A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation

Article13 June 2022Open Access Transparent process A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation Jan N Hansen Jan N Hansen orcid.org/0000-0002-0489-7535 Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Fabian Kaiser Fabian Kaiser Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis, Writing - review & editing Search for more papers by this author Philipp Leyendecker Philipp Leyendecker Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Birthe Stüven Birthe Stüven Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Jens-Henning Krause Jens-Henning Krause Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Fatemeh Derakhshandeh Fatemeh Derakhshandeh Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Jaazba Irfan Jaazba Irfan Mironid Ltd., SIPBS, Glasgow, Scotland, UK Contribution: Data curation Search for more papers by this author Tommy J Sroka Tommy J Sroka orcid.org/0000-0002-9016-5989 Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Kenley M Preval Kenley M Preval Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Data curation, Formal analysis Search for more papers by this author Paurav B Desai Paurav B Desai Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Data curation, Formal analysis Search for more papers by this author Michael Kraut Michael Kraut Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Data curation Search for more papers by this author Heidi Theis Heidi Theis Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Data curation Search for more papers by this author Anna-Dorothee Drews Anna-Dorothee Drews Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Elena De-Domenico Elena De-Domenico orcid.org/0000-0003-0336-8284 Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Kristian Händler Kristian Händler Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Gregory J Pazour Gregory J Pazour orcid.org/0000-0002-6285-8796 Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Conceptualization, Resources, Formal analysis Search for more papers by this author David J P Henderson David J P Henderson Mironid Ltd., SIPBS, Glasgow, Scotland, UK Contribution: Conceptualization, Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author David U Mick David U Mick orcid.org/0000-0003-1427-9412 Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Methodology, Writing - review & editing Search for more papers by this author Dagmar Wachten Corresponding Author Dagmar Wachten dwachten@uni-bonn.de orcid.org/0000-0003-4800-6332 Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jan N Hansen Jan N Hansen orcid.org/0000-0002-0489-7535 Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Fabian Kaiser Fabian Kaiser Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis, Writing - review & editing Search for more papers by this author Philipp Leyendecker Philipp Leyendecker Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Birthe Stüven Birthe Stüven Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Jens-Henning Krause Jens-Henning Krause Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Fatemeh Derakhshandeh Fatemeh Derakhshandeh Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Jaazba Irfan Jaazba Irfan Mironid Ltd., SIPBS, Glasgow, Scotland, UK Contribution: Data curation Search for more papers by this author Tommy J Sroka Tommy J Sroka orcid.org/0000-0002-9016-5989 Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Kenley M Preval Kenley M Preval Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Data curation, Formal analysis Search for more papers by this author Paurav B Desai Paurav B Desai Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Data curation, Formal analysis Search for more papers by this author Michael Kraut Michael Kraut Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Data curation Search for more papers by this author Heidi Theis Heidi Theis Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Data curation Search for more papers by this author Anna-Dorothee Drews Anna-Dorothee Drews Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Elena De-Domenico Elena De-Domenico orcid.org/0000-0003-0336-8284 Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Kristian Händler Kristian Händler Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany Contribution: Formal analysis, Methodology Search for more papers by this author Gregory