Cholesterol accessibility at the ciliary membrane controls hedgehog signaling
- Stanford University School of Medicine, United States
- University of Texas Southwestern Medical Center, United States
- Washington University School of Medicine, United States
- University of Oxford, United Kingdom
Figures
Figure 1
CRISPR screens identify lipid-related genes that influence hedgehog signaling.
(A) Flowchart summarizing the screening strategy. Screens for positive and negative regulators used a high, saturating concentration of SHH (HiSHH, 25 nM) or a low concentration of SHH (LoSHH, 3.2 …
Figure 2 with 1 supplement
Enzymes that generate cholesterol positively regulate hedgehog signaling.
(A) The post-squalene portion of the cholesterol biosynthetic pathway, with enzymes colored according to their FDR corrected p-value in our CRISPR screens (see Supplementary files 2 and 3). Two …
Figure 2—figure supplement 1
Abundance of sterols in Dhcr7-/- and Dhcr24-/- cells.
(A) Agarose gel electrophoresis showing amplification of relevant genomic DNA regions using primers that flank the two-guide CRISPR edits in the Dhcr7-/-, Dhcr24-/- and Lss-/- cell lines. A downward …
Figure 3 with 1 supplement
Enzymes that generate sphingomyelin negatively regulate hedgehog signaling.
(A) The pathway for the synthesis of SM, with enzymes colored according to their FDR corrected p-value in our CRISPR screens. Steps in the pathway blocked by myriocin and fumonisin B1 are denoted in …
Figure 3—figure supplement 1
Analysis of sphingomyelin levels and their effect on hedgehog signaling.
(A) Thin Layer Chromatography was used to measure SM and phosphatidylcholine (PC) levels in cells treated with myriocin. Purified egg and milk sphingomyelin were used as standards. (B) Flow …
Figure 4 with 1 supplement
Sphingomyelin depletion potentiates hedgehog signaling at the level of Smoothened.
(A) HH signaling triggered by HiSHH (25 nM) in the presence or absence of Vismodegib (2.5 µM) in NIH/3T3 cells treated with myriocin. (B) Human SMO in complex with cholesterol (PDB 5L7D) …
Figure 4—figure supplement 1
The effect of myriocin treatment on the abundances of hedgehog pathway components and on ciliary protein trafficking.
(A) Western Blot showing levels of HH signaling proteins (SMO, SUFU and GLI3-FL/GLI3R) and HH target genes (GLI1 and PTCH1) after treatment with myriocin and either LoSHH (2.5 nM), HiSHH (25 nM) or …
Figure 5
Reducing accessible cholesterol in myriocin-treated cells impairs hedgehog signaling.
(A) Cholesterol and SM form SM-cholesterol complexes in which cholesterol is sequestered and prevented from interacting with proteins like SMO. The ratio of SM to cholesterol determines the level of …
Figure 6
ALOD4 impairs hedgehog signaling by trapping accessible cholesterol.
(A) ALOD4 and MβCD reduce accessible cholesterol by different mechanisms. ALOD4 binds and traps accessible cholesterol in the outer leaflet of plasma membranes of intact cells, without changing …
Figure 7 with 1 supplement
Primary cilia have high sphingomyelin and low accessible cholesterol.
(A–C) Ratio of mean ciliary staining intensity to mean plasma membrane staining intensity (the C/P ratio, see text) for OlyA_E69A, OlyA, or PFO* (see Figure 5A) in NIH/3T3 cells left untreated (A) …
Figure 7—figure supplement 1
Sphingomyelin depletion increases accessible cholesterol at cilia.
(A–C) Ciliary staining intensity for OlyA_E69A, OlyA or PFO* in NIH/3T3 cells left untreated (A) or treated with either myriocin (80 μM) or cholesterol:MβCD complexes (B and C) (n > 28 cilia). …
Figure 8 with 1 supplement
PTCH1 decreases the pool of accessible cholesterol at primary cilia.
(A) Flow cytometry was used to measure plasma membrane PFO* staining in intact cells after HiSHH treatment (n > 4000 cells per violin). Fold changes of median values (SHH treated over untreated) are …
Figure 8—figure supplement 1
SHH- induced changes in accessible cholesterol levels at cilia are dependent on PTCH1 activity.
(A) PFO* staining at primary cilia in Ptch1-/- MEFs in the presence of HiSHH (50 nM, 15 min) in cells treated with myriocin or left untreated (n > 38 cilia). (B) PFO* staining at primary cilia in …
Additional files
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Supplementary file 1
List of genes and sgRNAs in the custom lipid library used for the screens in Figure 1.
The first tab lists all the sgRNAs used in the library and the second tab lists all the lipid-related genes targeted by the library.
- https://doi.org/10.7554/eLife.50051.015
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Supplementary file 2
Complete tabulated results for the HiSHH-Bottom10% screen shown in Figure 1B.
The first tab contains a description of the columns in the second tab, which contains the scores for each sgRNA output from the MAGeCK algorithm. In the MAGeCK algorithm, gene enrichment or depletion in the sorted population (compared to the unsorted) are denoted as ‘pos’ or ‘neg’ respectively. Genes are listed in rank order according to the MAGeCK criteria.
- https://doi.org/10.7554/eLife.50051.016
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Supplementary file 3
Complete tabulated results for the LoSHH-Top10% screen shown in Figure 1C.
Organized in a manner identical to Supplementary file 2, but for the LoSHH-Top5% screen.
- https://doi.org/10.7554/eLife.50051.017
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Supplementary file 4
Compiled lists of lipid pathway genes from the KEGG database used for the analysis in Figure 1D.
The first tab contains lists of genes identified in each lipid biosynthesis pathway found in the KEGG database for Mus musculus. Column A contains a legend used throughout the spreadsheet. For each lipid biosynthesis pathway, the name of the gene is given along with the FDR-corrected p-values from the HiSHH-Bottom10% and LoSHH-Top5% screens and the mRNA expression level in the NIH/3T3 (see Supplementary file 5). The second tab contains the same analysis focused on oxysterol-related genes manually curated from the literature.
- https://doi.org/10.7554/eLife.50051.018
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Supplementary file 5
Transcriptional profiling of NIH/3T3 cells using RNAseq.
A list of genes from NIH/3T3 cells and their respective mRNA abundances from two independent RNAseq experiments.
- https://doi.org/10.7554/eLife.50051.019
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Supplementary file 6
Key Resources Table.
This file describes reagents used in this study, including (when available or applicable) the type of reagent, the designation, the source and the catalogue numbers.
- https://doi.org/10.7554/eLife.50051.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.50051.021
