| Methodology |
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Cells and plasmids
BMMs were generated from femurs of C57BL/6J mice and cultured for 6–7 d, as described previously (Knapp and Swanson, 1990). All animal-related procedures were performed in compliance with the University of Michigan guidelines for the humane use of animals. MEFs and HEK293T cells were cultured as described previously (Yoshida et al., 2011). MEFs from TSC2-knockout (Zhang et al., 2003) and WT control mice were provided by D. Kwiatkowski (Harvard Medical School, Boston, MA). The plasmid pIRES-mCFP was constructed by replacing the EGFP sequence of pIRES-EGFP (BD Biosciences) and the PCR-amplified mCFP sequence between BstX1 and Not1. Rac-WT and Rac-N17 sequences were PCR amplified and subcloned into the pIRES-mCFP between Nhe1 and EcoR1 sites, resulting in the plasmids pRac1-WT-IRES2-mCFP and pRac1-N17-IRES2-mCFP, respectively. The plasmid pmCitrine-BtkPH-N1 was described previously (Kamen et al., 2007; Yoshida et al., 2015). In brief, the PCR-amplified BtkPH sequence was subcloned into the pmCitrine-N1 vector (Clontech) between XhoI and HindIII. The plasmid pC 1δ-YFP was a gift from T. Meyer (Stanford University, Palo Alto, CA). The plasmid pEGFP-N1 was from Clontech. The plasmids psPAX2 and pMD2.G were from Addgene. The plasmids pRac1-WT-IRES2-mCFP and pRac1-N17-IRES2-mCFP were derived from pIRES2-EGFP vector (BD Biosciences). The plasmids psPAX2 and pMD2.G were described previously (Suzuki et al., 2013).
Generation of Rac1 knockout MEFs using CRISPR/Cas9 genome editing
The 20-nt guide sequence targeting mouse Rac1 was designed using the CRISPR design tool. The guide RNA (gRNA) encoding DNA was cloned into a bicistronic expression vector (LentiCRISPR v2; a gift from F. Zhang (Broad Institute, Massachusetts Institute of Technology, Cambridge, MA); and plasmid 52961; Addgene) containing human codon–optimized Cas9 and the RNA components (Sanjana et al., 2014). The guiding sequence, with a 3-nt protospacer adjacent motif, targets exon 2 of mouse Rac1 gene: 5′-ACGGTGGGGATGTACTCTCCAGG-3′. As a control, a gRNA sequence targeting GFP was designed (5′-CATGCCTGAAGGTTATGTAC-3′). The LentiCRISPR vectors with different gRNAs were transfected into HEK293T cells with lentiviral packaging plasmid psPAX2 and envelope plasmid pMD2.G. The virus was collected and concentrated as described previously (Suzuki et al., 2013). MEF3 cells (a gift from L. Kotula, Upstate Medical School, Rochester, NY) were used as WT MEFs. 24 h after infection, the cells were selected for resistance to 5 µg/ml puromycin for 48 h. At day 6 after infection, the cells were examined for Rac1 expression and used for further experiments.
Cell treatments
For the biochemical assays, BMMs and MEFs were cultured in DMEM (low glucose; 11885; Life Technologies) without FBS overnight. BMMs were stimulated with M-CSF (6.9 nM) or PMA (100 nM) for the indicated times and lysates were prepared for Western blotting as described previously (Yoshida et al., 2011). For inhibitor treatment assays, BMMs were pretreated with U0126 (10 µM), A66 (3 µM), IC87114 (0.1–1 µM), MK2206 (2 µM), or EIPA (25 µM) for 30 min in DMEM, HBSS, or DPBS containing leucine or glucose. A combination of blebbistatin (75 µM for 35 min) and jasplakinolide (1 µM for 15 min) was also used. After treatments, cells were stimulated by M-CSF or PMA for 5 or 30 min, respectively. For calphostin C treatment, cells were pretreated with 500 nM calphostin C for 20 min in a CO2 incubator and transferred into a biological safety cabinet for light activation for another 10 min (Bruns et al., 1991). For leucine or glucose stimulation assays, BMMs were treated with DPBS with leucine or glucose at the indicated concentrations for 35 min (for blebbistatin and jasplakinolide experiments) or 50 min and stimulated by M-CSF or PMA for another 5 min or 30 min, respectively. MEFs were starved in DPBS with leucine (0.4 mM) or glucose (5.6 mM) for 30 min and stimulated with PDGF (2 nM) for 15 min (Gao et al., 2007). For inhibitor treatment assays, MEFs were pretreated 30 min with EIPA (25 µM) or the JB combination. For PDGF and leucine stimulation assays, MEFs were incubated in DPBS with glucose (5.6 mM) throughout, including 50 min starvation followed by stimulation for 10 min with PDGF and/or leucine (0.4 mM). For the dipeptide assay, MEFs were cultured in DPBS or DPBS with Ala-Leu (4.0 mM) for 30 min and stimulated by PDGF for another 30 min. For amino acid stimulation assays, BMMs or MEFs were incubated in HBSS (Life Technologies) for 50 min and then DMEM for 5 min with or without M-CSF (BMMs), or 10 min with or without PDGF (MEFs).
