Knockdown of Cep164 disrupts ciliary frequency. (E) Depletion of Cep164 by siRNA (F) causes a ciliary defect in 3D spheroid growth assays. IMCD3 cells transfected with either siCtrl or siCep164 were grown to spheroids in 72 hr and immunostained for acetylated tubulin (red). DAPI stains nuclei (blue). Doxycycline induced stably transfected NGFP-hCEP164-WT (green). Space bar represents 5 μm. (G) Nuclei and cilia were scored within a single spheroid to generate ciliary frequencies. siCep164 transfected cells manifest lower cilia frequencies (33%) compared to control transfected IMCD3 cells (49%). Induction of NGFP-hCEP164-WT in siCep164 transfected cells rescues this ciliary defect (57%). 50 spheroids per condition were analyzed in three independent experiments. Error bars represent SEM, n = 3, p value < 0.0002. (H) Ciliary frequency is not rescued by mutant CEP164. Ciliary frequencies are reduced in siCep164 transfected IMCD3 cells (39%) compared to control siCtrl transfected IMCD3 cells (54%). Induction of NGFP-hCEP164-Q525X does not rescue this ciliary defect (34%). 50 spheroids per condition were analyzed. Error bars represent SEM, ***p value < 0.0002 See also Figure S3. |
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