Methods

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

Cell culture, antibodies and other reagents

U2OS, HeLa and RPE cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s media (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (100 U/ml)/ streptomycin (100 μg/ml). Cas9-expressing K562 cells (Lu et al., 2018) were grown in RPMI 1640 Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate and penicillin (100 U/ml)/streptomycin (100 μg/ml). All cell lines were cultured at 37°C with 5% CO2. The NPC1 CRISPR knockout HeLa cell line was previously generated (Saha et al., 2020). Primary antibodies diluted in PBS- 1%BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were monoclonal Anti- LBPA (BMP) clone 6C4 1:1000 (Millipore, Cat# MABT837), rabbit polyclonal anti-LAMP2 1:500 (Invitrogen, Cat# PA1-655), mouse monoclonal anti-GST 1:1000 (Cell Signaling, Cat# 2624S), rabbit polyclonal anti-SNX13 1:1000 (Abcepta, Cat# AP12244b), mouse monoclonal anti- Tubulin 1:10000 (Sigma-Aldrich, Cat# T5168), rabbit monoclonal anti-HA Tag (Cell Signaling, Cat# 3724S). U18666A (Sigma-aldrich; Cat# U3633) was used at 1µM for 16h. Oleic acid (Sigma-Aldrich; Cat# O1008,) was conjugated with fatty-acid free BSA (Sigma-Aldrich; A8806) at a 6:1 molar ratio and used at 0.5 mM for 16h. The ACAT pharmacological inhibitor Sandoz 58-035 (Sigma-Aldrich; S9318) was used at 20 µg/mL for 16h.

Plasmids and transfections

Plasmids encoding SNX13-GFP and SNX14-GFP were a gift from Mike Henne (UT Sothwestern). The CFP-VAPA construct was a gift from Clare Futter (University College London). The WT-SNX13-2XHA plasmid was prepared as follows: First, the BioID2 insert from a target MCS-BioID2-HA plasmid (Addgene, Cat# 74224) was removed by restriction digestion at BspEI and HindIII sites and replaced with a 2XHA oligo sequence flanked by the same restriction sites. Next, the WT-SNX13 ORF sequence from the SNX13-GFP plasmid was shuttled into the NheI and BspEI restriction sites of the previously modified MCS- BioID2-HA. The SNX13 truncated constructs were cloned by Gibson assembly using WT- SNX13-2XHA as template (see primers in Supplementary Table 1). Transfections of U2OS cells plated on coverslips were carried out using GenJet Plus Reagent (SigmaGen Laboratories; Cat# SL10050) according to the manufacturer. For siRNA-mediated knockdown experiments, cells were transfected with siRNAs targeting human SNX13 (5’- CAGAAAGGCUCAACAGAAAUU-3’) or SNX14 (5’-GGAUGAAAGUAUUGACAAAUU-3’) using Lipofectamine RNAiMax (Invitrogen, Cat#13778-075) according to the manufacturer. Studies were conducted 48-72 h after siRNA transfection. A Scrambled siRNA was used as negative control (Ambion, Cat# AM4635).

Quantitative real-time PCR

Total RNA from SNX14 siRNA- or control siRNA-treated cells was extracted using QIAzol (Qiagen, Cat# 79306) as per the manufacturer. 1 μg isolated RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Bioscience, Cat# 4368814). qRT-PCR was performed using SYBRGreen-based technology GoTaq® qPCR master-mix (Promega, Cat# A600A). Specific SNX14 primers, and HPRT1 primers (reference gene) were used (see Table 1). PCRs were run in a LightCycler 96® Real-time PCR system (Roche). Transcript levels relative to HPRT1 were calculated using the ΔCt method.

Flow cytometry analysis of Cholesterol and BMP

K562 cells were treated ± 1µM U18666A. After 16h cells were harvested and washed before fixation in 2% paraformaldehyde, then permeabilized in 0.1% Saponin and stained with PFO*-Alexa647 or anti-BMP and Alexa Fluor 647 goat anti–mouse antibodies (Invitrogen). Quantification of cholesterol and BMP total fluorescence was performed using a BD Accuri C6 flow cytometer and data were analyzed using Flowjo software (Tree Star).

