Fig 1: Interaction and MT-Associated Co-localization of CEP104 and CSPP-L(A–C) Immunodetection of indicated endogenous centrosomal and/or ciliary proteins co-purified with full-length or truncated GFP-tagged CEP104 (A and B) and CSPP-L variants (C). GFP-fusion proteins were expressed in Hek293T cells and purified using paramagnetic GFP-trap beads. The N-terminal CEP104-domain (aa 1–200) confers interaction with the C-terminal CSPP-L (aa 842–1,221) domain. CSPP-L but not CEP104 co-purifies PCM1.(D and E) Schematic drawing of CEP104 (D) and CSPP-L (E) proteins and tested truncation variants, including functional domains and allocated interaction partners identified here and previously (Hauge et al., 2007, Patzke et al., 2010, Jiang et al., 2012). Predicted coiled-coil domains (UniProt) are indicated as black boxes. Bio-informatic analysis identifies structural homologies (blue boxes) to IFT25 (CEP104 aa 1–158 to hIFT25 aa 17–151: probability 99.9%; E value 1.1E−21, p value 7.2E−27) and a single chTOG domain (CEP104 aa 415–637 to hCLASP aa 28–266: probability 99.7%; E value 2E−15, p value 1.3E−20).(F) IFM of hTERT-RPE1 cells transiently expressing mCherry-CEP104 (red) and GLAP3-CSPP-L (green) and co-stained for α-tubulin (a-acetylated-tubulin, white). At increasing expression levels, mCherry-CEP104 and GLAP3-CSPP-L decorate centrosome originating MTs (see also Figure S4).(G and H) Live cell microcopy of hTERT-RPE1 cells co-transfected with mCherry-CEP104 and CSPP-L(294-842)-EGFP (G) or CSPP-L(842-1221)-EGFP (H), respectively, showing dependence on the C-terminal domain of CSPP-L for the recruitment of mCherry-CEP104 along microtubules.(I) IFM of hTERT-RPE1 cells transiently expressing mCherry-CEP104 (red) and GLAP3-CSPP-L (green) and co-stained for ARL13B and CEP164 to label the TZ and the axoneme (a-ARL13B and a-CEP164, white). mCherry-CEP104 (red in cilia sketch) and GLAP3-CSPP-L (green in cilia sketch) co-localize at the tip of the primary cilium and occasionally at the axoneme (arrow in ii).
Fig 2: CEP131 knockout Jurkat cells display an apoptosis and mitochondrial signature.a Schematic overview of the mass spectrometry analysis performed on five biological replicates of wild-type (WT) or CEP131 KO#1 Jurkat cells. The volcano plot illustrates the 7285 detected proteins of which 542 are downregulated and 309 are upregulated. Proteins were classified as downregulated or upregulated if the pvalue < 0.05 and |log2(fold change)|>0.5. b Heatmaps representing the abundance of centriolar satellite components based on the densitometric analysis performed on immunoblotting of lysates from WT, CEP131KO#1, and KO#2 Jurkat cells (mean of n = 3 biological replicates) and on the proteomic analysis of WT and CEP131 KO#1 Jurkat cells (mean of n = 5 biological replicates). c Comparison of the CEP131 KO#1 Jurkat cells proteome to the centrosomal proteome (GO:0005813). Upregulated and downregulated proteins among the 385 detected centrosomal ones are shown in blue and percentages are indicated ( | log2(FC)|>0.5). d Comparison of the proteome of CEP131 KO#1 Jurkat cells with the one of PCM1 knockout IMCD3 cells10. The top Venn diagram shows total differential proteins and the bottom Venn diagram is focused on centrosomal differential proteins (pvalue < 0.05, |log2(FC) | > 0.5). e The top 10 KEGG pathways enriched for the proteins significantly downregulated in CEP131 KO#1 Jurkat cells (pvalue < 0.05, log2(FC) < −0.5). f The top 10 GO Component enrichment for the proteins significantly upregulated in CEP131 KO#1 Jurkat cells is presented (pvalue < 0.05, log2(FC) > 0.5). g–i Workflow of the data mining performed (g). The top 100 genes that presented co-dependencies (based on CRISPR screening) with CEP131 or PCM1 were extracted from the depmap portal database. Analysis of the enrichment was done on the STRING database and compartments enriched in the top 100 co-dependencies with CEP131 (h) or PCM1 (i) are presented. Data information: (e, f, h, i) data are represented using the false discovery rate (FDR).
