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ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C

Abstract

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER–lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann–Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann–Pick type C (NPC). Thus, ER–lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.

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Fig. 1: Cholesterol accumulates at the limiting membrane of NPC1-deficient lysosomes in an OSBP-dependent manner.
Fig. 2: OSBP is required for cholesterol-dependent lysosomal recruitment and activation of mTORC1.
Fig. 3: OSBP regulates mTORC1 upstream of the Rag GTPases.
Fig. 4: Both the lipid-transporting and lysosome-targeting functions of OSBP are required for mTORC1 activation by cholesterol.
Fig. 5: Excess lysosomal PtdIns4P after OSBP inhibition does not cause mTORC1 inhibition.
Fig. 6: VAPA and VAPB, the ER anchors for OSBP, are essential for cholesterol-dependent mTORC1 activation.
Fig. 7: OSBP inhibition abrogates aberrant mTORC1 signalling in NPC1-deficient cells.
Fig. 8: OSBP inhibition restores autophagic function in NPC1-deficient cells.

Data availability

Mass spectrometry data that support the findings of this study are provided in Supplementary Table 1 and have been deposited in ProteomeXchange with accession number PXD014733. Statistics source data for Figs. 14 and 68 and Supplementary Figs. 16 are provided in Supplementary Table 2. All data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank all of the members of the Zoncu laboratory for their insights; R. Perera and M. Rape for reading the manuscript; M. D. Shair (Harvard University) for providing us with the OSW-1 compound; G. Fairn (University of Toronto) for the D4H cholesterol probe cDNA; G. Hammond (University of Pittsburgh) and S. Grinstein (Hospital for Sick Children) for the P4M reporter and the scSac1 catalytic domain cDNAs; H. Arai and N. Kono (University of Tokyo) for the anti-OSBP rabbit polyclonal antibody. C.-Y.L. is the recipient of an American Heart Association postdoctoral fellowship (18POST34070059). This work was supported by the NIH Director’s New Innovator Award (1DP2CA195761-01), the Pew–Stewart Scholarship for Cancer Research, the Damon Runyon-Rachleff Innovation Award, the Edward Mallinckrodt Jr. Foundation Grant and the Packer Wentz Endowment (to R.Z.), a National Institutes of Health grant (R01 HL067773; to D.S.O).

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Authors

Contributions

C.-Y.L. and R.Z. conceived the study. C.-Y.L., O.B.D., H.R.S. and R.Z. designed experiments. C.-Y.L., H.R.S., O.B.D. and J.Z. performed experiments. X.J., C.A.B. and J.L.C. performed mass spectrometry measurements. D.K.N. and D.S.O. provided advice on experimental design and data analysis. C.-Y.L. and R.Z. wrote the manuscript. All of the authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Roberto Zoncu.

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Competing interests

R.Z. is a co-founder, consultant and stockholder of Frontier Medicines Corporation. D.K.N. is a co-founder, stock-holder and scientific adviser for Artris Therapeutics and Frontier Medicines Corporation.

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Integrated supplementary information

Supplementary Figure 1 OSBP-delivered cholesterol accumulates on the surface of NPC1-null lysosomes.

a, Coomassie blue staining of recombinant GST-tagged D4H*-mCherry. b, NPC1-deleted HEK-293T cells were treated with 1 μM LysoSensor Green DND-189 for 30 min, fixed, permeabilized with liquid nitrogen (LN2) where indicated, and subjected to cholesterol labeling by GST-D4H*-mCherry and filipin. Scale bar, 10 μm. c, Accumulation of D4H*-mCherry on the plasma membrane of non-permeabilized cells. Control HEK-293T cells were subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP2. Scale bar, 10 μm. d, NPC1 knockout MEFs display cholesterol accumulation at the lysosomal membrane, in addition to the lysosomal lumen. Wild type and NPC1 knockout MEFs were fixed, breached with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP1. Scale bar, 10 µm. e, NPC1 inhibition via U18666A treatment induces cholesterol deposition at the limiting membrane as well as in the lumen of lysosomes. Control human fibroblasts were treated with U18666A (5 µg/ml) for 6h and subjected to cholesterol labeling and LAMP2 staining. Scale bar, 10 µm. f, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in control human fibroblasts treated with either DMSO or U18666A (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 11), U18666A (n = 10), two-tailed, unpaired t-test. ****P = 5.35648 x 10-13 vs. U18666A). g, Depletion of OSBP significantly suppresses the accumulation of lysosomal membrane cholesterol in human NPC1 patient-derived fibroblasts. Human NPC1 patient-derived fibroblasts depleted of ORPs via shRNA were subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, then stained for endogenous LAMP2. Scale bar, 10 µm. See quantitation in Fig. 1f. Experiments in ad, and g performed independently two times. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 2 OSBP inactivation via OSW-1 treatment abrogates the accumulation of lysosomal membrane cholesterol in NPC1-deficient cells.

