Journal: International Journal of Molecular Sciences

Article Title: BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

doi: 10.3390/ijms22031345

Figure Lengend Snippet: Investigation of the primary cilium in fibroblast obtained from patients with bardet biedl syndrome (BBS) and in the hTERT-immortalized retinal pigment epithelial cell-line, (RPE-1) transfected with small interfering RNA (siRNA) against the BBS genes. ( A ) IFM analysis of primary cilia. Primary cilia were labeled with anti-ARL13B antibody (green). Nuclei were visualized with DAPI staining (blue). Controls (Ctrl, here CtrlD) are also shown. Scale bars 10 µM. ( B ) Percentage of patient fibroblasts with cilia. Compared to percentage of ciliated cells in a pool of control fibroblasts (Ctrl.pool = CtrL.A + Ctrl.D + Ctrl.E; 92.60%, n = 798 cells), the percentage of ciliated cells was significantly lower in BBS5 −/− (B) (79.54%, n = 308 cells, *** p =5.13 × 10 −10 ), BBS10 −/− (A) (89.16%, n = 369 cells, * p = 0.0494) and BBS10 −/− (B) (85.78%, n = 225 cells, ** p = 0.0015) fibroblasts. No significant differences were observed for BBS1 −/− (94.98%, n = 239, p = 0.202) and BBS5 −/− (A) (92.77%, n = 249, p = 0.9301) compared to the pool of control fibroblasts. There was a significant difference in percentage of ciliated cells between the three controls (Ctrl). The percentage was significantly lower in Ctrl.D (89.18%, n = 305 cells) compared with Ctrl.A (95.72%, n = 234 cells; Ctrl.D/Ctrl.A: * p = 0.0054) and Ctrl.E (93.82%, n = 259 cells; Ctrl.D/Ctrl.E: * p = 0.037). No significant difference was observed between CtrlA and CtrlE (CtrlA/CtrlE: p = 0.34). ( C ) Percentage of siBBS RNA-treated RPE1 cells with cilia. The cilia were labeled with anti ARL13B antibody and nuclei were visualized with DAPI staining. For siRNA efficacy, see . Compared to percentage of ciliated cells in siSCR transfected RPE1 cells (86.81%, n = 379 cells), the percentage of ciliated cells were significantly decreased in RPE1 cells transfected with siBBS5 (60.20%, n = 294 cells, *** p = 2.2802 × 10 −15 ) and siBBS10 (76.40%, n = 322 cells, *** p = 0.000347). No significant differences compared to siSCR transfected cells were obtained for siBBS1 (84.49%, n = 361 cells, p = 0.36789). ( D ) Quantification of primary cilia length in patient fibroblasts. No significant difference was obtained by comparing the three control fibroblasts lines (Ctrl.A, n = 224 cells; Ctrl.D, n = 270 cells; Ctrl.E n = 243 cells. Ctrl.A/Ctrl.D: p = 0.0918; Ctrl.A/Ctrl.E: p = 0.675; Ctrl.D/Ctrl.E: p = 0.207). A pool of all the controls ( n = 737 cells) was used for comparison with the five BBS patient fibroblasts lines. Compared to the controls, BBS1 −/− ( n = 222, p = 3.77 × 10 −11 ) had significantly shorter cilia, whereas BBS5 −/− (A) ( n = 228, *** p = 1.83 × 10 −13 ), BBS5 −/− (B) ( n = 242, *** p = 8.77 × 10 −8 ), BBS10 −/− (A) ( n = 333, *** p = 2.00×10 −16 ) and BBS10 −/− (B) ( n = 202, * p = 0.0165) all had significantly longer cilia. ( E ) Cilia length variation visualized as a boxplot of (C) showing the length variation for all cell-lines. Compared to control cell-lines, BBS5 −/− (A), BBS5 −/− (B) and BBS10 −/− (A) had broader length distributions. ( F ) Quantification of primary cilia length in siBBS-treated RPE1 cells. The cilia length was significant shorter in RPE1 cells treated with siBBS1 ( n = 361, *** p = 2.29 × 10 −8 ), and siBBS10 ( n = 322, *** p = 3.54 × 10 −19 ) compared to siSCR-treated control RPE cells, whereas no significant difference was observed between siBBS5- ( n = 294, p = 0.21) and siSCR-treated cells ( n = 329). SiBBS transfection led to a substantial reduction in expression of the BBS genes . In ( B , C ), the number of cells ( n ) investigated consist of pooled data from three separate experiments and significance was determined using the χ 2 -test. In ( D , F ), the number of cilia ( n ) measured consist of pooled data from three separate experiments. p -values and significance were determined using Student’s t -test, two-tailed. Significance levels p < 0.05, * p < 0.05, and *** p < 0.0005 were used. Error bars represent the standard deviation.

