Truong et al., 2021 - Vertebrate cells differentially interpret ciliary and extraciliary cAMP. Cell   184(11):2911-2926.e18 Full text @ Cell

Fig. 1 (A) Immunofluorescence imaging of somites 12–14 in 24 hpf Gli:mCherry transgenic embryonic somites without bPAC (control), expressing Cyto-bPAC or expressing Cilia-bPAC raised in the dark or stimulated with light. Scale bar, 40 μm. (B) Quantification of Gli:mCherry-expressing cells per somite. n = 8–10 embryos collected over two independent clutches. Cells in somites 12 through 15 were counted and an average value of cells per somite was determined for each embryo. The average values per embryo were used as individual data points in Figure 2 graphs and statistical analyses. (C) Schematic of how HH signaling affects somitic cell fate. Muscle pioneer cells (MPs, green) express high levels of En and are specified by high levels of HH signaling. Superficial slow fibers (SSFs) express Prox1 and are specified by lower levels of HH signaling. Modest attenuation of HH signaling attenuates MP formation, and more severe attenuation of HH signaling attenuates SFF formation. (D) Immunofluorescence imaging of En (green) and Prox1 (magenta) in wild-type and Cyto-bPAC- or Cilia-bPAC-expressing embryos with or without light stimulation. Scale bar, 40 μm. (E) Quantification of the average number of En-expressing cells per somite. (F) Quantification of the average number of Prox1-expressing cells per somite. n = 9–20 embryos for each condition. Each data point represents the average number of En or Prox1 expressing cells per somite 12 through 15 per 24 hpf embryo. Significance was determined via two-way ANOVA followed by Tukey’s multiple comparison test. A p value <0.05 was considered statistically significant and is denoted as follows: ∗∗∗∗p < 0.0001. Data are represented as means ± SD.

Fig. 2 (A) Immunofluorescence imaging of wild-type and transgenic NIH/3T3 cells stably expressing GFP-tagged Cyto-bPAC or Cilia-bPAC under the control of a minimal δ-crystallin promoter stained for GFP-tagged bPAC (green), cilia (TUBAC, red), basal bodies (γTUB, grayscale) and nuclei (blue). Arrowheads indicate cilia depicted in insets. Insets display overlay (top), cilia and basal bodies alone (middle), and GFP-tagged bPAC alone (bottom). Scale bars, 10 μm and 2 μm (inset). (B) Immunoblot of wild-type and transgenic NIH/3T3 cells stably expressing GFP-tagged Cyto-bPAC and Cilia-bPAC under the control of the EF1α promoter. Whole-cell protein lysates were immunoblotted with antibodies to GFP and GAPDH loading control. (C) Quantification of cAMP in wild-type or transgenic cells expressing Cyto-bPAC or Cilia-bPAC under the control of a minimal δ-crystallin promoter by ELISA. Cells were stimulated with pulsed 0.14 mW/cm2 470 nm blue light or kept in the dark in the presence of 100 μM IBMX for 30 min. cAMP concentration was determined by ELISA and normalized to total protein content. n = 4 biological replicates. Significance was determined via two-way ANOVA followed by Tukey’s multiple comparison test. (D) Quantitation of Ciliary Pink Flamindo fluorescence in Cyto-bPAC-expressing (red) or Cilia-bPAC-expressing (blue) cells either with (solid circles) or without blue light (open circles). Cells were stimulated with 100-ms pulses of 52.7 mW/cm2 blue light every 3 s for 1 min. At the end of that minute, 100 μM forskolin was added. We calculated the ratio of Pink Flamindo to mIFP fluorescence normalized to the ratio at t = 0. Each trace represents n > 12 cells from three independent experiments. The maximum Ciliary Pink Flamindo fluorescence on blue light stimulation is also shown. Significance was assessed using two-way ANOVA followed by Tukey’s multiple comparison test. (E) qRT-PCR measurement of Gli1 expression by wild-type, Cyto-bPAC-expressing cells, or Cilia-bPAC-expressing cells stimulated for 4 h with vehicle (DMSO), 200 nM SAG, or 200 nM SAG with pulsed 0.14 mW/cm2 470 nm blue light. n = 3 biological replicates. Significance was assessed using one-way ANOVA followed by Tukey’s multiple comparison test. (F) Ratios of Gli1 expression in wild-type and Cyto-bPAC-expressing or Cilia-bPAC-expressing cells treated with 200 nM SAG and blue light to Gli1 expression treated with 200 nM SAG alone. Significance was assessed using one-way ANOVA followed by Tukey’s multiple comparison test. (G) Immunoblots of GLI3 and GAPDH of whole-cell lysates from wild-type, Cyto-bPAC-expressing, or Cilia-bPAC-expressing cells stimulated as in (E). (H) Quantification of GLI3 repressor (GLI3R) normalized to GAPDH (loading control). n = 3 biological replicates. Significance was assessed using one-way ANOVA followed by Tukey's multiple comparison test. For all panels, p values are indicated as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. Data are represented as means ± SD.