PFO* purification

Recombinant PFO was purified as previously described (Li, Lee and Pfeffer, 2017). Briefly, expression was induced in Rosetta 2 cells with 1 mM isopropyl β-p-thio- galactopyranside for 4 h at 37 °C. Cells were resuspended in buffer A PBS, 10% (vol/vol) glycerol, protease inhibitors and lysed using an Emulsiflex C-5 homogenizer (Avestin). Clarified lysate was incubated with Ni-NTA agarose (Qiagen, Cat# 30210) for 1 h at 4 °C and after washing with buffer A + 50 mM imidazole, bound protein was eluted with buffer A + 300 mM imidazole. The eluate was concentrated with an Amicon Ultra 10-kDa cutoff centrifugal filter (Millipore, Cat# UFC901024) and then exchanged into buffer A + 1 mm EDTA. PFO was directly conjugated to NHS ester Alexa Fluor 647 dye as per the manufacturer (Life Technologies). For immunofluorescence analysis of cholesterol, GST-PFO* was used (Meneses-Salas et al. 2020).

CRISPR/Cas9-bEXOmiR screens in K562 cells

The Genome-wide K562 CRISPR knockout library line was generated as previously described (Morgens et al., 2017). Briefly, a whole- genome library of exon-targeting sgRNAs (10 sgRNA per gene; Morgens et al., 2017) was synthesized and cloned into a lentivirus vector (Addgene, Cat# 89359) which together with third- generation lentiviral packaging plasmids (pVSVG, pRSV and pMDL) were transfected into HEK293T cells to generate lentiviral particles. Then ∼300 million Cas9-expressing K562 cells were infected at low MOI (<1). Transduced cells were selected and expanded in puromycin- supplemented media over 5–7 days before conducting experiments 10 days post-infection. All screens were performed as independent replicates. Two independent screens were performed for each lipid (cholesterol and BMP) and condition (±U18666A). For each screen, 600 million cells were stained. 16h before staining, cells were treated with 1µM U18666A or vehicle (control). Next day, cells were first pelleted and washed twice in cold PBS followed by fixation in 2% paraformaldehyde-PBS for 30 min at 4°C. Cells were then washed twice in PBS and permeabilized/blocked in 0.1%Saponin-1%BSA-PBS for 10 min. Cells were then incubated with 10 µg/ml Alexa-647 labeled Perfringolysin O* (Li et al., 2017) for 45 min in the cold. Alternatively, for BMP screens, staining was performed with mouse anti-BMP antibody for 1 h at 4°C, followed by 1 h incubation with Alexa 647-conjugated secondary antibody (Life Technologies) used at 1:2,000. Finally, cells were washed once in cold PBS and then kept in 15 mL PBS-0.5%BSA at 4°C for 16h before sorting. Next day, cells were separated into 10% high or 10% low PFO/BMP fluorescence populations by sorting on a BD FACSAriaTMII. Around 20 million cells were recovered from each gated population. Sorted cells were then sedimented by centrifugation, and the cell pellet was frozen at -80°C before genomic DNA isolation. Approximately 200 million unsorted cells (1000x coverage per library element) were saved for screen data normalization. Genomic DNA was extracted using Qiagen Blood Midi or Maxi kits (Qiagen, Cat# 51183; 51194) for sorted or unsorted cells respectively, as per the manufacturer. To prepare sequencing libraries, the sgRNAs sequences were PCR-amplified from genomic DNA and the number of PCR reactions was scaled to use 40-60 µg isolated genomic DNA. Each of these 1st PCR reactions contained: 10 µg of genomic DNA, 2 µL Herculase II Fusion DNA polymerase (Agilent Technologies, Cat# 600677), 20 µL 5X Herculase buffer, 1 µL 100 mM dNTPs, 1 µL 100 µM oMCB_1562 (forward primer), 1 µL 100 µM oMCB 1563 (reverse primer) and water to adjust the volume to 100 µL. PCR amplification was conducted as follows: 1x 98°C/2 min, 18x 98°C/30 s, 59.1°C/30 s, 72°C/45 s, 1x 72°C/3 min. 1st PCR reactions from each sample were pooled and then 2nd PCR reactions were set up for each sample as follows: 5 µL from 1st PCR pooled amplicons, 2 µL Herculase II Fusion DNA polymerase, 20 µL 5X Herculase buffer, 2 µL of 100 mM dNTPs, 0.8 µL 100 µM oMCB_1439 (forward primer), 0.8 µL 100 µM of barcoded CRISPR KO reverse primer, and 69.4 µL H2O. PCR protocol for this 2nd PCR reaction was: 1x 98°C/2 min, 20x 98°C/30 s, 59.1°C/30 s, 72°C/45 s, 1x 72°C/3 min. Finally, 50 µL of 2nd PCR reaction was separated by running on a 2% TBE-agarose gel. The PCR products were excised and purified using a QIAquick Gel Extraction Kit (Qiagen, Cat# 28704) according to the manufacturer’s instructions. The sgRNA libraries were analyzed by deep sequencing on an Illumina NextSeq 500 using a custom sequencing primer (oMCB_1672), with ∼40 million reads per condition (∼200x coverage per library element). Computational analysis and comparison of sgRNA composition of sorted versus unsorted cells were performed using casTLE v.1.0 (https://bitbucket.org/dmorgens/castle) as previously described (Lu et al., 2018; Morgens et al., 2016). Briefly, sgRNA distribution was compared between the sorted and unsorted cell samples and sgRNA enrichments were calculated as log ratios between sorted and unsorted cells. A maximum likelihood estimator was used to estimate the phenotypic effect size for each gene and the log-likelihood ratio (confidence score) by comparing the distribution of the 10 different sgRNAs targeting each gene to the distribution of negative control sgRNAs. P values were determined by permuting the gene-targeting sgRNAs in the screen and comparing to the distribution of negative controls using casTLE. For genome-wide cholesterol screens, we used a threshold of 5% FDR (calculated using the Benjamini-Hochberg procedure) to define hits. For the BMP screens, the top 100 ranked genes in the analysis were considered as hits. Because cholesterol is functionally linked to BMP (Chevallier et al 2008), despite lower statistical significance for some of the BMP screen hits, we found that a significant number of genes that passed this cutoff overlapped with those identified as hits in the cholesterol screens. See Supplementary Table 1 for complete genome-wide screen datasets.