Article Snippet: RPE1 (hTERT-RPE1) cells were a kind gift from Lotte Bang Pedersen (University of Copenhagen, Department of Biology, Section for Cell Biology and Physiology), originally obtained from ATCC (hTERT RPE-1 ATCC ® CRL-4000TM).

Techniques: Transfection, Small Interfering RNA, Labeling, Staining, Control, Comparison, Expressing, Two Tailed Test, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

doi: 10.3390/ijms22031345

Figure Lengend Snippet: Investigation of Hedgehog (Hh) signaling in control and BBS-defective cells. ( A ) Investigation of Hh signaling fibroblast. Expression profile of GLI1 mRNA was normalized to the amount of endogenous GADPH mRNA. Pur-induced expression of GLI1 was significantly increased in control cells compared to patient fibroblasts (BBS1 −/− : * p = 0.015711, BBS5A −/− : * p = 0.012758, BBS5B −/− : * p = 0.014811, BBS10A −/− : * p = 0.014944, BBS10B −/− : * p = 0.013701). Compared to unstimulated cells, a significant increase in GLI1 expression as an effect of pur stimulation was observed in the control (Ctrl), BBS1 −/− and BBS10A −/− fibroblasts (Ctrl: ** p = 0.001787, BBS1 −/− : * p = 0.047977, BBS10A −/− = * p = 0.039275) but not in the other cell types (BBS5A −/− : p = 0.148291, BBS5B −/− : p = 0.232452, BBS10B −/− : p = 0.098018). ( B ) Investigation of Hh signaling in siRNA transfected RPE1 cells. Expression profile of GLI1 was normalized to the amount of endogenous GADPH . Cells transfected with siSCR showed a significant increase in GLI1 expression after pur stimulation (siSCR * p = 0.038869). The increase in siBBS1 and siBBS10 transfected cells was smaller, but still significant, compared to unstimulated cells (siBBS1: * p = 0.020942, siBBS10: * p = 0.046011). No significant effect was observed in siBBS5 transfected cells (siBBS5: p = 0.323142). SiRNA transfection led to a substantial reduction in expression of the BBS genes . ( C ) Ciliary localization of SMO in fibroblast lines. The cells were labeled with anti-AC-TUB (cilia marker, red) and anti-SMO antibody (green). Nuclei were visualized with DAPI staining (blue). Scale bars 10 µM. Arrows indicate ciliary localization. In contrast to control fibroblasts, SMO was observed in a large number of cilia in all the BBS patient fibroblasts. In the control cells, SMO was only observed in a large number of cilia after pur stimulation . ( D ) Quantification of cilia with SMO. The fibroblasts were grown under serum-reduced conditions (0.05% FCS) for 48 h in the presence or absence of 5 µM pur for the final 24 h, as indicated. SMO was present in a large number of the cilia in all BBS patient fibroblasts, both in the presence and the absence of pur. Significantly more SMO was present in the BBS fibroblast cells compared to a pool of control cells (Ctrl.pool: Ctrl.A + Ctrl.D, n = 315) in the absence of pur (BBS1 −/− : *** p = 1.28 × 10 −12 , n = 156, BBS5A −/− : *** p = 2.76 × 10 −8 , n = 145, BBS5B −/− : *** p = 6.95 × 10 −5 , n = 136, BBS10A −/− : *** p = 2.26 × 10 −5 , n = 134, and BBS10B −/− : *** p = 0.00014, n = 141). The control cells had a significant increase in ciliary SMO localization after pur stimulation (*** p = 4.58 × 10 −9 , n = 349/426). All BBS fibroblasts showed a significantly higher basal level of ciliary SMO that only increased significantly in BBS10A after pur stimulation (BBS1 −/− : p = 0.960, n = 222, BBS5A −/− : p = 0.371, n = 228, BBS5B −/− : p = 0.802, n = 242, BBS10A −/− : * p = 0.045, n = 333, BBS10B −/− : p = 0.219, n = 202). All included data were pooled from three independent experiments ( n = 3). In ( A , B ), Student’s t -test, one-tailed, with p < 0.05 significance level was performed. In ( D ), the χ 2 -test was performed with significance levels * p < 0.05, and *** p < 0.0005. Error bars represent the standard deviation.