J Pazour Gregory J Pazour orcid.org/0000-0002-6285-8796 Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA Contribution: Conceptualization, Resources, Formal analysis Search for more papers by this author David J P Henderson David J P Henderson Mironid Ltd., SIPBS, Glasgow, Scotland, UK Contribution: Conceptualization, Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author David U Mick David U Mick orcid.org/0000-0003-1427-9412 Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Methodology, Writing - review & editing Search for more papers by this author Dagmar Wachten Corresponding Author Dagmar Wachten dwachten@uni-bonn.de orcid.org/0000-0003-4800-6332 Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Jan N Hansen1, Fabian Kaiser1, Philipp Leyendecker1, Birthe Stüven1, Jens-Henning Krause1, Fatemeh Derakhshandeh1, Jaazba Irfan2, Tommy J Sroka3, Kenley M Preval4, Paurav B Desai4, Michael Kraut5, Heidi Theis5, Anna-Dorothee Drews5, Elena De-Domenico5, Kristian Händler5, Gregory J Pazour4, David J P Henderson2, David U Mick3 and Dagmar Wachten *,1 1Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany 2Mironid Ltd., SIPBS, Glasgow, Scotland, UK 3Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany 4Program in Molecular Medicine, University of Massachusetts Chan Medical School, Biotech II, Worcester, MA, USA 5Precise Platform for Single Cell Genomics and Epigenomics, Department of Systems Medicine, German Center for Neurogenerative Diseases, Bonn, Germany *Corresponding author. Tel: +49-228-287-51978; E-mail: dwachten@uni-bonn.de EMBO Reports (2022)23:e54315https://doi.org/10.15252/embr.202154315 AbstractSynopsis Introduction Results Discussion Material and Methods Data availability Acknowledgement Author contributions Disclosure and competing interests statementSupporting InformationReferencesPDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. Metrics1MetricsTotal downloads3,251Last 6 Months1,601Total citations1Last 6 Months1View all metrics ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease. Synopsis This study reveals a cAMP signalosome in primary cilia that evokes a distinct gene expression program via PKA-dependent CREB phosphorylation in the cilium. Chronic stimulation of this pathway remodels renal epithelial cells and drives cystogenesis. The ciliary signalosome relies on an increase in ciliary cAMP level and subsequent PKA-dependent phosphorylation of CREB in the cilium. Chronic stimulation of this signalosome remodels renal epithelial cells from tubules into cysts. Phosphodiesterase 4 (PDE4) long isoforms maintain the ciliary cAMP compartment. Activation of PDE4 long isoforms counteracts chronic ciliary cAMP signaling and inhibits cyst growth. Introduction Primary cilia are microtubule-based protrusions of the plasma membrane, which are present on most vertebrate cells. The primary cilium receives environmental stimuli and transduces this information into an intracellular response. In turn, ciliary signaling controls cellular functions, such as cell proliferation or differentiation (Pazour & Witman, 2003; Anvarian et al, 2019; Nachury & Mick, 2019; Wachten & Mick, 2021). Signal transduction in the primary cilium is temporally and spatially compartmentalized. This is maintained by (i) the transition zone, which prevents lateral protein diffusion, (ii) the intraflagellar transport (IFT) machinery, which provides anterograde and retrograde protein transport along the axoneme in and out of the cilium, and (iii) the BBSome, which is part of the IFT (Nachury, 2018). Thereby, ciliary signaling controls cellular functions, such as cell proliferation or differentiation, independently of the rest of the cell (Pazour & Witman, 2003; Anvarian et al, 2019; Nachury & Mick, 2019; Truong et al, 2021; Wachten & Mick, 2021). Highlighting the relevance of primary cilia for tissue and organ function, primary cilia dysfunction leads to severe diseases commonly referred to as ciliopathies with a broad band of phenotypes (Hildebrandt et al, 2011; Pazour et al, 2020; Richards et al, 2021). However, how ciliary signals are transduced into a cellular response and the molecular mechanisms underlying disease development are not well understood. Cyclic AMP (cAMP) signaling is an important second messenger for ciliary signaling. Components of the cAMP signaling cascade are enriched in primary cilia (Mick et al, 2015; Bachmann et al, 