a, OSW-1 treatment in human NPC1 patient-derived fibroblasts abolishes cholesterol deposition on the lysosomal limiting membrane. Human patient cells were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP2. Scale bar, 10 μm. b, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in human NPC1 patient-derived cells treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 11), OSW-1 (n = 10), two-tailed, unpaired t-test. ****P = 1.07121 x 10-12 vs. DMSO). c, OSW-1 treatment in NPC1 knockout MEFs significantly reduces cholesterol levels on the lysosomal limiting membrane. NPC1 knockout MEFs were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP1. Scale bar, 10 μm. d, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1 knockout MEFs treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 30), OSW-1 (n = 34), two-tailed, unpaired t-test. ****P = 8.39052 x 10-15 vs. DMSO). e, OSW-1 treatment in NPC1-null cells significantly removes cholesterol levels on the lysosomal limiting membrane. NPC1-deleted HEK-293T cells expressing FLAG-GFP-Tmem192 were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, and subjected to cholesterol labeling by GST-D4H*-mCherry and filipin. Scale bar, 10 μm. f, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1-null cells treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 66), OSW-1 (n = 73), two-tailed, unpaired t-test. ****P = 8.69018 x 10-11 vs. DMSO). Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 3 Lysosomal localization of a subset of OSBP in cells.

a, A pool of OSBP is localized to LAMP2-positive lysosomes. (left) HEK-293A cells stably expressing wild type or the indicated variants of FLAG-GFP-OSBP were subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 µm. (middle) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per genotype; n represents cell number: WT (n = 13), ΔELSK (n = 11), ΔPH (n =13), ANOVA with Dunnett’s multiple comparison test. ****Adjusted P = 0.0001 vs. WT cells). (right) Expression levels of the wild type FLAG-GFP-OSBP in relative to those of the endogenous OSBP in HEK-293A cells. Cells were harvested and subjected to immunoblot analysis. Experiment repeated two times. b, A pool of OSBP is localized to LAMP2-positive lysosomes where mTOR and p18 are present. (left) HEK-293A cells stably expressing either wild type or PH domain-deleted FLAG-GFP-OSBP were subjected to immunofluorescence for endogenous LAMP2, mTOR and p18, respectively. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-, mTOR-, p18-labeled vesicles that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per genotype; n represents cell number: LAMP2 for WT (n = 9), ΔPH (n = 12), two-tailed, unpaired t-test. ****P = 1.07095 x 10-6 vs. WT; mTOR for WT (n = 10), ΔPH (n = 12), two-tailed, unpaired t-test. ****P = 3.89303 x 10-7 vs. WT; p18 for WT (n = 11), ΔPH (n = 9), two-tailed, unpaired t-test. ****P = 1.54976 x 10-7 vs. WT). c, OSW-1 treatment triggers clustering of OSBP on lysosomes. (left) HEK-293A cells stably expressing FLAG-GFP-OSBP were treated with either DMSO or with the OSBP inhibitor OSW-1 (10 nM) for 2h and subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per treatment; n represents cell number: DMSO (n = 11), OSW-1 (n = 10), two-tailed, unpaired t-test. ****P = 3.47655 x 10-8 vs. DMSO). d, Depletion of VAPA and VAPB increases the lysosomal localization of OSBP. (left) Cells expressing the indicated shRNAs were subjected to immunofluorescence for endogenous OSBP and LAMP2. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per genotype; n represents cell number: shLuc (n = 18), shVAPA/B (n = 17), two-tailed, unpaired t-test. ****P = 9.24193 x 10-11 vs. shLuc). (bottom) knockdown of VAPA/B was validated by immunoblotting. Experiments repeated two times. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 4 OSBP inhibition causes cholesterol sequestration in the ER and its depletion from lysosomes.