Article Snippet: RPE1 (hTERT-RPE1) cells were a kind gift from Lotte Bang Pedersen (University of Copenhagen, Department of Biology, Section for Cell Biology and Physiology), originally obtained from ATCC (hTERT RPE-1 ATCC ® CRL-4000TM).

Techniques: Control, Expressing, Transfection, Labeling, Marker, Staining, One-tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Intrinsic Diversity in Primary Cilia Revealed Through Spatial Proteomics

doi: 10.1101/2024.10.20.619273

Figure Lengend Snippet: Spatially profiling the primary cilia proteome A-B) New staining concept. A) Ciliated cells (i.e., starved hTERT-RPE1) cells are co-stained with markers for nuclei (DAPI), cilia (ARL13B antibody), basal bodies (PCNT antibody) and for a protein of interest (i.e., with a polyclonal antibody against IFT43 from the Human Protein Atlas). B) Confocal z-stacks are generated, which allow 3D analysis of the whole cell including the cilia, even when they stick out of the cell to the top or bottom (Blue: Nuclei, Red: Cilia, Yellow: Basal Body, Green: Protein of Interest). C) Three cell lines are investigated: Starved hTERT-RPE1 cells from the retina, RPTEC/TERT1 cells from the kidney, and ASC52telo cells from the adipose tissue. Example images from all three cell lines are shown with magnified insets, from a co-staining of DAPI (labeling nuclei), ARL13B (labeling cilia), and the protein of interest IFT20. Gender of the cell line indicated. D) CiliaQ analysis of all images underlying this study to determine the ciliation rate and cilia length distribution of the studied cell line cultures. In total 463,454 cells with 218,410 cilia were included. Top: Each dot represents all images from one individual experiment day. Bars and error bars represent median and quantiles, respectively. Bottom: Normalized histograms of the cilia lengths for all cilia analyzed, distinguished by cell line. E-H) Example images of special cilia observations in the three different cell lines. Only the Nucleus (DAPI), cilia (ARL13B), and Basal Body (PCNT) channels are displayed. E) Mitotic ASC52telo cells retaining a cilium. F) RPTEC/TERT1 cells with two adjacent cilia. G) Starved hTERT-RPE1 cell with two adjacent cilia. H) Two adjacent RPTEC/TERT1 cells whose cilia are connected and form a “bridge”. I) Sunburst plot of all data sets accumulated to form the cilia candidate list of 9634 human genes. Different data sets and studies, as well as additional educated guesses were convolved (see Materials & Methods). Each study is colored by species. J) Candidate list of 1,935 genes for which a staining with Human Protein Atlas antibodies was conducted, including a substantial portion of genes that had not been associated with cilia before (“new”, purple). Scale bars indicated.

Article Snippet: hTERT-RPE1 cells (Evercyte GmbH, Vienna, Austria) were acquired from ATCC and cultivated in DMEM/F12 (Thermo Fisher Scientific Inc, GibcoTM, Cat. no. 11320033) + 10% FBS (Merck KGaA, Sigma-Aldrich, Cat. no. F7524) at 37°C and 5% CO2.

Techniques: Staining, Generated, Labeling

Journal: bioRxiv

Article Title: Intrinsic Diversity in Primary Cilia Revealed Through Spatial Proteomics