Fig. 4 (A) Schematic of tools to control a Gɑs-coupled designer GPCR at distinct subcellular locations. PM-DREADD localizes to the plasma membrane. Cilia-DREADD, a fusion with the ciliary protein ARL13B, localizes to cilia. These Gɑs-coupled DREADDs induce cAMP generation on stimulation with their ligand, the otherwise pharmacologically inert drug CNO. (B) Immunofluorescence imaging of wild-type and transgenic NIH/3T3 cells stably expressing GFP-tagged PM-DREADD or Cilia-DREADD. Images depict cells stained for the GFP-tagged DREADDs (GFP, green), cilia (TUBAC, red), basal bodies (γTUB, grayscale), and nuclei (Hoechst, blue). Insets indicate amount of GFP-tagged DREADD localization to the cilium (defined by TUBAC and γTUB). Scale bars, 10 μm and 1 μm (inset). (C) Quantification of cAMP in wild-type and PM-DREADD- or Cilia-DREADD-expressing NIH/3T3 transgenic cell lines. Cells were stimulated with either vehicle (DMSO) or 100 nM CNO in the presence of 10 μM IBMX for 3 h. cAMP concentration was determined by ELISA and normalized to total protein content. n = 4 biological replicates. Significance was determined via two-way ANOVA followed by Tukey’s multiple comparison test. Data are represented as means ± SD. (D) Expression of Gli1 by wild-type and PM-DREADD- or Cilia-DREADD-expressing cells stimulated with vehicle (DMSO), 200 nM SAG, or 200 nM SAG and 5 nM CNO for 5 h before measurement by qRT-PCR. n = 4–5 biological replicates. Significance was determined via one-way ANOVA followed by Tukey’s multiple comparison test. (E) Ratios of Gli1 expression in wild-type and PM-DREADD- or Cilia-DREADD-expressing cells treated with 200 nM SAG and 5 nM CNO to Gli1 expression treated with 200 nM SAG alone. Significance was determined via one-way ANOVA followed by Tukey’s multiple comparison test. n = 4–5 biological replicates. (F) Immunofluorescence imaging of wild-type and transgenic NIH/3T3 cells stably expressing SSTR3-GFP, a ciliary Gαi-coupled GPCR, fused to GFP. Images depict cells stained for SSTR3-GFP (GFP, green), cilia (ARL13B, red), basal bodies (FOP, grayscale), and nuclei (Hoechst, blue). Cells were treated either vehicle (DMSO) or 10 μM somatostatin (SST) for 5 h. Insets depict SSTR3-GFP localization to the cilium (defined by ARL13B and FOP). Scale bars, 10 μm and 1 μm (inset). (G) Quantification of SSTR3-mediated inhibition of cAMP production. Wild-type and SSTR3-GFP-expressing NIH/3T3 cells were stimulated with either vehicle (DMSO), 10 μM SST, 10 μM FSK, or 10 μM FSK and 10 μM SST in the presence of 10 μM IBMX for 30 min. cAMP concentration was measured by ELISA and normalized to total protein content. n = 3–6 biological replicates. Significance was determined via one-way ANOVA followed by Tukey’s multiple comparison test. Data are represented as means ± SD. (H) Expression of Gli1 by wild-type and SSTR3-GFP-expressing NIH/3T3 cells treated with vehicle (DMSO), 3 nM SAG, or 10 μM SST for 5 h and measured by qRT-PCR. n = 4 biological replicates. Significance was determined via one-way ANOVA followed by Tukey’s multiple comparison test. Data are represented as means ± SD. For all panels, a p value <0.05 was considered statistically significant and is denoted as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig. 5 (A) Modeling differential ciliary membrane localized PKA activation on cAMP generated at either the ciliary membrane or at the plasma membrane. Left panel: simulated distribution of PKA localization (magenta) to the plasma membrane and cilium (inset). Scale bar, 5 μm. Right panel: the percent of activated ciliary membrane localized PKA upon either ciliary membrane or plasma membrane cAMP generation. (B) Schematic of tools to inhibit PKA at distinct subcellular locations. Dominant-negative PKA (dnPKA), a form of PKA-R that constitutively binds and inhibits PKA-C, was fused to RAB23 S23N to prevent it from localizing to cilia (Extraciliary dnPKA). dnPKA was fused to RAB23 Q68L to localize it to cilia (Ciliary dnPKA). dnPKA was fused to 2x PACT to localize it to the basal body (Basal Body dnPKA). (C) Immunofluorescence imaging of zebrafish somites either uninjected or expressing GFP-tagged untargeted dnPKA, Extraciliary dnPKA, Basal Body dnPKA, or Ciliary dnPKA. Images depict 24 hpf somites stained for the GFP tag on dnPKA (green), cilia (TubAC, red), basal bodies (γTUB, grayscale), and nuclei (Hoechst, blue). Arrowheads indicate cilia depicted in inset. Insets display overlay of dnPKA, cilia, and basal bodies (left), overlay of cilia and basal bodies (middle), and dnPKA alone (right). Scale bars, 4 μm and 1 μm (inset). (D) Immunofluorescence imaging of Gli:mCherry-transgenic somites expressing GFP alone or the indicated dnPKAs. Images depict 24 hpf somites stained for En (green) and mCherry (magenta). Scale bar, 40 μm. (E) Immunoblot of lysates from 24 hpf zebrafish embryos expressing indicated GFP-tagged forms of dnPKA. Blotting for β-actin controls for loading. (F) Relative GFP fluorescence of control uninjected embryos, Extraciliary dnPKA-expressing embryos, and Ciliary dnPKA-expressing embryos. Fluorescence was normalized to the mean of uninjected embryos. (G) Quantification of Gli:mCherry-expressing cells per somite of uninjected embryos, and those expressing GFP, untargeted dnPKA, Extraciliary dnPKA, Basal Body dnPKA, or Ciliary dnPKA. Each point represents the number of mCherry-expressing cells averaged over four somites per embryo. Cells in somites 12 through 15 were counted and an average value of cells per somite was determined for each embryo. The average values per embryo were used as individual data points in graphs and statistical analyses in (G) and (H). (H) Quantification of En-expressing MPs per somite of uninjected embryos, and those expressing GFP, untargeted dnPKA, Extraciliary dnPKA, Basal Body dnPKA, or Ciliary dnPKA. For Basal Body dnPKA-expressing embryos, n = 18 and for all other conditions, n > 30 from three independent injections. Significance was determined via one-way ANOVA followed by Tukey’s multiple comparison test. A p value <0.05 was considered statistically significant and is denoted as follows: ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Data are represented as means ± SD throughout the figure.

