Fig 2: Unique functions for Talpid3, C2CD3, and OFD1 in assembling distal-end structures. a Localization of CEP350/FOP/CEP19 module, SDA proteins, and b, c the organization of cytoplasmic actin and centriolar satellites (CS) was examined in control, C2CD3-/-, OFD1-/-, and Talpid3-/- cells. Localization of Talpid3, C2CD3, and OFD1 was examined in control, CEP350-/-, FOP-/-, and CEP128-/- cells. Cells were serum-starved for 24 h and then visualized with indicated antibodies. Cumulative data from two or three independent experiments are shown in a, as indicated by data dots. For each group, a minimum of 100 cells/experiment was averaged. Data from one experiment are shown in b, with 40 cells per group in the left panel and 45 cells per group in the right panel. Experiments were repeated independently two times with similar results. d Schematic of the unique functions for Talpid3, C2CD3, and OFD1 in assembling distal-end structures, CS and actin network (-, defective; +, normal; ++, enhanced). All data are presented as mean ± SD. *p < 0.05 (unpaired t-test). Scale bars = 2 µm

Fig 3: Mapping the centriole localization and ciliogenesis domain in CEP19. (a) Schematics of various GFP-tagged truncation fragments generated for CEP19. (b) IF micrographs representing the localization of GFP-tagged CEP19 deletion fragments in cycling WT RPE-1 cells. Main merged panels are immuno-labelled with GFP (green), GT335 (red) and counterstained with DAPI (blue), corresponding top and bottom insets represent GFP and GT335, respectively. Scale bar, 5 µm. Insets 1 µm. (c) Co-IPs showing the interaction of GFP-tagged CEP19 fragments with endogenous FOP and CEP350. (d) IF micrographs of expressed GFP-tagged CEP19 deletion fragments in serum-starved CEP19 KO cells to monitor ciliogenesis. Main merged panels are labelled with GFP (green), GT335 (red) and counterstained with DAPI (blue), corresponding left and middle insets represent GFP and GT335, respectively. Scale bar, 15 µm. Insets 1 µm. (e) Quantifications of percentage of ciliated cells in the presence of indicated GFP-tagged deletion fragments in serum-starved RPE-1 cells. (f) Summary of different CEP19 fragments generated and their ability to rescue ciliogenesis in CEP19 KO cells and interact with FOP and CEP350.

Fig 4: Three-dimensional SIM-based localization of CEP19, FOP and CEP350 at the distal end of the centrioles. (a) Axial distance of indicated proteins with respect to GFP-CEP19, or CEP164 (the median together with the 25% and 75% quantiles are shown for each category; n > 10). (b) Schematic overview of relative localization of indicated proteins along the length of the centriole in HeLa cells. (c) Three-dimensional SIM micrographs of centrioles immuno-labelled with GFP-CEP19 (in green, imaged at 488 nm), indicated centriolar proteins (in red, imaged at either 568 or 642 nm) and merged channels. Scale bar, 250 nm. (d) Table summarizing the outer ring diameter of the indicated proteins.

Fig 5: CEP19, FOP and CEP350 are required for ciliogenesis. (a) CEP19, FOP and CEP350 localize to the basal body in serum-starved WT RPE-1 cells. Scale bar, 5 µm. (b) IF micrographs of CEP19, FOP and CEP350 KO and WT RPE-1 cell lines upon serum starvation. Main merged panels are immuno-labelled with PCNT (green), ARL13B (red) and counterstained with DAPI (blue), corresponding top and bottom insets represent PCNT and ARL13B, respectively. Scale bar, 15 µm. Insets 1 µm. (c) Expression of GFP-tagged CEP19, FOP and CEP350 rescues the defect in ciliogenesis in their respective cell lines. *p < 0.01 by Student's t-test, n > 200.