Immunofluorescence

To visualize endolysosomes in suspension K562 cells (Figure 1B-D), cells were attached to glass coverslips using a cytospin (Shandon) at 800 rpm for 5 min. Cells were fixed with 3.7% (v/v) paraformaldehyde for 15 min, washed and permeabilized/blocked with 0.1% saponin/1% BSA-PBS except for cells labeled with GST-PFO*, which were permeabilized for 3 min in 0.1% Triton X-100 and blocked with 1% BSA in PBS. Primary antibodies were diluted in PBS-1%BSA and incubated for 1h at RT. Highly cross-adsorbed H+L secondary antibodies (Life Technologies) conjugated to Alexa Fluor 488, 568, or 647 were used at 1:2,000 in PBS-1%BSA and incubated at RT for 45 min. Nuclei were stained using 0.1μg/ml DAPI (Sigma-Aldrich,Cat# D9542) and coverslips were mounted on glass slides with Mowiol. Microscopy images were acquired using a Zeiss LSM880 laser scanning spectral confocal microscope (Carl Zeiss, Germany) equipped with an Axio Observer 7 inverted microscope, blue diode (405nm), Argon (488nm), diode pumped solid state (561nm) and HeNe (633nm) lasers and a Plan Apochromat 63x numerical aperture (NA) 1.4 oil-immersion objective lens was used. DAPI, Alexa 488, Alexa Fluor 555 and Alexa Fluor 647 images were acquired sequentially using 405, 488, 561and 633 laser lines, AOBS (Acoustic Optical Beam Splitter) as beam splitter and emission detection ranges 415- 480, 500-550 nm, 571-625 nm and 643-680nm respectively. Confocal pinhole was set at 1 Airy units. All images were acquired in a 1024 x 1024 pixel format. In some experiments (Figure 1B-D, Supp. Figure 4B), images were obtained using Metamorph software with a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge-coupled device (EMCCD) camera (Andor) and a 100x 1.4 NA oil-immersion objective. Typical exposure times of 100–300 ms were used. 3D-rendered images (Supplementary Figure 4E) were generated using IMARIS software (Bitplane AG). All image quantifications were performed using CellProfiler (Carpenter et al., 2006).