Fig. 6 (A) Immunofluorescence imaging of Cilia-bPAC transgenic embryos expressing GFP, untargeted dnPKA, Extraciliary dnPKA, Basal Body dnPKA, or Ciliary dnPKA and either raised in the dark or stimulated with light. Images depict 24 hpf somites stained for MPs (En, green) and nuclei (Hoechst, blue). Scale bar, 40 μm. (B) Quantification of En-expressing cells per somite. n = 12–20 embryos per condition, from three independent injections. Statistical significance was assessed using a two-tailed unpaired t test to compare dark- and light-treated embryos for each construct. A p value <0.05 was considered statistically significant and is denoted as follows: ∗∗∗∗p < 0.0001. Data are represented as means ± SD. (C) Schematic model of how ciliary cAMP and PKA regulate HH signal transduction. Ciliary cAMP regulated by ciliary GPCRs locally activates a pool of ciliary PKA, which phosphorylates GLI to generate its transcriptional repressor form (GLIR). Equivalent amounts of cAMP produced by GPCRs in the plasma membrane do not activate ciliary PKA. Thus, upon HH stimulation and in the absence of ciliary PKA activity, GLI assumes its transcriptional activator form (GLIA) and induces HH target gene expression.

Acknowledgments:
ZFIN wishes to thank the journal Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Cell, 184(11), Truong, M.E., Bilekova, S., Choksi, S.P., Li, W., Bugaj, L.J., Xu, K., Reiter, J.F., Vertebrate cells differentially interpret ciliary and extraciliary cAMP, 2911-2926.e18, Copyright (2021) with permission from Elsevier. Full text @ Cell