doi: 10.1101/2024.10.20.619273

Figure Lengend Snippet: Spatial map of the primary cilia proteome A) Schematic drawing of a primary cilium and its substructures. B) Protein localization to four ciliary substructures was annotated, for which example images are shown: Primary cilium, if the protein was overlapping with the ciliary marker ARL13B; Primary cilium Tip (Tip), if the protein of interest channel showed a signal at the distal end of the cilium; Basal Body, if the protein of interest channel showed signals overlapping or accumulating close to the basal body marker PCNT; Primary cilium transition zone (Transition zone) if the protein of interest channel showed an enrichment between basal body and cilium. C) Venn diagram showing the number of genes observed at one or multiple of the four locations specified in B. D) Example images (single confocal slice images) of additional subpatterns observed in primary cilia. Gray interrupted line distinguishes two individual images placed adjacently for showing two cilia. E) Example images (single confocal slice images) of stainings for proteins that are established as localizing to cilia - gene names are shown. F) Comparing the list of genes encoding proteins that were annotated as localizing to at least one of the four cilium locations (B) to existing datasets. 71 proteins were not found in other datasets. G-H) Pie charts showing the number of occurrences in other datasets for each gene that encoded a protein found in one of the four annotated cilia compartments. G) Comparing the list of revealed proteins to datasets derived from Human samples only. H) Comparing the list of revealed proteins to datasets from any species. I) Example images of proteasomal subunits observed in cilia. J) Example images (single confocal slice images) for proteins observed in cilia that were previously only detected in individual proximity-labeling-based MS approaches. K) Motile cilia proteins detected in the cilia of RPTEC/TERT1 cells. Images were derived from stainings of different cell lines: Starved hTERT-RPE1 cells: DNAL4, ZNRF1, EFCAB7, EVI2A, TRIM46, SMAD7, RAB7A, SEPTIN2, RAB23, ARL15; RPTEC/TERT1 cells: CFAP300, TRIO, C2CD4C, TMEM237, IFT22, ARL6, PSMD10, PSME3, PSMA6, HYDIN, IQCA1, CATSPER2; ASC52telo cells: KIF7, CEP164, TCTN2, KIF7, ADCY3, ZNF330, RSL1D1.

Article Snippet: hTERT-RPE1 cells (Evercyte GmbH, Vienna, Austria) were acquired from ATCC and cultivated in DMEM/F12 (Thermo Fisher Scientific Inc, GibcoTM, Cat. no. 11320033) + 10% FBS (Merck KGaA, Sigma-Aldrich, Cat. no. F7524) at 37°C and 5% CO2.

Techniques: Marker, Derivative Assay, Labeling

Journal: bioRxiv

Article Title: Intrinsic Diversity in Primary Cilia Revealed Through Spatial Proteomics

doi: 10.1101/2024.10.20.619273

Figure Lengend Snippet: The primary cilium is a signaling hub A) Comparative Gene Ontology enrichment analysis of Biological Process terms between the two major cilia locations primary cilium (including tip) and basal body (including transition zone). The Venn diagram shows the number of terms found to be significantly enriched (adj. P value < 0.01 and q value < 0.01) for only primary cilium (n=125) or basal body (n=129), as well as for both locations (n=198). Significant terms were clustered by semantic similarity, and for each resulting GO term cluster, a word cloud is shown summarizing the biological functions of the terms in the cluster based on keywords enrichment of term names. The font size in the word clouds correlates with -log(P value) of the key word enrichment, i.e. the larger, the more enriched a keyword is compared against background GO vocabulary. The color in the heat maps represents the semantic similarity. B) Protein localization of example signaling proteins to the primary cilium. All images show single confocal slice images except for PDGFRA, where a maximum intensity projection of 5 confocal slices is shown to show the whole cilium. The inset for PDGFRA shows the same position with displaying only the PDGFRA channel. C) Multilocalization of cilia-related proteins with the other 35 annotated subcellular compartments. Abbreviations: Centriolar Sat. = Centriolar Satellites; Cyt. bridge = cytokinetic bridge; Int. Fil. = Intermediate Filaments; Mitotic chr. = Mitotic chromosome; Nuc. bodies = Nuclear bodies; Nucleoli fib. cent. = Nucleoli fibrillar center; Nuc. Membrane = Nuclear membrane; Nuc. speckles = Nuclear speckles. D) Example transcription factors revealed in cilium and nucleus. E) Example enzymes revealed in cilia. Images were derived from stainings of different cell lines: Starved hTERT-RPE1 cells: PTK2, PP2R3C, CNGA2, TULP3, CRX, ATF3, CREB1, HSD3B1/HSD3B2, LANCL1, OCRL, SPK1; RPTEC/TERT1 cells: AKT1, AKT3, TAOK1, PIK3CA, PIK3R4, CAMK1G, MIB1, OPRL1, PDGFRA, AGBL5, UGGT1; ASC52telo cells: FLT4, TGFBR1.

