Fig 2: FSD1 forms a ring structure at the subdistal region of both centrioles. a Schematic of centrosome structure. The centrosome is composed of a pair of centrioles, and each centriole is divided into three portions, including proximal end (red), middle region (green), and distal end (blue). The older centriole of the pair (the mother) is often decorated with distal appendages and subdistal appendages. During ciliogenesis, the transition zone extends from the distal end of the mother centriole. b FSD1 was visualized with centriolar proximal (C-Nap1), distal (Cep162, Centrin2, and IFT88), subdistal appendage (ODF2), and distal appendage (Cep164) markers. ODF2, IFT88, and Cep164 localize to mother centrioles. Scale bar, 500 nm. c The centriolar localization of FSD1 was observed in ciliated and non-ciliated cells stained with indicated antibodies. Cep162, the centriole distal end marker. ARL13B, the primary cilia marker. Scale bars, 5 µm (main image) and 500 nm (magnified region). d The centriolar localization of FSD1 was observed in ciliated cells stained with indicated antibodies. Cep162, the centriole distal end marker. MKS1 and NPHP8, the ciliary transition zone markers. Scale bar, 500 nm. e RPE-1 cells were transfected with mCherry-Centrin2 plasmids and stained with the indicated antibodies. The samples were then imaged using the stimulated emission depletion microscopy (STED). Cep164 and FBF1 localize to the distal appendage of mother centrioles. Scale bars, 500 nm. f Schematic representation of the centrosome illustrates that FSD1 forms a ring structure at the middle region encircling both centrioles

Fig 3: (a) Harvested calf thymocytes were fixed chemically, resin-embedded and ultrathin-sections were obtained. Some sections contain centrioles (black arrow) with an amorphous density (red arrowhead) similar to those described in cryo-ET images of isolated thymus centrosomes. (b) Resin inclusion of the centrosome pellet sedimented after lysis in which a similar amorphous density is evident. Note that this density is usually present at only one of the centrioles and it appears to be attached to the centriolar wall by linkers. This density is immunolabeled for SMC4 (c), C-NAP1 (d), SMC1 (e) and NCAPD2 (f).

Fig 4: CEP250 -/- mice show defect in E-cadherin polarity change during cell cycle progression at P2, related to Fig 6 Representative images of P2 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), E-cadherin (green), and DAPI (blue) showing GSCs in interphase. Scale bars: 5 µm.Quantification of (A). Percentage of GSCs displaying different polarity status is indicated. CEP250 +/+ n = 3 mice, CEP250 -/- n = 3 mice. At least 300 cells were analyzed in each mouse and the average was calculated for each mouse.Representative images of P2 mouse ST 10-µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prophase. PH3 staining marks the mitotic chromatin. Scale bars, 5 µm.Quantification of (C). Percentage of GSCs displaying different polarity status is indicated. CEP250 +/+ n = 3 mice, CEP250 -/- n = 3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse.Representative images of P2 mouse ST 10-µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prometaphase. PH3 staining marks the mitotic chromosome condensation. Scale bars: 5 µm.Quantification of (E). Percentage of GSCs displaying different polarity status is indicated. CEP250 +/+ n = 3 mice, CEP250 -/- n = 3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In A, C, and E, white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin toward the basement membrane. In (B, D, F), data are presented as mean ± SEM. n.s., not significant, *P < 0.05, ***P < 0.005 (the unpaired Student's t-test).

Fig 5: Loss of CEP250 leads to premature differentiation AImmunofluorescence staining for PLZF (red) with DAPI staining (blue) of 10 µm cryo-sections of testes from CEP250 +/+ and CEP250 -/- mice at P5, P10, and P14. Scale bars: 20 µm.BQuantification of (A). Quantification of PLZF+ cells per seminiferous (ST) section of CEP250 +/+ and CEP250 -/-, testes at P5, P10, and P14. Dots (colors reflect STs from different mice) represent value per ST, CEP250 +/+ (P5) n = 3 mice, CEP250 -/- (P5) n = 3 mice, 50 STs were analyzed for each mouse; CEP250 +/+ (P10) n = 3 mice, CEP250 -/- (P10) n = 3 mice, at least 30 STs were analyzed for each mouse; CEP250 +/+ (P14) n = 3 mice, CEP250 -/- (P14) n = 3 mice, at least 50 ST were analyzed for each mouse.CImmunofluorescence staining for SCP1 (green), PLZF (red), and DAPI (blue) of 10 µm cryo-sections of testes from CEP250 +/+ and CEP250 -/- mice at P2, P3, P5, and P7. Arrowheads highlight PLZF- SCP1+ (green) cells, which represent differentiated spermatogonia. Scale bars: 20 µm.DIllustration of sequential spermatogonia differentiation and formation of seminiferous tubules.E–HQuantification of (C). The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF-, and SCP1+ cells normalized to total cell number per ST section in CEP250 +/+ and CEP250 -/- testes at P2, P3, P5, and P7, respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250 +/+ (P2-P7) n = 3 mice, CEP250 -/- (P2-P7) n = 3 mice, at least 40 STs were analyzed for each mouse.ISummary of proportion of PLZF+ SCP1-, PLZF+ SCP1+, PLZF-, and SCP1- cells per ST section in CEP250 +/+ and CEP250 -/- testes from P2-P7. Data information: In (B, E–H), data are presented as mean ± SEM. n.s., not significant, *P < 0.05, ***P < 0.005, ****P < 0.0001 (the Mann–Whitney test).