Fig 2: CEP162 interacts with CEP290 and mediates its association with MTsa, Endogenous CEP162 or CEP290 in HEK293T cell extracts were immunoprecipitated, and co-precipitated proteins were probed with indicated antibodies by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. b, Mapping the CEP290 binding domain of CEP162. 293T cells expressing various Flag-tagged fragments of CEP162 were subjected to immunoprecipitations and western blot with antibodies as indicated. Uncropped images of immunoblots are presented in Supplementary Fig. S1. c, RPE1 cells at different cell cycle stages were processed for immunofluorescence with anti-CEP290 (red), anti-CEP162 (green), and anti-centrin (blue) antibodies. DNA (DAPI, blue). d, Spindle association of CEP290 depends on CEP162. RPE1 cells transfected with control, CEP162, or CEP290 siRNA for 72 hours were stained with indicated antibodies against acetylated-tubulin (green), CEP162 (red), and CEP290 (blue). DNA (DAPI, blue). e, Overexpression of CEP162 induced MT bundles recruiting endogenous CEP290. U2OS cells transiently expressing HA-tagged CEP162FL or HA-tagged CEP162tNC1C2 were stained with antibodies against CEP162 (green), CEP290 (red) and DAPI (DNA, blue). f, Overexpression of CEP162 but not CEP290 induced MT bundles. NIH3T3 cells transiently expressing HA-tagged human CEP162FL or GFP-tagged mouse CEP290FL were stained with antibodies against α-tubulin (Red), CEP162 (HA, green) or CEP290 (GFP, green). g, Centriolar localizations of CEP290 and CEP162 are independent of each other. RPE1 cells were transfected with control, CEP162, or CEP290 siRNA for 96 hours. Cells were stained with anti-centrin (green), anti-CEP162 (red), and anti-CEP290 (blue) antibodies.

Fig 3: Untethered CEP162 promotes ectopic recruitments of TZ components to cilia tipsa–d, Ectopic recruitments of TZ components via CEP162ΔCC3 and CEP162tNC1C2. HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was expressed in RPE1 cells followed by serum starvation for 48hr and stained with antibodies against CEP290 (a), TCTN1 (b), Tmem67 (c) or RPGRIP1L (d) in red, acetylated-tubulin (AcTu, green) and HA (blue). Nuclei were visualized with DAPI (blue). e, Centriole-bound components, centrin (top) and CEP164 (bottom), remained localized at the cilia base in CEP162ΔCC3-expressing cells.

Fig 4: Cilia tips modified by CEP162 swell exceedingly and discharge ciliary contentsa, RPE1 cells expressing GFP-tagged CEP162tNC1C2 were traced by time-lapse fluorescence microscopy. Images were taken at 2-min time interval. Existing cilia tips were marked by arrows, and newly formed tips were marked with arrowheads. b–e, HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was conditionally expressed in RPE1 cells followed by serum starvation for 48hr. Cells were stained with antibodies against CEP290 (b), RPGRIP1L (c), IFT88 (d), Arl13b (e) in red, acetylated-tubulin (green) and HA (blue). DNA was stained with DAPI. Arrows indicate the CEP162-positive structures released from cilia tips. f, Correlative light-scanning electron microscopy images of control cells (left), or RPE1 cells expressing GFP-tagged CEP162ΔCC3 (middle & right). GFP-CEP162ΔCC3 (green) marked cilia tip (middle & right). Primary cilia in control cells (left) were labeled with acetylated-tubulin (green). Arrows indicate cilia tips. Merged images were shown under low SEM magnification. g, Correlate LM-SEM images of the swollen structure released from cilia tips in RPE1 cells expressing GFP-tagged CEP162ΔCC3. Released structures from cilia tip were marked with GFP (green). Merged images were shown under low SEM magnification.

Fig 5: The maintenance of Cep290 by FSD1 at CS is required for TZ assembly. a Non-ciliated (+Serum) or ciliated (−Serum) RPE-1 cells were stained with indicated antibodies as shown. Scale bars, 5 μm (main image) and 1 μm (magnified region). b Effects of FSD1 depletion on the localization of Cep290 at centriolar satellites in cycling cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with anti-Cep290 (green) and anti-Centrin2 (red) antibodies. Scale bars, 5 μm (main image) and 1 μm (magnified region). c Effects of FSD1 depletion on the PCM1 localization at centriolar satellites in cycling RPE-1 cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with indicated antibodies. Scale bars, 5 μm (main image) and 1 μm (magnified region). d Effects of FSD1 or PCM1 depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. e, f Effects of FSD1 or PCM1 depletion on the relative intensity of the transition zone components TMEM67 (e) and NPHP8 (f) at mother centrioles in quiescent cells. g Model accounting for the TZ assembly defects caused by FSD1 depletion due to the disruption of CS Cep290. TZ transition zone, CS centriolar satellite. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. **P < 0.01, ***P < 0.001