Cell culture and transfection
HEK293A cells and derivatives were maintained in full medium (FM): DMEM with 10% fetal calf serum (FCS) as previously described (Kochl et al., 2006
; Chan et al., 2007
). To induce autophagy, cells were incubated for 2 h in starvation medium: Earle's balanced salt solution (EBSS). When indicated, bafilomycin A1 (Calbiochem, La Jolla, CA) in dimethyl sulfoxide was used at 100 nM. The 293/GFP-LC3 cells were previously described (Kochl et al., 2006
). The 293/GFP-DFCP1 (clone 2O1; Axe et al., 2008
) was a kind gift from N. T. Ktistakis (Brabaham Institute, Cambridge, United Kingdom) and was were maintained in the presence of G418 at 400 μg/ml. The Atg9−/−
and matched wild-type immortalized MEFs (Saitoh et al., 2009
) were a kind gift from T. Saitoh and S. Akira (Osaka University, Osaka, Japan) and were maintained in FM. The 293/GFP-LC3/mRFP-Atg9 and 293/mRFP-Atg9 were generated by transfection of 293/GFP-LC3 and HEK293 cells with pCDNA4-TO-mRFP-Atg9 or pEGFP-mRFP-Atg9 plasmids and selected with 750 μg/ml of Zeocin (Invitrogen, Carlsbad, CA) or 800 μg/ml of G418 (Life Technologies, Carlsbad, CA), respectively, followed by clonal dilution. Clones were screened by Western blot and confocal microscopy for low-level expression, in which mRFP-Atg9 distribution resembled that of endogenous mAtg9. The 293/GFP-LC3/mRFP-Atg9 clone 9B9 was maintained in the presence of Zeocin at 400 μg/ml. The 293/mRFP-Atg9 clone 1F10 was maintained in the presence of G418 at 400 μg/ml.
For RNA interference, cells were transfected using Oligofectamine (Invitrogen), followed by a second transfection 24 h later with Lipofectamine 2000 (Invitrogen). Cells were analyzed 72 h after the first transfection. The final concentration of siRNA oligos ranged between 20 and 50 μM, depending on the target protein: D-001220-01 (RISC-Free, control), D-014294-02 or -04 (Atg9), D-020521-09 or -12 (WIPI2), or D-005049-04 (ULK1; Dharmacon, Lafayette, CO). Custom siRNA oligo UGA UGG GGA AAC CAG GAA AUU was used to knock down Vps26. Transferrin receptor was knocked down using siRNA oligos (pools of two) as previously described (Herbison et al., 2009
Lipofectamine 2000 was used for transient transfection of DNA. For biochemical analysis, 1 μg of plasmid per milliliter of transfection mix was used, whereas for immunofluorescence (IF) and live-cell imaging the plasmid was diluted 1:5 with a carrier vector.
The following primary antibodies were used: rabbit anti-mAtg9 (STO215, STO219; Young et al., 2006
); Armenian hamster anti-mAtg9 (clone 14F2; Webber and Tooze, 2010a
); mouse anti-Atg16L (TMD-PH-AT16; CosmoBio, Carlsbad, CA); rabbit anti–β-tubulin (Abcam, Cambridge, MA); mouse anti-LC3 (clone 5F10; 0231-100; NanoTools, Teningen, Germany); rabbit anti-LC3 (ab48394; Abcam); rabbit anti-WIPI2 (Polson et al., 2010
); mouse anti-TfR (13-6800; Invitrogen); sheep anti-TGN46 (AHP00G; AbD Serotec, Raleigh, NC); rabbit anti-CIMPR (STO52; Dittie et al., 1999
); mouse anti-EEA1 (610457; BD Biosciences PharMingen, San Diego, CA); polyclonal anti-ULK1 (sc-33182; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-SOD1 (16831; Abcam); mouse anti-GFP (clone 3E1; Cancer Research UK, London, United Kingdom); mouse anti-Rab11 (610657; BD Biosciences PharMingen); rabbit anti-PDI (SPA-891; Bioquote, York, United Kingdom); mouse anti-tubulin (7291, Abcam); rabbit anti–syntaxin-13 (110 132; Synaptic Systems, Göttingen, Germany); rabbit anti-Vps26 was a kind gift of M. Seaman (Cambridge Institute for Medical Research, Cambridge, United Kingdom); COPI was a kind gift of F. Wieland (University of Heidelberg, Heidelberg, Germany). The gold-conjugated anti-TfR antibody was a kind gift of Clare Futter (Institute of Ophthalmology, London, United Kingdom).