2016; Hilgendorf et al, 2016; Mykytyn & Askwith, 2017; Wachten & Mick, 2021). Remarkably, adenylyl cyclase 3 (AC3) is commonly used as a primary cilia marker in different cell types (Bishop et al, 2007). Recent reports demonstrating that mislocalization of ciliary cAMP signaling is associated with ciliopathies and their symptoms further substantiate the central role of cAMP in ciliary signaling (Siljee et al, 2018; Somatilaka et al, 2020; Wang et al, 2021). This applies to the most common ciliopathy, autosomal-dominant polycystic kidney disease (ADPKD), which occurs with an incidence of 1:500 to 1:1,000 (Solazzo et al, 2018) and where the role of cAMP remains enigmatic. ADPKD is caused by mutations in PKD1 or PKD2, encoding for the ciliary proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively (Bergmann, 2017). Levels of cAMP are high in ADPKD patients, and the only approved therapy for the treatment of rapidly progressive ADPKD, tolvaptan, reduces renal cAMP levels and reduces cyst progression in ADPKD (Torres & Harris, 2014; Blair, 2019). Thus, there is a strong association between cAMP signaling and kidney cyst growth, but how cAMP signaling promotes cyst development and its spatial organization in the cell, in particular the contribution of the cilium, is not well defined. Here, we have applied an integrated approach using optogenetics and next-generation RNA sequencing (RNA-seq) and reveal the molecular basis for compartmentalized cAMP signaling and its role in kidney cyst development. By specifically targeting the photoactivated adenylyl cyclase bPAC to primary cilia and contrasting this against bPAC action in the cell body, we identify a novel cAMP signalosome that is functionally distinct from the cytoplasm. In contrast to HH signaling, it promotes a specific gene expression program upon increasing ciliary cAMP levels. We identify the genes and pathways targeted by the ciliary cAMP signalosome, shed light on the underlying mechanisms and downstream signaling, and show that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelial morphology from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules that activate long isoforms of phosphodiesterase 4 (PDE4) inhibits cyst development. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner. The identification of these new molecular components in cilia-specific epithelial cell remodeling might provide new insights into the pathomechanism underlying one of the most common genetic diseases, polycystic kidney disease. Results Spatial manipulation of cAMP levels using optogenetics To investigate the role of ciliary cAMP signaling in epithelial remodeling, we used mIMCD-3 cells as a model system. We generated mIMCD-3 cell lines stably expressing the photoactivated adenylyl cyclase mNphp3(201)-bPAC-mCherry in the cilium (cilia-bPAC) (Fig 1A). We have previously shown that bPAC fused to mNphp3(201) can be used to moderately increase ciliary cAMP levels when stimulated by light (Hansen et al, 2020). By comparing cilia-bPAC cells to cells stably expressing bPAC-mCherry, which localizes to the cytoplasm (cyto-bPAC, Fig 1B), we aimed to differentially manipulate ciliary versus cytoplasmic cAMP levels. We first verified the light-stimulated cAMP synthesis in our cell lines: Photoactivation of cyto-bPAC significantly increased total cAMP levels, similar to stimulation with the transmembrane AC activator Forskolin (Fig 1C). In contrast, photoactivation of cilia-bPAC did not significantly change total cAMP levels (Fig 1C), which is in line with our earlier findings using a nanobody-based approach (Hansen et al, 2020). We verified the light-induced increase in ciliary cAMP levels using the genetically encoded biosensor Pink Flamindo (Harada et al, 2017). In line with previous reports (Truong et al, 2021), photoactivation of cilia-bPAC increased ciliary cAMP levels to a greater extent than photoactivation of cyto-bPAC cells (Fig 1D and E). Thus, cilia- and cyto-bPAC are perfectly suited to reveal the molecular basis for compartmentalized cAMP signaling and its role in kidney cyst development. Figure 1. Spatial manipulation of cAMP levels using optogenetics A, B. Stable mIMCD-3 cell lines expressing (A) mNphp3(1-201)-bPAC-mCherry (magenta, cilia-bPAC), which localizes to the primary cilium, or (B) bPAC-mCherry (magenta, cyto-bPAC), which resides in the cytoplasm. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (green) to label cilia, and a gamma-Tubulin antibody (gTub, cyan) to label the microtubule-organizing centers. The box indicates the position of the magnified view shown at the bottom left. Magenta arrow indicates the direction and the length of the shift of the respective fluorescence channel. Scale bars are indicated. C. ELISA-based measurements of total cAMP levels from wild-type (WT), cilia-bPAC, or cyto-bPAC mIMCD-3 cells kept in the dark or light-stimulated (1 h, 465 nm, 38.8 µW/cm²) and WT cells stimulated with DMSO (as control) or 10 µM of Forskolin (1 h). Each data point represents an individual biological replicate (n = 4–6 biological replicates). Bars display mean ± SD. D, E. Measurements of ciliary cAMP levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia (cilia-bPAC or cyto-bPAC cells) expressing the cilia-Pink Flamindo biosensor after light stimulation. Scale bars are indicated. cAMP levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); P-value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia (biological replicates) from n = 31 independent experiments. Download figure Download PowerPoint Ciliary cAMP signaling drives cyst growth To investigate the role of ciliary cAMP levels in epithelial cyst development, we applied a matrix-based 3D culture system, which has been widely used as an in vitro model (Elberg et al, 2012). We confirmed that a chronic increase of cAMP levels using Forskolin, which non-selectively activates ACs, promoted cyst growth (Fig 2A) (Elberg et al, 2012). Using immunofluorescence labeling, we verified that the 3D approach maintains the integrity of epithelial cell polarity and adherens junctions. It also promotes primary cilia formation on the luminal apical membrane (Fig 2B and C), recapitulating the in vivo situation. Without stimulation, cells formed tubular structures, whereas in the presence of Forskolin, large cysts formed with primary cilia facing the lumen (Fig 2B and C). Thus, chronic stimulation of AC activity and cAMP signaling emulate the disease drive in ADPKD, providing a relevant cellular in vitro model for cAMP-dependent cyst growth with the limitation that it only includes one cell type, that is, mIMCD-3 cells. Figure 2. Ciliary cAMP signaling drives cyst growth A. Wild-type (WT) mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst number and area is shown on the right (quantification approach is illustrated in Appendix Fig S1). Data are shown as mean ± SD, each datapoint corresponds to an independent experiment, P-values calculated using an unpaired, two-sided Student’s t-test are indicated. B, C. Immunocytochemistry of mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Cells were labeled with DAPI (blue) to label the DNA, with an ARL13B antibody (magenta, ciliary marker) to label cilia, and a ZO-1 antibody (green) to label tight junctions of the epithelium (green). Scale bars are indicated. (B) Maximum intensity projection of a z-stack through the entire tubulus (top) or cyst (bottom). (C) Projection of confocal slices acquired from a cyst, illustrating that cilia are facing the lumen. D. mIMCD-3 cells stably expressing cilia-bPAC or cyto-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark and incubated with 10 µM of Forskolin. Exemplary images are shown (n ≥ 3). E. Quantification of the data exemplified in (D). Data are shown as mean ± SD, each datapoint corresponds to an independent experiment; P-values calculated using an unpaired, two-sided Student’s t-test are indicated. Download figure Download PowerPoint When applying cilia-bPAC or cyto-bPAC in mIMCD-3 cells cultured in 3D, we were astonished by the results: in contrast to the long-standing supposition that whole-cell cAMP elevation drives cystogenesis (Wallace, 2011), we observed that photoactivation of cilia-bPAC, but not cyto-bPAC, triggered cystogenesis, as determined by an increase in cyst number and the area covered by cysts (Fig 2D and E). Our results uncover that compartmentalized cAMP signaling underlies cyst development. Importantly, we verified that both cell types are in principle responsive to a pharmacological cAMP stimulus in the dark: Forskolin stimulated cyst development in both cell lines (Fig 2D and E). Furthermore, in cysts induced by photoactivation of cilia-bPAC, primary cilia were also facing the lumen, similar to the organization observed after Forskolin stimulation (Fig 