Western blotting
Cells were lysed 10 min in ice-cold lysis buffer (40 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 0.3% CHAPS, and a mixture of protease inhibitors; Roche) as reported previously (Yoshida et al., 2011). Lysates were centrifuged at 13,000 g for 15 min at 4°C, and the supernatant was mixed with 4× SDS sample buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and applied to Western blotting with the indicated antibodies. At least two independent experiments were performed to confirm the results of pilot studies.
Microscopy
Phase-contrast and fluorescence microscopic images were collected in an Eclipse TE-300 inverted microscope with a 60× NA1.4, oil-immersion PlanApo objective lens (Nikon) and a Lambda LS xenon arc lamp for epifluorescence illumination (Sutter Instruments). Fluorescence excitation and emission wavelengths were selected using a 69008 set (Chroma Technology) and a Lambda 10–2 filter wheel controller (Shutter Instruments) equipped with a shutter for epifluorescence illumination control. Images were recorded with a Photometrics CoolSnap HQ cooled charge-coupled device camera (Roper Scientific).
For live cell imaging, cells plated onto glass-bottom, 35-mm diameter dishes (MatTek Corp.) were preloaded by endocytosis of TRDx (0.5 mg/ml × 2–3 h) followed by a 2- to 4-h chase in unlabeled medium. Cells were first imaged in Ringer’s buffer (RB; 10 mM Hepes, pH 7.2, 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, and 10 mM glucose; 23°C), then stimulated with 200 ng/ml M-CSF in RB to stimulate macropinocytosis, with added LY, rinsed, transferred to the microscope, and imaged at 20-s intervals. Images were processed as video or still sequences using MetaMorph software. To confirm the results of live cell imaging, three or more independent experiments were performed.
Immunofluorescence staining for BMMs was performed as described previously (Racoosin and Swanson, 1993). In brief, cells were washed three times with 37°C RB and fixed for 30 min at 37°C with fixation buffer (20 mM Hepes, pH 7.4, 2% PFA, 4.5% sucrose, 70 mM NaCl, 10 mM KCl, 10 mM MgCl2, 2 mM EGTA, 70 mM lysine-HCl, and 10 mM sodium periodate). The fixed cells were rinsed with washing buffer (TBS buffer: 20 mM Tris-HCl, pH 7.4, 150 mm NaCl, 4.5% sucrose) for 3 × 5 min, permeabilized with ice-cold methanol for 20 s and then incubated with blocking buffer (TBS buffer with 2% goat serum) for 30 min at RT. Immunofluorescence staining for MEFs was performed as described previously (Yoshida et al., 2011). The fixed cells were rinsed with DPBS for 3 × 5 min, permeabilized with 0.01% saponin in DPBS for 10 min at RT then blocked with blocking buffer (TBS buffer; 0.01% Triton X-100, and 2% BSA) for 30 min at RT. Samples were incubated with primary antibodies at a dilution of 1:100 in blocking buffer at RT for 2 h. After rinsing three times with TBS buffer, samples were incubated with secondary antibodies at a dilution of 1:200 in blocking buffer at RT for 2 h. After rinsing three times with TBS buffer, samples were mounted on microscope slides using Prolong Gold (Life Technologies). To confirm qualitative observations, quantitative analysis was applied to 10 or more immunofluorescence images.
Macropinosome assay
To measure macropinocytosis, cells on coverslips were pulse labeled for the indicated times with LY (1 mg/ml) or FDx70 (1.2 mg/ml) in medium containing M-CSF, PDGF, or PMA and then were rinsed, fixed, and observed using a Nikon TE300 fluorescence microscope. Uningested probes were removed by gently washing with DPBS before cells were fixed for 30 min at 37°C with fixation buffer. Phase-contrast and FDx70 or LY fluorescence images of fixed cells were captured and merged after reducing background signal using MetaMorph (version 6.3; Molecular Devices). The number of induced macropinosomes per cell was determined by counting FDx70- or LY-positive vesicles on the merged images; >25 cells were observed for each assay.