Article Snippet: hTERT-RPE1 cells (Evercyte GmbH, Vienna, Austria) were acquired from ATCC and cultivated in DMEM/F12 (Thermo Fisher Scientific Inc, GibcoTM, Cat. no. 11320033) + 10% FBS (Merck KGaA, Sigma-Aldrich, Cat. no. F7524) at 37°C and 5% CO2.

Techniques: Membrane, Derivative Assay

Journal: bioRxiv

Article Title: Intrinsic Diversity in Primary Cilia Revealed Through Spatial Proteomics

doi: 10.1101/2024.10.20.619273

Figure Lengend Snippet: Cellular diversity and spatiotemporal dynamics of the primary cilia proteome A) Venn diagram of the genes for the proteins identified in primary cilia (including tip and transition zone), distinguishing by cell line. Only genes included for which an approved staining was obtained from all three cell lines. (Abbreviations: RPTEC = RPTEC/TERT1, RPE1 = starved hTERT-RPE1) B) Example images (single confocal slice images) for stainings across the three cell lines, showing heterogeneity in ciliary localization across cell types. C) Comparative Gene Ontology enrichment analysis of Biological Process terms between the three included cell lines to investigate cell-line differences and commonalities. The Venn diagram shows the overlap of significantly enriched terms (adj. P value < 0.01 and q value < 0.01) for each cell line. Significant terms of each section of the Venn diagram were clustered by semantic similarity, and for each resulting GO term cluster, a word cloud is shown summarizing the biological functions of the terms in the cluster based on keywords enrichment of term names. The font size in the word clouds correlates with -log(P value) of the key word enrichment, i.e. the larger, the more enriched a keyword is compared against background GO vocabulary. D) Comparative Gene Ontology enrichment analysis of Biological Process terms between proteins annotated as displaying intensity variation in one of the four ciliary locations, and annotated as stable as described in C). E) Example images (single confocal slice images) for stainings of the two ciliogenesis-related proteins RAB8A and RAB34. F) Example images (single confocal slice images) of stainings revealing proteins with heterogeneous localization to primary cilia. G) Example images (maximum intensity projections of whole stack) of CKAP2 staining in the three cell lines.Cell lines for images (if not indicated): Starved hTERT-RPE1 cells: LPAR3, SSTR3, 5HTR7, ADRB2, HSD3B1 HSD3B2, SPHK1, RAB8A, SEPTIN3; RPTEC/TERT1 cells: AGBL5; ASC52telo cells RAB34.

Article Snippet: hTERT-RPE1 cells (Evercyte GmbH, Vienna, Austria) were acquired from ATCC and cultivated in DMEM/F12 (Thermo Fisher Scientific Inc, GibcoTM, Cat. no. 11320033) + 10% FBS (Merck KGaA, Sigma-Aldrich, Cat. no. F7524) at 37°C and 5% CO2.

Techniques: Staining

Journal: bioRxiv

Article Title: Intrinsic Diversity in Primary Cilia Revealed Through Spatial Proteomics

doi: 10.1101/2024.10.20.619273

Figure Lengend Snippet: Clinical relevance of the newly revealed ciliary proteome A) Flow chart for splitting the 654 genes into three groups based on their OMIM annotations. Additionally the genes were classified by being established ciliopathy proteins. Example genes for each group are written on the right, and shown in B. B) Example images for staining the proteins for genes pointed out in A. Cell lines for images: Starved hTERT-RPE1 cells: NRCAM, DDC, NYAP2, CAMK2N1, ZNRF1, AXIN1, KIF26B, ANO3; ASC52telo cells: ABITRAM. C) Example images from stainings of CREB3 in RPE1 cells (serum-starved), ASC52telo cells, or RPTEC/TERT1 cells. The white interrupted line distinguishes two images that were placed adjacently to show two cilia. Abbreviations: TZ = transition zone. BB = Basal Body. The basal body PCNT marker was not shown to better visualize the localizations of CREB3 - The PCNT marker was however used to correctly mark BB and TZ positions with arrows.

Article Snippet: hTERT-RPE1 cells (Evercyte GmbH, Vienna, Austria) were acquired from ATCC and cultivated in DMEM/F12 (Thermo Fisher Scientific Inc, GibcoTM, Cat. no. 11320033) + 10% FBS (Merck KGaA, Sigma-Aldrich, Cat. no. F7524) at 37°C and 5% CO2.

Techniques: Staining, Marker