a, Western blot analysis of immuno-isolated lysosomes used for mass spectrometry-based measurement of lysosomal cholesterol content. Lysosomes were purified via anti-HA magnetic beads from cells expressing Tmem192-mRFP-3xHA that have been previously treated with either DMSO or 20 nM OSW-1 for 8h. Samples for lysosomes and post-nuclear supernatant (PNS) were immunoblotted for the indicated proteins. See lysosomal cholesterol measurement in Fig. 2a. Experiments repeated two times. b, (left) Depletion of OSBP induces a significant increase in cholesteryl esters but not total cholesterol. HEK-293T cells were depleted of OSBP by doxycycline-induced shRNA, and total lipid extracts were subjected to mass spectrometry analysis. ‘– Dox’ ‘+ Dox’ indicate control and OSBP-depleted cells (mean ± s.d., n = 5 biologically independent samples per group, two-tailed, unpaired t-test; total cholesterol, ****P = 0.996460171; cholesteryl esters, ****P = 3.69304 x 10-5; cholesteryl oleate, ****P = 0.000215477). (right) OSBP knockdown efficiency was validated by immunoblotting. Experiments repeated two times. c, Lipidomic profiling of HEK-293T cells presented in heat map indicating the relative levels of all the measured lipids normalized to their respective controls (– Dox group as control, n = 5 biologically independent samples per group). d and e, OSBP inactivation induces cholesterol accumulation in the ER. Cells were depleted of OSBP via shRNA (d) or treated with either DMSO or 20 nM OSW-1 for 8h (e), fixed and stained with filipin to visualize intracellular cholesterol distribution. Three independent fields of cells per condition are shown. Scale bar, 10 μm. Experiments repeated two times. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 5 Exogenously supplied cholesterol is transported to the lysosome via an OSBP-dependent route.

a, HEK-293T cells stably expressing Tmem192-mRFP-3xHA (T192-mRFP) were treated with DMSO or 20 nM OSW-1 for 2h, labeled with TopFluor-cholesterol (TF-Chol) complexed with MCD, chased at the indicated times, fixed, and imaged on a spinning disk confocal microscope. Scale bar, 10 µm. b, Quantitation of co-localization of TF-Chol with T192-mRFP-labeled lysosomes in cells treated with DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, data expressed as the fraction of T192-mRFP-labeled lysosomes that are positive for TF-Chol, 10 fields of view for each treatment/time point; n represents cell number: DMSO_18 min (n = 106), DMSO_30 min (n = 85), DMSO_60 min (n = 78), DMSO_90 min (n = 77), DMSO_180 min (n = 87), OSW-1_18 min (n = 100), OSW-1_30 min (n = 82), OSW-1_60 min (n = 85), OSW-1_90 min (n = 83), and OSW-1_180 min (n = 107). Total peak area derived from the Area Under Curve (AUC) analysis: DMSO, 60.94 ± 3.111, OSW-1, 14.86 ± 1.849). Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 6 OSBP regulates cholesterol-induced lysosomal recruitment and activation of mTORC1.

