Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
- Department of Biochemistry and Medicine, Stanford University School of Medicine, United States
- Department of Physiological Chemistry, Genentech, United States
- Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, United Kingdom
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
PTCH1 activity increases inner leaflet cholesterol.
(A) Total internal reflection fluorescence microscopy (TIRFM) was used to measure the recruitment of two different fluorescent cholesterol sensors (TdTomato-PFOD4H or GFP-GRAM1b) to the inner …
Figure 1—figure supplement 1
PTCH1 activity decreases outer leaflet cholesterol.
(A) Steady-state mNeon-ALOD4 binding to the outer leaflet of the plasma membrane of HEK293T cells expressing PTCH1 under the control of a Dox-inducible promoter. SHH (1 μM) was added to inactivate …
Figure 2 with 1 supplement
Measurement of outer leaflet accessible cholesterol in live cells with total internal reflection fluorescence microscopy (TIRFM).
(A) Extraction of outer leaflet accessible cholesterol by MβCD results in detachment of TdTomato-PFOD4H from the inner leaflet of the plasma membrane due to flip-flop of cholesterol between the two …
Figure 2—figure supplement 1
Characterization of a kinetic assay to measure outer leaflet accessible cholesterol.
(A) Steady-state TdTomato-PFOD4H fluorescence at the basal plasma membrane measured by TIRFM in cells incubated with or without ALOD4 (2 μM or 5 μM for 45 min prior to assay). Each circle represents …
Figure 3 with 2 supplements
PTCH1 depletes outer leaflet accessible cholesterol.
(A) Cartoon representations of PTCH1 bound to SHH and PTCH1-ᐃL2. Deletion of the second extracellular loop (L2) in PTCH1-ᐃL2 abolishes both SHH binding and a putative tunnel through PTCH1 that has …
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Figure 3—source data 1
Dotted lines mark the cropped region of the immunoblot that was used to generate panel Figure 3B.
As denoted on the left immunoblot PTCH1 (ptc) samples were run in duplicate.
- https://cdn.elifesciences.org/articles/70504/elife-70504-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Effect of PTCH1-ᐃL2 on membrane cholesterol accessibility.
(A) Immunoblotting was used to measure the abundance of cytoplasmically expressed TdTomato-PFOD4H in HEK293T cells incubated in media without Dox or with Dox to induce PTCH1 expression. See Figure …
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Figure 3—figure supplement 1—source data 1
Uncropped original scans of films used for immunoblots shown in Figure 3—figure supplement 1A.
Dotted lines mark the cropped region of the immunoblot that was used to generate panel Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/70504/elife-70504-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
PTCH1 and PTCH1-ᐃL2 reduce outer leaflet cholesterol accessibility.
(A, B) Curves showing data without normalization to baseline fluorescence values corresponding to Figure 3C and I.
Figure 4
Opposing effects of PTCH1 and sphingomyelin (SM) depletion on outer leaflet accessible cholesterol.
(A) Cartoon showing that depletion of SM with myriocin increases accessible cholesterol in the membrane outer leaflet, increasing the transport load on PTCH1 and preventing it from depleting …
Figure 5
PTCH1 cation binding site mutants fail to reduce accessible cholesterol.
(A) A view of the potential ion binding site in the middle of the PTCH1 transmembrane (TM) domain observed in the human SHH-PTCH1 complex (PDB 6DMY4) (Gong et al., 2018). The blue mesh represents …
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Figure 5—source data 1
Uncropped original scans of films used for immunoblots shown in Figure 5B.
Dotted lines mark the cropped region of the immunoblot that was used to generate panel Figure 5B.
- https://cdn.elifesciences.org/articles/70504/elife-70504-fig5-data1-v2.pdf
Figure 6 with 1 supplement
PTCH1 activity requires the cellular potassium gradient.