Indirect IF was performed as previously described (Young et al., 2006
). For visualization of endogenous LC3, cells were either fixed in 3% paraformaldehyde and permeabilized in methanol and blocked in 5% bovine serum albumin. Secondary antibodies were all Alexa Fluor conjugated (488, 555, or 647) from Invitrogen, with the exception of Cy3-conjugated goat anti–Armenian hamster (Jackson ImmunoResearch Laboratories, West Grove, PA).
Cells were imaged with either an LSM 510 or an LSM 710 laser scanning confocal microscope equipped with a 63×, 1.4 numerical aperture (NA), Plan Apochromat oil immersion objective (Carl Zeiss MicroImaging, Jena, Germany). Confocal sections for colocalization analysis were 0.8 μm thick. Images were processed using LSM 510 and Zen software.
Quantification of Atg9
All quantifications were performed with Imaris software (Bitplane, Zurich, Switzerland), setting an appropriate threshold, which was kept constant during the whole analysis. Images for quantification were taken under the same conditions, and about five images per experiment were quantified. The values of all images from a single experiment were averaged together, and this value was used as n = 1 for statistical analysis with Excel (Microsoft, Redmond, WA) and Prism (GraphPad Software, La Jolla, CA) software.
For “total Atg9” the signal of Atg9 and the relative marker in the whole cell were compared. For “local” colocalization, at least 20 spots that were positive for the marker of interest were picked in blind per experiment. The colocalization between Atg9 and the marker was then evaluated only in a squared area of approximately twice the diameter around each spot.
For live-cell imaging, cells were grown on MatTek dishes (MatTek Corporation, Ashland, MA). Full-medium imaging medium consisted of DMEM, 10% FCS, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, low bicarbonate, minus phenol red, minus riboflavin, and minus folic acid. Starvation imaging medium was EBSS and 30 mM HEPES, pH 7.4.
Cells were imaged at 37°C using a widefield inverted Nikon Eclipse TE2000-E microscope and a 60×, 1.40 NA, oil immersion objective (Nikon, Melville, NY). Image sequences were then processed using MetaMorph (Molecular Devices, Sunnyvale, CA) and exported as .avi files (Microsoft Video 1 compression).
For CLEM, GFP-LC3/mRFP-Atg9 cells were grown on photoetched gridded coverslips and incubated for 2 h in starvation medium. For the CLEM experiments with Tfn and TnR, cells were starved for 2 h in EBSS and the Tnf-Alexa 647 ligand and anti-TnR gold conjugate (10-nm gold) was added to the EBSS. Cells were then fixed in 8% formaldehyde/0.2 M HEPES, pH 7.2, added directly to the cell medium for 10 min, followed by fixation in 4% formaldehyde/0.2 M HEPES, pH 7.2, for 1 h. Cells of interest were identified and imaged by phase contrast and fluorescence microscopy, using a 63× water objective and taking 0.8-μm-thick z-stacks at 0.4-μm intervals. Cells were then fixed in 2.5% glutaraldehyde/4% formaldehyde in 0.1 M phosphate buffer (PB) for 1 h. The samples were postfixed in reduced osmium tetroxide, stained with tannic acid, dehydrated stepwise to 100% ethanol, and embedded in Epon. Serial sections (~70 nm) were cut using an Ultracut UCT ultramicrotome (Leica Microsystems UK, Milton Keynes, United Kingdom), collected on formvar-coated slot grids, and poststained with lead citrate.
EM immunolocalization of Atg9 was performed by cryosectioning and immunolabeling (Tokuyasu, 1973
). Briefly, cells were fixed in 8% formaldehyde/0.4% gluteraldehyde/0.1 M PB, added directly to the cell medium at 37°C for 10 min, and followed by fixation in 4% formaldehyde/0.2% gluteraldehyde/0.1 M PB for 30 min at room temperature. The cells were embedded in 2% gelatin, cryoprotected in 2.3 M sucrose, mounted onto pins, and plunge-frozen in liquid nitrogen. Ultrathin cryosections were cut using an FC6 cryo-ultramicrotome (Leica Microsystems UK). Cryosections were immunolabeled with rabbit polyclonal anti-Atg9 (1:10; Young et al., 2006
) and protein A conjugated to 10-nm gold (Cell Microscopy Center, University Medical Center Utrecht, Utrecht, Netherlands).