a, OSBP drives sterol-dependent lysosomal recruitment of mTORC1. HEK-293T cells expressing the indicated shRNAs were subjected to cholesterol depletion, followed by restimulation with MCD:cholesterol where indicated, and subjected to immunofluorescence for endogenous mTOR and LAMP2. Representative confocal microscopic images are shown. Scale bar, 10 μm. See quantitation in Fig. 2c. Experiments repeated two times. b, OSW-1 abolishes sterol-induced lysosomal recruitment of mTORC1. HEK-293T cells were subjected to cholesterol starvation and restimulation in the presence of DMSO or OSW-1 at the indicated concentrations and subjected to immunofluorescence for endogenous mTOR and LAMP2. Scale bar, 10 µm. c, Quantitation of co-localization between mTOR and LAMP2-positive lysosomes in cells subjected to the indicated treatments (mean ± s.d., 10 fields of view per treatment; n represents cell number: – chol (n = 150), + chol (n = 123), 1 nM OSW-1 + chol (n = 155), 10 nM OSW-1 + chol (n = 163), 20 nM OSW-1 + chol (n = 172), 50 nM OSW-1 + chol (n = 180), ANOVA with Dunnett’s multiple comparison test. **** Adjusted P = 0.0001 vs. + chol). d, Depletion of OSBP inhibits mTORC1 lysosomal localization in cells under full media conditions. HEK-293T cells depleted of OSBP were subjected to immunofluorescence for endogenous mTOR and LAMP2. Representative confocal microscopic images are shown. Scale bar, 10 μm. e, Quantitation of co-localization between mTOR and LAMP2-positive lysosomes in cells cultured in full media (mean ± s.d., 10 fields of view per genotype; n represents cell number: shLuc (n = 103), shOSBP (n = 118), two-tailed, unpaired t-test. ****P = 5.68202 x 10-15 vs. shLuc). f, knockdown of OSBP impairs basal state mTORC1 signaling. Cells expressing the indicated shRNAs were harvested and analyzed by immunoblotting for the indicated proteins and phospho-proteins. Experiments repeated two times. g, Depletion of OSBP has a modest effect on mTORC1 activation by amino acids. HEK-293T cells expressing the indicated shRNAs were either starved of amino acids for 50 min, or starved and restimulated with amino acids for 10 min. Cell lysates were immunoblotted for the indicated proteins and phospho-proteins. Fold changes of the protein phosphorylation are indicated. Experiments repeated two times. h, Loss of OSBP does not affect lysosomal localization of the Ragulator-Rag GTPase complex. HEK-293T cells depleted of OSBP were subjected to cholesterol starvation and restimulation, followed by immunofluorescence for endogenous p18, RagC and LAMP2, respectively. Scale bar, 10 μm. See quantitation in Fig. 3a. Experiment performed once. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 7 OSBP-mediated mTORC1 regulation requires its membrane tethering and lipid transfer activities.

a, The PH domain and FFAT motif are essential for proper intracellular targeting of OSBP. (left) Domain organization for OSBP full length and the truncations employed in this study. (right) HEK293A cells stably expressing the full length and the indicated truncations of FLAG-GFP-OSBP were subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 μm. b, HEK-293T cells were depleted of OSBP via shRNA or depleted of OSBP followed by lentivirus-mediated re-expression of FLAG-GFP-OSBP, either wild type or the indicated truncations. Cells were starved of cholesterol for 2h, or starved and restimulated with cholesterol for 2h. Cell lysates were immunoblotted for the indicated proteins and phospho-proteins. c, (upper) OSBP mutants and their corresponding loss of function are shown in the table. (bottom) HEK-293T cells were depleted of OSBP, reconstituted with OSBP wild type and mutants as indicated, and evaluated for cholesterol-dependent mTORC1 signaling. Cell lysates were immunoblotted for the indicated proteins and phospho-proteins. d, HEK-293A cells stably expressing wild type, PH domain-deleted and organelle-targeted FLAG-GFP-OSBP isoforms were subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 μm. Experiments in a (right), b, c (bottom) and d repeated two times with similar results.