(A) The sodium and potassium gradients present across the plasma membrane of mammalian cells. In each of the panels a red ‘X’ is used to mark the gradient that is disrupted by the treatment used in …
Figure 6—figure supplement 1
Effect of rubidium and cesium ions on PTCH1 activity.
(A) Effect of isotonic media in which 150 mM NaCl is replaced with an equal concentration of choline-Cl (see also Figure 6D). (B) Time course of the change in TdTomato-PFOD4H membrane fluorescence …
Figure 7
Models for how PTCH1 inhibits SMO by reducing membrane cholesterol.
(A) Three proposed mechanisms by which PTCH1 depletes outer leaflet accessible cholesterol (see text for details). In models 1 and 2, inner leaflet cholesterol is predicted to be reduced, but in …
Author response image 1
Anti-PTCH1 antibodies were used to measure the fluorescence of PTCH1 at primary cilia in mouse embryonic fibroblasts (MEFs) stably expressing PTCH1 (left) or at the plasma membrane of HEK293T cells …
Author response image 2
Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition to HEK293T cells treated with increasing concentrations of Dox to induce the expression of progressively greater amounts of PTCH1.
The immunoblot (top) shows whole cell PTCH1 abundance after treatment with increasing concentrations of Dox.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Escherichia coli) | BL21 Rosetta DE3 pLYS | MilliporeSigma | Cat# 71403-3 | |
| Cell line (Homo sapiens) | 293T-REx Flp-In | Invitrogen | Cat# R780-07;RRID:CVCL_U427 | |
| Cell line (H. sapiens) | 293T-REx Flp-In: PTCH1-1D4 | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Cell line (H. sapiens) | 293T-REx Flp-In: PTCH1-ᐃL2-1D4 | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Cell line (H. sapiens) | 293T-REx Flp-In: PTCH1-1D4, D513Y | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Cell line (H. sapiens) | 293T-REx Flp-In: PTCH1-1D4, D513A/514 A | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Antibody | Anti-PTCH1(Rabbit polyclonal) | PMID:17641202 | Dr. Rajat Rohatgi (Stanford University)WB (1:500) | |
| Antibody | Anti-RFP (Mouse monoclonal) | Thermo Fisher Scientific | Cat# MA5-15257; RRID:AB_10999796 | WB (1:2000) |
| Antibody | Anti-P38(Rabbit polyclonal) | Abcam | Cat# ab7952; RRID: AB_306166 | WB (1:2000) |
| Antibody | Anti-mouse IgG (H + L)(Peroxidase AffiniPure Donkey) | Jackson ImmunoResearch Laboratories | Cat# 715-035-150; RRID: AB_2340770 | WB (1:10,000) |
| Antibody | Anti-rabbit IgG (H + L) (Peroxidase AffiniPure Donkey) | Jackson ImmunoResearch Laboratories | Cat# 111-035-144; RRID: AB_2307391 | WB (1:10,000) |
| Recombinant DNA reagent | mNeon-ALOD4 in pRSETB (plasmid) | PMID:33712199 | Dr. Arun Radhakrishnan (University of Texas, Southwestern Medical Center) | |
| Recombinant DNA reagent | mNeon-ALOD4- mutant in pRSETB (plasmid) | PMID:33712199 | Dr. Arun Radhakrishnan (University of Texas, Southwestern Medical Center) | |
| Recombinant DNA reagent | pcDNA5-FRT-TO Flp-In (plasmid) | Thermo Fisher Scientific | Cat# V652020 | |
