Fig 1: Longitudinal distribution of distal appendage proteins. a A comparative wide-field and 3D STORM analysis of the SDA component Odf2 and various DAPs on longitudinally-oriented older mother centrioles in interphase. The average ± s.d. thickness of the signals is noted. n = 11 centrioles for Odf2, 10 for CCDC41, 22 for SCLT1, 16 for FBF1, 26 for Cep164, and 10 for ANKRD26. b Two-color SIM images illustrating the lateral shift between the DAPs. The average ± s.d. distance between centers of each signal’s intensity peaks is indicated. CCDC41 signal localizes most distally, while SCLT1 almost colocalizes with Cep164. FBF1 signal appears slightly shifter towards the distal end. ANKRD26 and Cep164 colocalize. n = 8 centrioles for Odf2, 11 for CCDC41, 17 for SCLT1, 15 for FBF1 and 10 for ANKRD26. c Two-protein STORM analysis of longitudinally-oriented centrioles showing that CCDC41 and FBF1 are laterally shifted for ~100 nm, and CCDC41 and SCLT1 and CCDC41 and ANKRD26 for ~80 nm, corroborating values obtained in (b) by SIM. d A 3D model illustrating the organization of DAs electron densities, and the localization of the DAPs with respect to the electron densities. Scale bars: 1 µm for all wide-field images of centrioles and 200 nm for STORM images
Fig 2: PIPKIγ and INPP5E coordinate the association between TTBK2 and CEP164 by regulating a centrosomal PtdIns(4)P pool.(a,b) HEK293T cells transfected with indicated siRNAs (a) or plasmids (b) were subjected to immunoprecipitation (IP) with anti-TTBK2 antibody or normal rabbit IgG (IgG). Flag-INP, Flag-INPP5E. The resulting precipitates and cell lysates (Input) were immunoblotted with indicated antibodies. α-Tub, α-tubulin. (c) IMCD3 cells were transfected with empty vector (Control), HA-PIPKIγΔCT or Flag-INPP5E. Without (+FBS) or with 6- h serum starvation (−FBS, most cells were not yet ciliated under this condition), each group of cells was subjected to IF microscopy to visualize PtdIns(4)P (PI(4)P, green), CEP135 (red), HA-tag (far-red) and nuclei (blue, DAPI staining). Stars indicate transfected cells. (d) In each group in c, the intensity of centrosomal PtdIns(4)P signal in >20 cells was quantified and plotted from at least three independent experiments. (e,f) RCTE cells stably expressing GFP-TTBK2 were transfected with HA-tagged PACT, PACT-P4MSidM (PACT-P4M) or PACT-PHPLCδ (PACT-PH) for 24 h. Cells were then processed for IF microscopy with antibodies against TTBK2 (green), HA-tag (red) and CEP83 (far-red, pseudo-colored blue). (f) The fluorescence intensity of centrosomal TTBK2 was quantified in >20 cells of each group. Results from three independent experiments were plotted. Error bars represent s.e.m. ***P<0.001. Scale bars, 5 μm (green); Scale bars, 0.5 μm (white).
Fig 3: Sdccag8 is required or cilia formation and Hh signaling.(A,B) Sdccag8gt/gt cells have shorter cilia. Immunofluorescence staining of cultured mouse embryonic fibroblasts derived from wild type (A) and Sdccag8gt/gt (B) mice using antibodies against distal appendage marker CEP164 (red) and cilia marker acetylated tubulin (green), demonstrate shorter cilia in Sdccag8gt/gt cells (B). (C) Quantitation of the percentage of ciliated cells for wild type and Sdccag8gt/gt cells after serum starvation (48 hrs). Significantly less Sdccag8gt/gt cells grow cilia compared to wild type cells (18% vs. 93%, n = 100 for both genotypes, **p = 0.0018). (D) Sdccag8gt/gt MEFs have significantly shorter cilia (1.9 ± 0.2 µm, n = 20) compared to wild type cells (2.9 ± 0.0 µm, n = 142, ****p<0.0001). (E) Sdccag8gt/gt MEFs have attenuated response to Hh signal agonist SAG. qRT-PCR analysis demonstrates reduced levels of Hh pathway target gene Gli1 in SAG treated Sdccag8gt/gt MEFs compared to wild type cells (N = 3). (F) Knockdown of SDCCAG8 causes a reduction in cilia formation in hTERT-RPE1 cells. Only 43% of SDCCAG8 knockdown cells grew cilia compared to 94% wild type cells (**p = 0.0092). (G) Cilia length is significantly reduced in SDCCAG8 knockdown cells (1.9 ± 0.1 µ, n = 63) compared to wild type cells (2.8 ± 0.1 µ., n = 80, ****p<0.0001). Scale bar: (A,B) 7.5 µm. wt, Sdccag8 wild type allele; gt, Sdccag8 gene-trap allele. (H,I,J,K) Immunofluorescence staining of control siNS hTERT-RPE1 cells (H,J) and siSDCCAG8 hTERT-RPE1 cells (I,K) with antibodies against acetylated tubulin (green) and distal appendage markers CEP83 (red) (H,I) or FBF1 (red) (J,K), reveals no abnormalities in the formation of centriolar distal appendages despite the loss of cilia in SDCCAG8 knockdown cells (I,K) compared to control cells transfected with non-specific siRNA (F,H).