Sections were viewed using a Tecnai G2 Spirit 120-kV transmission electron microscope (FEI Company, Eindhoven, Netherlands) with either an Orius or an Ultrascan 1000 charge-coupled device camera (Gatan UK, Abingdon, United Kingdom).
Rat hepatocytes were prepared and infected with mRFP-Atg9 and GFP-LC3 viruses as previously described (Kochl et al., 2006
; Young et al., 2006
) before immuno-EM immunolocalization as described.
Cells were lysed in ice-cold TNTE buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.3% wt/vol Triton X-100, 5 mM EDTA) containing EDTA-free Complete Protease Inhibitor cocktail (Roche, Indianapolis, IN). Lysates were cleared by centrifugation and resolved by SDS–PAGE. For LC3 lipidation assays NuPage 4–12% Bis-Tris gels in 2-(N-morpholino)ethanesulfonic acid buffer were used (Invitrogen). For gradient fractions 20 × 20 cm format 8% Laemmli SDS–PAGE was performed. Gels were transferred onto polyvinylidene fluoride membrane using Bio-Rad Semidry apparatus (Bio-RAD, Hercules, CA). Blots were revealed by horseradish peroxidase–conjugated secondary antibodies, followed by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). When needed, membranes were stripped using Restore PLUS (Thermo Scientific, Waltham, MA).
Immunoprecipitation and mass spectrometry analysis
Cells were lysed in ice-cold CHAPS buffer (1% CHAPS, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA) containing Complete Protease Inhibitor (Roche). Lysates cleared by centrifugation were incubated with protein G– or protein A–Sepharose beads (Sigma-Aldrich, St. Louis, MO) coupled to one of the following antibodies: mouse anti-HA (clone 12CA5; Cancer Research UK), rat anti-HA (11 867 423 001; Roche), mouse anti-GFP (clone 4E1; Cancer Research UK), rabbit affinity-purified anti-Atg9 (STO215), and hamster anti-Atg9 (14F2). After three washes in TNTE, proteins were eluted using 5× SDS sample buffer and heated to 65°C for 5 min.
For mass spectroscopy determination of mAtg9 interactors, HEK293 cells were transfected with either HA-tagged mAtg9 or empty vector. Cell lysates were immunoprecipitated as described. To reduce the risk of nonspecific binding, we performed two independent determinations using antibodies from different species: mouse or rat anti-HA antibodies. Proteins were resolved on NuPage 4–12% Bis-Tris gels and stained with GelCode Blue (Pierce, Thermo Fisher Scientific, Rockford, IL). Entire lanes were analyzed. Interactors were considered significant if they were present in both experiments.
Cells were homogenized in basic homogenization buffer (HBB; 250 mM sucrose, 25 mM HEPES, pH 7.2; ), or rich homogenization buffer (HRB: 250 mM sucrose, 25 mM HEPES, pH 7.2, 25 mM KCl, 2.5 mM Mg acetate), both containing Complete Protease Inhibitor. Postnuclear supernatants were loaded on 1–22% Ficoll gradients, 20 mM TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), pH 7.4, and 250 mM sucrose, on a 45% Nycodenz cushion and prepared in a 12.5 Quick-Seal tube (342413; Beckman Coulter, Brea, CA). Samples were centrifuged for 36 min at 50,000 rpm, using a VTi65.1 rotor in a Beckman Coulter Optima L-100 XP centrifuge.
We collected 500-μl fractions (~24 or 25 per sample), and the refractive index of each fraction was determined using a refractometer (Bellingham + Stanley Ltd., Tunbridge Wells, United Kingdom). The fractions in the linear range (usually numbers 6–24) were analyzed by SDS–PAGE.
To determine the distribution profile of the different markers, Western blots were quantified using ImageJ (National Institutes of Health, Bethesda, MA) and plotted against the refractive index of the corresponding fraction. To compare the data across experiments, each profile was fitted to a Gaussian curve (least square fit) using GraphPad Prism, version 5.0d, determining the mean value of the control full-medium sample, to which all other values were compared.
Spots (GFP-DFCP1, GFP-LC3, WIPI2, Atg16) were counted using Imaris and expressed as spots/cell or spots/area unit. LC3-I and LC3-II blots were quantified using ImageJ. A two-tailed unpaired t test or one-way analysis of variance followed by a Tukey posttest was performed using GraphPad Prism, version 5. To measure mAtg9 dispersion, images were blinded and scored by two operators. From 50 to 90 cells were counted per condition in two independent experiments.