| Recombinant DNA reagent | PTCH1-1D4 in pcDNA5-FRT-TO Flp-In (plasmid) | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Recombinant DNA reagent | dL2-PTCH1-1D4 in pcDNA5-FRT-TO Flp-In (plasmid) | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Recombinant DNA reagent | PTCH1-1D4, D513Y in pcDNA5-FRT-TO Flp-In (plasmid) | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Recombinant DNA reagent | PTCH1-1D4, D513A/D514A in pcDNA5-FRT-TO Flp-In (plasmid) | This paper | Dr. Rajat Rohatgi (Stanford University) | |
| Recombinant DNA reagent | TdTomato-PFO-D4H in pGEX-6P1 (plasmid) | PMID:25663704 | Dr. Gregory Fairn (University of Toronto) | |
| Recombinant DNA reagent | GFP-Gram1b in pEGFP-C1 (plasmid) | PMID:31724953 | Dr. Yasunori Saheki (Nanyang Technological University, Singapore) | |
| Recombinant DNA reagent | POG44 Flp-Recombinase expression vector (plasmid) | Thermo Fisher Scientific | Cat# V600520 | |
| Peptide, recombinant protein | Sonic Hedgehog | PMID:19561611 | Dr. Christian Siebold (Oxford University) | |
| Chemical compound, drug | Methyl beta cyclodextrin | Sigma-Aldrich | Cat# C4555-5G | |
| Chemical compound, drug | Myriocin | Cayman Chemicals | Cat# 35891-70-4 | |
| Chemical compound, drug | Doxycycline | Sigma-Aldrich | Cat# D9891 | |
| Chemical compound, drug | Monensin | Thermo Fisher Scientific | Cat# 461450010 | |
| Chemical compound, drug | Nigericin | Sigma-Aldrich | Cat# 481990 | |
| Chemical compound, drug | Dulbecco’s modified Eagle’s medium | Thermo Fisher Scientific | Cat# SH30081FS | |
| Chemical compound, drug | Fetal bovine serum | Sigma-Aldrich | Cat# S11150 | |
| Chemical compound, drug | Sodium pyruvate | Gibco | Cat# 11-360-070 | |
| Chemical compound, drug | L-glutamine | Gemini Bio-products | Cat# 400106 | |
| Chemical compound, drug | Penicillin/ streptomycin | Gemini Bio-products | Cat# 400109 | |
| Chemical compound, drug | Nonessential amino acids | Gibco | Cat# 11140076 | |
| Chemical compound, drug | Essential amino acids | Thermo Fisher Scientific | Cat# 11130051 | |
| Chemical compound, drug | Vitamins | Thermo Fisher Scientific | Cat# 11120052 | |
| Chemical compound, drug | Glucose | Thermo Fisher Scientific | Cat# A2494001 | |
| Chemical compound, drug | HEPES | Lonza | Cat# 17-737E | |
| Chemical compound, drug | Sodium pyruvate | Gibco | Cat# 11-360-070 | |
| Chemical compound, drug | HT supplement | Thermo Fisher Scientific | Cat# 11067030 | |
| Chemical compound, drug | Optimem | Thermo Fisher Scientific | Cat# 31985-070 | |
| Chemical compound, drug | Polyethylenimine | Polysciences | Cat# 23966-1 | |
| Chemical compound, drug | Matrigel | Thermo Fisher Scientific | Cat# CB-40234A | |
| Chemical compound, drug | SigmaFast Protease inhibitor cocktail, EDTA-free | Sigma-Aldrich | Cat# S8830 | |
| Chemical compound, drug | Hygromycin B | VWR Life Science | Cat# 97064-454 | |
| Chemical compound, drug | Nikon Immersion Oil, Type NF 50 cc, nd = 1.515 (23°C) | Nikon | Cat# MXA22024 | |
| Chemical compound, drug | Cesium chloride | Sigma-Aldrich | Cat# C4036 | |
| Chemical compound, drug | Rubidium chloride | Sigma-Aldrich | Cat# R2252 | |
| Chemical compound, drug | NMDG chloride | Sigma-Aldrich | Cat# 66,930 | |
| Chemical compound, drug | Choline chloride | Research Products International | Cat# C41040 | |
| Others | Chambered #1.5 German Coverglass, 8 well | Lab-Tek II | Cat# 155409 |