Supplementary Figure 8 OSBP inhibition abolishes mTORC1 signaling and promotes clearance of p62 aggregates in NPC1-deficient cells.

a, Increased ER-lysosome contacts in NPC1-deficient cells. Human fibroblasts co-expressing the lysosomal and ER markers FLAG-GFP-TMEM192 and mCherry-Sec61b, respectively, were seeded on 35mm-glass-bottom dishes and subjected to time-lapse imaging on a spinning disk confocal microscope. The two channels were sequentially acquired at 1 sec intervals. Scale bar, 10 μm; Insets show magnified time-lapse images of the ER-lysosome contacts. Scale bar, 2 μm. b, Electron microscopy (EM) images of lysosomes/autophagic vacuoles in control and NPC1 patient-derived fibroblasts. Scale bar, 0.5 μm; scale bar in insets, 0.2 μm. Arrows indicates the close apposition between lysosomal vesicles and ER tubules. c, OSW-1 treatment eliminates p62/SQSTM1 accumulated in human NPC1 patient-derived fibroblasts in a dose-dependent manner. Cells were treated with OSW-1 at the indicated concentrations for 8h and subjected to immunoblotting for the indicated proteins. d, OSW-1 treatment eliminates p62/SQSTM1 accumulated in Npc1-/- MEFs in a dose-dependent manner. Cells were treated with OSW-1 at the indicated concentrations for 24h and subjected to immunoblotting for the indicated proteins, including p62 and LC3B-II. e, Bafilomycin A1 blocks OSW-1-induced p62 clearance. Npc1-/- MEFs were treated with either 20 nM OSW-1 for 24h, 400 nM Bafilomycin A1 for 4h, or in combination as indicated. f, Combined treatment of Torin1 and OSW-1 has no additional effects on p62 clearance in Npc1-/- MEFs. Cells were treated with either 20 nM OSW-1, 250 nM Torin1, or in combination as indicated for 24h. Cell lysates were immunoblotted for the indicated proteins. Experiments in af repeated two times with similar results.

Supplementary Figure 9 Unprocessed images of all gels and blots.

Lyso-IP (upper, Fig. 1e). OSBP depletion (lower left, Fig. 2d) and OSW-1 (lower right, Fig. 2e) inhibits sterol-induced mTORC1 signaling. OSBP acts upstream of the Rag GTPases (upper, Fig. 3d), GATOR1 (lower left, Fig. 3e) and KICSTOR (lower right, Fig. 3f). OSBP activates mTORC1 via lysosomal contact (upper, Fig. 4d) and lipid transport (lower, Fig. 4e). Excess PI4P does not inhibit mTORC1 in OSBP-depleted cells (Fig. 5d). VAPA/B (upper, Fig. 6c), but not STARD3 (lower left, Fig. 6d) and ORP1L (lower right, Fig. 6e), regulate sterol-dependent mTORC1 signaling. NPC1 regulates sterol-induced OSBP-VAP binding (left, Fig. 7b). OSBP depletion abolishes hyperactive mTORC1 in NPC1-null cells (right, Fig. 7c). OSBP inhibition by OSW-1 (left, Fig. 8b) and shRNA (right, Fig. 8c) restores autophagy in NPC1 fibroblasts. GFP-OSBP expression in HEK-293A cells (left, Supplementary Fig. 3a). Validation of VAPA/B knockdown (right, Supplementary Fig. 3d). Western blots of Lyso-IP (left, Supplementary Fig. 4a). Validation of OSBP knockdown (right, Supplementary Fig. 4b). Basal state (left, Supplementary Fig. 6f) and amino acid-induced (right, Supplementary Fig. 6g) mTORC1 signaling in OSBP-depleted cells. Truncation (left, Supplementary Fig. 7b) and mutation (right, Supplementary Fig. 7c) analysis of OSBP on mTORC1 activation by sterol. OSW-1 activates autophagy in NPC1 fibroblasts (left, Supplementary Fig. 8c; middle, Supplementary Fig. 8d) in a BafA1-sensitive manner (upper right, Supplementary Fig. 8e) via mTORC1 pathway (lower right, Supplementary Fig. 8f).

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Supplementary Information

Supplementary Figures 1–9, titles and legends for Supplementary Tables 1–3.

Reporting Summary

Supplementary Table 1

Full lysosomal proteomics data.

Supplementary Table 2

Statistics source data.

Supplementary Table 3

List of antibodies.

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Lim, CY., Davis, O.B., Shin, H.R. et al. ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C. Nat Cell Biol 21, 1206–1218 (2019). https://doi.org/10.1038/s41556-019-0391-5

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