Fig 3: Characterization of CEP131 knockout Jurkat cells.a Schematic representation of the CEP131 gene, with positioning of the CRISPR sequence guide (blue) in exon 3. Genomic sequences in wild-type (WT) and two bi-allelic clones (KO#1 and KO#2; mutations in red) are shown. b The RNA expression level of CEP131 was assessed by qPCR in WT, CEP131KO#1, and KO#2 cells (mean ± SEM, n = 3 biological replicates, fold change using ACTB and HPRT1 as housekeeping genes for normalization, one-way ANOVA, ****p < 0.0001). c, d Lysates from WT, CEP131 KO#1, and KO#2 Jurkat cells were prepared and analyzed by immunoblotting with antibodies specific to the indicated proteins. e Representative immunofluorescence images of PCM1 (green) and γ-tubulin (red) in WT, CEP131 KO#1, and KO#2 Jurkat cells. Nuclei were counterstained with 4’−6-diamidino-2- phenylindole (DAPI). Scale bars, 10 µm. f, g Radial profile analysis of PCM1 in a 29 µm circle whose center is defined by γ-tubulin, in WT, CEP131 KO#1, and KO#2 Jurkat cells (mean ± SEM of n = 10 cells/condition). h Cell lysates from WT, CEP131 KO#1, and KO#2 Jurkat were immunoprecipitated (IP) with antibodies against PCM1 or with non-relevant Ig (negative control). Samples were then analyzed by immunoblotting as indicated. Inputs are the total lysates collected before the IP. i WT, CEP131 KO#1, and KO #2 Jurkat cells were treated with 10 µM nocodazole for 1 h, prior to washing. Microtubule regrowth was assessed after 5 min by confocal microscopy analysis of α-tubulin. Representative images of three independent experiments (left) (scale bars, 10 µm), and quantification of α-tubulin intensity of one representative experiment (right) (n = 70 cells/condition; one-way ANOVA, ****p < 0.0001). Data information: (c, d, h) GAPDH served as a loading control. Molecular weight markers (Mr) are shown. Data are representative of three independent experiments.
Fig 4: (See previous page). Tctex1d2 and Wdr60 localize to microtubule organizing centers. (A and B) Immunofluorescence microscopy of fixed HeLa cells stained with Hoechst 33342 DNA dye, and anti-α-tubulin, anti-Tctex1d2 (A), or anti-Wdr60 (B) antibodies. Images show the cell cycle subcellular localization of Tctex1d2 (A) and Wdr60 (B). Note that both proteins localize to the microtubule-organizing center (MTOC) in interphase, the spindle poles during mitosis (prometaphase, metaphase) and cytokinesis, and near the cytokinetic bridge during cytokinesis. Bar= 5 μm. (C and D) Immunofluorescence microscopy of fixed interphase HeLa cells stained with Hoechst 33342 DNA dye, anti-γ-tubulin, anti-Centrin, anti-Tctex1d2 (C), or anti-Wdr60 (D) antibodies. Bar= 5 μm. Lower panels show a zoom view of the MTOC region. Bar= 2 μm. (E and F) Immunofluorescence microscopy of fixed interphase HEK293 cells expressing LAP-Tctex1d2 fixed and stained with Hoechst 33342 DNA dye, anti-α-tubulin, anti-PCM1 (E), or anti-Wdr60 (F) antibodies. Bar= 5 μm. Lower panels show a zoom view of the MTOC region. Bar= 2 μm. (G and H) Immunofluorescence microscopy of fixed interphase HeLa cells treated with or without Nocodazole, fixed, and stained with Hoechst 33342 DNA dye, anti-γ-tubulin, anti-α-tubulin, anti-Tctex1d2 (G), or anti-Wdr60 (H) antibodies. Bar= 5μm. Rightmost panels show a zoom view of the MTOC region. Bar= 2 μm. (I) Analysis of Tctex1d2 and Wdr60 protein levels throughout the cell cycle. HeLa cells were synchronized in G1/S, released into the cell cycle and cells were harvested at the indicated time points. Protein extracts were prepared, resolved by SDS PAGE, transferred to a PVDF membrane and immunoblotted with indicated antibodies. Note that Tctex1d2 and Wdr60 protein levels remain constant throughout the cell cycle. See also Fig. S2.
Fig 5: Analysis of centriolar satellites in the talpid3 choroid plexus.An area clear of electron-dense condensations was observed around the basal body in wildtype cells (area outlined by dots; A), electron-dense condensations were observed adjacent to talpid3 centrioles (indicated by arrows, D). Quantified in (G). Immunostaining for a centriolar satellite marker in the choroid plexus, PCM1 (magnified area outlined by dashed line; PCM1 = red, γ tubulin, green B, B′, C, C′, C′′, E, E′, F, F′, F′′). KIAA0586 protein does not colocalize with AZI1, a satellite protein in human RPE1 cells (KIAA0586 = red, AZI1 = green H, I, J). Scale bars: A, D = 500 nm; B, E 10 = μm; C, F = 2 μm H, I, J 5 μm.DOI: http://dx.doi.org/10.7554/eLife.08077.008
Supplier Page from Abcam for Anti-PCM1 antibody