1, EV1). Thus, our results demonstrate for the first time that a specific increase in ciliary cAMP levels is sufficient to induce cyst growth in a relevant cellular model for renal cyst development. Click here to expand this figure. Figure EV1. Ciliary cAMP signaling drives cyst growth A, B. Immunocytochemistry of mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, with an ARL13B antibody (magenta, ciliary marker) to label cilia, and a ZO-1 antibody (green) to label tight junctions of the epithelium. Scale bars are indicated. (A) Maximum intensity projection of a z-stack through the entire tubule (top) or cyst (bottom). (B) Projection of confocal slices acquired from a cyst. Scale bars are indicated. Download figure Download PowerPoint Ciliary cAMP signaling controls cell proliferation via mTOR signaling Epithelial cell proliferation is key for cyst development in ADPKD (Reif & Wallace, 2019). Thus, we investigated whether an increase in ciliary cAMP levels promotes cell proliferation and performed a fluorescent assay using the proliferation marker Ki-67. Photoactivation of cilia-bPAC cells, but not cyto-bPAC cells, significantly increased Ki-67 labeling compared to control cells (Fig 3A and B). In 3D culture, cysts formed by photoactivation of cilia-bPAC also showed Ki-67 high cells throughout cyst development (Fig 3C). Thus, ciliary cAMP signaling controls cell proliferation. Figure 3. Ciliary cAMP signaling controls cell proliferation via mTOR signaling A–C. Analysis of Ki-67 expression in wild-type (WT) and cilia-bPAC mIMCD-3 cells, kept in the dark or stimulated with light (1 h light/1 h dark, 72 h, 465 nm, 38.8 µW/cm²). (A) Cells were labeled with DAPI (blue) to label the DNA and a Ki-67 antibody (look-up table indicated). The DAPI channel was used as a mask (determined single-cell ROIs indicated) for the Ki-67 signal. Arrows indicate cells considered as Ki-67 high cells in the quantification shown in (B). Scale bars are indicated. (B) Quantification of Ki-67 high cells. The fraction of Ki-67 high cells was calculated in wild-type (WT), cilia-bPAC, and cyto-bPAC mIMCD-3 cells, kept in the dark or after light stimulation and the fold change compared to WT dark was calculated. Data are shown as mean ± SD, n = 9, 134–5,509 (median: 702) cells were analyzed per condition and experiment, each datapoint corresponds to an individual experiment; P-values were calculated using an unpaired, two-sided Student’s t-test. (C) Immunocytochemistry of mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during continuous exposure to light for 3 days (top) or 9 days (bottom) (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, with an ARL13B antibody (magenta) to label cilia, and a Ki-67 antibody (shown on right, look-up-table indicated). Arrows indicate Ki-67 high cells. D. Immunoblotting of lysates from wild-type (WT) and cilia-bPAC mIMCD-3 cells for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²). E. Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± SD, n > 3; P-values were calculated using a paired, two-sided Student’s t-test. Each data point shows an independent experiment. F. Quantification of Ki-67 high cells. The fraction of Ki-67 high cells was calculated in cilia-bPAC mIMCD-3 cells kept in the dark or stimulated with light and in the presence or absence of 10 nM rapamycin. The fold change compared to cilia-bPAC dark was calculated. Data are shown as mean ± SD, n = 9, 134–16,781 (median: 1,309) cells were analyzed per condition and experiment, each data point corresponds to an individual experiment; P-values were calculated using an unpaired, two-sided Student’s t-test. Data for cilia-bPAC in the dark and after light stimulation (both without rapamycin) has been taken from (B) but normalized as indicated here. G. mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with or without 10 nM rapamycin. Light exposure started 2 days later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3). H. Quantification of the data exemplified in (G). Data are shown as mean ± SD, each datapoint corresponds to an independent experiment; P-values calculated using a paired, two-sided Student’s t-test are indicated. Download figure Download PowerPoint Cell proliferation in ADPKD has been proposed to be driven by mTOR signaling (Ye et al, 2017). We hypothesized that ciliary cAMP signaling controls cell proliferation via stimulating mTOR signaling. To test this