Immunoblotting

Cells were lysed in lysis buffer (50 mM HEPES, 150 mM KCl, 1% Triton X- 100, 5 mM MgCl2, pH 7.4) supplemented with a protease/phosphatase inhibitor cocktail (1mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). Lysates were boiled in 1x sample buffer, resolved on SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Cat# 1620115) using a Bio-Rad Trans-blot system. Membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 for 60 min at RT. Primary antibodies were diluted in blocking buffer and incubated either 1 h at RT or overnight at 4°C. HRP-conjugated secondary antibodies (Bio-Rad or Abcam) diluted in blocking buffer at 1:5,000 were incubated for 60 min at RT and developed using enhanced chemiluminescence EZ-ECL (Biological Industries). Blots were imaged using an ImageQuant LAS 4000 system (GE Healthcare) and quantified using ImageJ software.

Thin Layer chromatography

Total Lipids were extracted from control or SNX13-depleted cells as follows. Briefly, cells were washed with PBS and resuspended in 1 volume of Methanol:Chloroform (1:2). Next 1/2 volume chloroform and 1/2 volume of H2O were added and tubes were centrifuged at 5000 RPM for 5 min. The organic phase was collected, dried, resuspended with chloroform and spotted on on TLC silica gel 60 plates (Millipore, Cat# 1055530001), and dried for several minutes. Plates were run in a solvent system of hexane/diethyl ether/acetic acid (70:30:1) until 3/4 of the total length of the plate was reached. Lipids were stained using a phosphomolybdic acid (ACROS organics, Cat# 206385000)-ethanol solution.

Bis(monoacylglycero)phosphate (BMP) lipidomics

Targeted high resolution UPLC-MS/MS was used to accurately quantitate the three geometrical isoforms (2,2’-, 2,3’-, and 3,3’-) of di-22:6- BMP and di-18:1-BMP in control or SNX13 siRNA-treated cells ±U18666A. Lipidomics analyses were conducted by Nextcea, Inc. (Woburn, MA) as previously described (Liu et al., 2014) using a SCIEX TripleTOF 6600 mass spectrometer equipped with an IonDrive Turbo V source (SCIEXm Framingham, MA). Standard curves were prepared using authentic BMP reference standards.

Functional Network analysis

Top hits from both cholesterol and BMP screens were collectively queried in STRING (http://string-db.org/) to search for experimentally-confirmed interactions with a high confidence score (0.7). The resulting STRING graphics file reporting interactions was then manually curated using Adobe Illustrator software (Adobe) to create a visually comprehensive functional interactome. Interactions were represented as edges whose thickness was proportional to a calculated score derived from STRING analysis. Additional relevant interactions not reported by STRING but confirmed by the BioGRID database (http://thebiogrid.org/) were also displayed as green edges. The final interactome map was further manually curated to include additional screen hits (nodes) that despite not being reported to interact, could still be clustered in a common functional category revealed by the screens performed.

Statistics

Results are expressed as mean ± SEM unless otherwise specified. Means were compared using Student’s t test where two experimental conditions were compared. When three or more experimental conditions were compared statistical significance was assessed via multiple t test by Holm-Sidak method with α = 0.05 or two-way ANOVA with Tukey’s post hoc test using Graph Pad Prism 9. Two-tailed P values < 0.05 were considered statistically significant (Lord et al., 2020).

Article TitleCRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

Abstract

We report here two genome-wide CRISPR screens carried out to identify genes that when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also carried out under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight co- regulation of cholesterol and bis(monoacylglycero)phosphate levels in cells, and reveal an unexpected role for the ER-localized, SNX13 protein as a negative regulator of lysosomal cholesterol export. In the absence of NPC1 function, SNX13 knockout decreases lysosomal cholesterol, and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

SUMMARY Genome-wide CRISPR screens carried out with and without NPC1 function identify shared pathways that coordinately control lysosomal cholesterol and bis(monoacylglycero)phosphate. ER-localized SNX13 protein plays an unexpected regulatory role in modifying NPC1 function to regulate cellular cholesterol localization.


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