Quantitative analysis of mTOR-LAMP colocalization
Phase-contrast and mTOR and LAMP-1 immunofluorescence images were taken of BMMs containing tubular endolysosome structures. More than 49 BMM images from five independent experiments were observed per condition, and the frequency of the cells showing mTOR–LAMP-1 was determined by comparing mTOR, LAMP-1, and merge images using MetaMorph. Data were analyzed by the t test. To analyze colocalization in MEFs, pilot experiments were followed by one experiment in which >10 MEF images per condition were randomly captured, and LAMP-1 and mTOR images were compared using the “Measure Colocalization” command in MetaMorph, after thresholding each image to reduce background signals. We calculated the area of mTOR-LAMP colocalization divided by the area of LAMP, and the result was considered as the frequency of mTOR–LAMP-1 colocalization in this study. Identical methods were used to quantify colocalization of mTOR with LY. All data were analyzed by the t test.
Ratiometric imaging
Cells were prepared for live cell imaging as described by Yoshida et al. (2009). A ratiometric imaging approach was used to measure the ratios of two fluorescent chimeras in BMMs, using MetaMorph software as described previously (Swanson, 2002; Hoppe and Swanson, 2004; Yoshida et al., 2009; Welliver and Swanson, 2012). Ratio images reported the concentrations of YFP-BktPH relative to CFP, to localize PIP3, and of YFP-C1δ relative to CFP, to localize DAG, thereby correcting for variations in optical path length owing to cell shape. Ratio images of LY and TRDx reported the relative distributions of the two dyes in endocytic compartments. All observations were repeated >10 times.
LY endocytosis
LY was used to investigate the trafficking of small solutes ingested by macropinocytosis. For time course experiments, cells were incubated in DMEM or HBSS with 1 mg/ml LY for 1, 5, or 10 min, with or without M-CSF, and then rinsed three times before fixation. For colocalization assays, cells were stained for immunofluorescence with anti–LAMP or anti–mTOR antibodies. For Rac1 expression experiments, BMMs were transfected with plasmids encoding pRac1-WT-IRES2-mCFP or pRac1-N17-IRES2-mCFP, using a Mouse Macrophage Nucleofector kit (Amaxa). Cells were incubated for 24 h and used for the LY assay. For starvation assays, cells were incubated in HBSS for 30 min before 10-min M-CSF stimulation. To quantify the amount of ingested LY, phase-contrast and LY images were taken and shade/bias corrections were applied (Hoppe, 2007). More than 10 cell images per condition were captured on three separate days, and the total intensity of LY inside each cell was obtained using the “Measure” tool in MetaMorph, after thresholding each image to subtract background signals. To quantify the frequency of mTOR-LY colocalization, phase-contrast, mTOR, and LY images were taken and shade/bias corrections were applied. More than 10 cell images per condition were captured. The frequency of the integrated signal of mTOR (or LY) overlapping of LY (or mTOR) inside of a cell was measured as the frequency of mTOR-LY colocalization using the “Measure Colocalization” tool in MetaMorph. Because we used CFP as a marker for cells overexpressing Rac1 or Rac1(N17), we corrected the LY fluorescence image (IF: excitation 430/424; emission 535/530) for crossover signal from CFP (ICFP: excitation 430/424; emission 470/424). Crossover fluorescence was corrected by measuring the coefficient β from cells expressing CFP only (IF/ICFP) and isolating the LY fluorescence from cells containing LY and CFP, using the equation ILY = IF − βICFP (Hoppe, 2007). All data were analyzed by the t test.
Detail of statistical methods
All experimental replicates subsequent to pilot studies are presented here. In scoring for macropinosome formation (Fig. 2, B–F; Fig. 3, A–D and H; Fig. 4, A–G; Fig. 5 B; Fig. 6 I; Fig. S2 C; Fig. S3, C and E; and Fig. S4 F), a one-tailed, paired t test was applied to data obtained from three technical replicates of samples with 15 or more cells for each condition. For mTOR-LAMP colocalization analysis in BMMs (Fig. S4 C), a two-tailed, paired t test was applied to five technical replicates of samples with seven or more cells for each condition. For analysis of mTOR recruitment to macropinosomes (Fig. S4 G), a one-tailed, paired t test was applied to data obtained from three technical replicates of samples with 25 or more macropinosomes for each condition. For mTOR-LAMP colocalization analysis in MEFs (Fig. S5, B, E, and G) and mTOR-LY colocalization analysis in BMMs (Fig. 6, H, K, and M), a one-tailed, two-sample unequal variance t test was applied to images of 10 or more cells for each condition. For analysis of integrated intensity of LY in BMMs (Fig. 6, D, J, and L), a one-tailed, two-sample unequal variance t test was applied to images of 10 or more cells for each condition.
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