Fig 4: Removal of specific DCPs is a prerequisite for DA assembly. a WT RPE1 cells were transduced with lentiviruses expressing Myc-PACT–CEP120, Myc-PACT–Centrobin, or Myc-PACT-Neurl4, as indicated. Two days after transduction, cells were serum-starved for 48 h and examined by IF with antibodies against DA markers, CEP83 and CEP164. b Control and Talpid3−/− cells were transfected with siRNAs against CEP120 (Supplementary Figures 2a, b) and Centrobin. Two days after transfection, cells were serum-starved for 24 h and examined by IF and WB using indicated antibodies. c WT RPE1 cells were transfected with siRNAs against CEP83. Two days after transfection, cells were serum-starved for 24 h and were visualized with indicated antibodies to check DA and DCPs. d WT RPE1 cells were transfected with siRNAs against CEP120 and Centrobin. Two days after transfection, cells were serum-starved for 24 h and then visualized with indicated antibodies to check the localization of DCPs, which are recruited as schematized (right). e Schematic of the relationship between removal of DCPs and DA assembly. Cumulative data from three independent experiments are shown. For each group, a minimum of 100 cells/experiment was averaged. All data are presented as mean ± SD. *p < 0.05 (unpaired t-test). Scale bars = 2 μm
Fig 5: PIPKI? is required for the removal of CP110 from and the recruitment of TTBK2 to the M-centriole/basal body.(a,c) RCTE cells were treated with control (siNC) or PIPKI?-specific (siPIPKI?) siRNA for 48 h, serum starved (-FBS) for additional 24 h and stained with indicated antibodies for IF microscopy. Percentage of cells (n>200) with CP110 on both centrioles (a) or with TTBK2 at the M-centriole/basal body (c) was quantified from three independent experiments. Error bars represent s.d. (b) Serum-fed (+FBS) RCTE cells were treated with siNC, siPIPKI? or siPIPKI? plus CP110-specific (siCP110) siRNA for 72 h, and then subjected to IF microscopy to visualize polyglutamylated tubulin and CP110. (d) CP110 and TTBK2 protein levels are not changed in PIPKI?-depleted cells. Cell lysates from control (siNC) or PIPKI?-depleted (siPIPKI?) cells were immunoblotted using indicated antibodies. (e) Serum-fed RCTE cells stably expressing GFP-TTBK2 were transfected with empty vector (Control), wild-type (WT) or kinase-dead (KD) HA-PIPKI??CT for 8 h. Then cells were subjected to IF microscopy to visualize TTBK2, HA tag and CEP83. (f) The fluorescence intensity of centrosomal TTBK2 in >20 control, HA-PIPKI??CT-WT or HA-PIPKI??CT-KD expressing cells was quantified from three independent experiments and plotted. Error bars represent s.e.m. (g) Cell lysates from cells described in e were immunoblotted using indicated antibodies. a-Tub, a-tubulin. (h) CP110 was visualized by IF microscopy in serum-fed control or HA-PIPKI??CT-WT expressing cells. (i) Percentage of cells (n>40) showing diminished CP110 signal at M-centrioles was quantified in each group and results from three independent experiments were plotted. Error bars represent s.d. Arrowheads indicate M-centrioles/basal bodies. ***P<0.001. Scale bars, 0.5 µm.
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