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SNARE priming is essential for maturation of autophagosomes but not for their formation

  1. Zvulun Elazara,1
  1. aDepartment of Biomolecular Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel;
  2. bDepartment of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel;
  3. cLife Sciences Core Facilities, Weizmann Institute of Science, 76100 Rehovot, Israel
  1. Edited by Sharon Anne Tooze, Francis Crick Institute, London, United Kingdom, and accepted by Editorial Board Member Pietro De Camilli October 17, 2017 (received for review April 6, 2017)

  1. Fig. 2.

    Autophagosome formation does not require SNARE priming. (A, Left) Depiction of the protection assay scheme described in Materials and Methods. (A, Right) HeLa cells stably expressing GFP-LC3 were transfected with nontargeting (control) siRNA (non tgt) or αSNAP siRNA (siαSNAP) using DharmaFECT 1 transfection reagent. After 48 h, the cells were incubated for 4 h in the absence or presence of 0.1 μM BafA and were then homogenized, centrifuged, and subjected to Proteinase K and Triton X-100 treatment as described in Materials and Methods. Protein levels were quantified using Image Studio Lite software. (B, Top immunofluorescent analysis) HeLa cells were transfected with nontargeting (control) siRNA (non tgt), αSNAP siRNA (siαSNAP), the siRNA of three GABARAP family members (siGABARAPs), or both siαSNAP and siGABARAPs, using DharmaFECT 1 transfection reagent. After 48 h, the cells were fixed with 100% methanol, immunostained with anti-LC3 and anti-WIPI2 antibodies, and analyzed by an Olympus confocal microscope. (Scale bar, 20 μm.) Large magnification of WIPI staining is presented in the Right column. (B, Bottom scheme) Depiction of the autophagy-related bodies detected under the different treatments. (C, Top images) HeLa cells were transected as in A. After 48 h the cells were fixed with 100% methanol, immunostained with anti-LC3 and anti-p62 antibodies, and subjected to Dual Color 3D STORM imaging as described in Materials and Methods. (C, Bottom graph) Ratio quantification of LC3- and p62-puncta morphologies; n = 25. (D, Left) HeLa cells stably expressing GFP-LC3 were transfected as in A. Cryosections of fixed cells were immunolabeled with anti-GFP antibodies and analyzed by TEM as described in Materials and Methods. (D, Right) Analysis of the GFP-labeled structures by ImageJ software; n > 40, ***P < 0.004. Values are means ± SE.

  2. Fig. 3.

    αSNAP is essential for priming autophagy-related SNARE complexes. (A, Left) HeLa cells were transfected with nontargeting (control) siRNA (non tgt) or αSNAP siRNA (siαSNAP) using DharmaFECT 1 transfection reagent. After 48 h, the cells were fixed with 100% methanol, immunostained with anti-LC3 and anti-LAMP1 antibodies, and then analyzed by an Olympus confocal microscope. (Scale bar, 20 μm.) Large magnification of stained cells is presented in the Right column. (A, Right) Intensity profiles of the marked lines in the large magnification column were obtained by Olympus FluoView FV 1000 analysis software. (B) HeLa cells were transfected and treated as in A, lysed with RIPA extraction buffer, heated to the indicated temperatures, and analyzed by Western blotting. nt, nontargeting (control) siRNA. (C) HeLa cells were transfected as in A, treated with or without NEM and DTT, lysed with SNARE lysis buffer, and immunoprecipitated with Vamp8 antibodies. (Left) Immunoprecipitation scheme as detailed in Materials and Methods. (Right Top) IP with Vamp8 antibodies. (Bottom) TCL, total cell lysates; Ab, antibodies with no lysate incubation; IP, immunoprecipitation. Further details are provided in Materials and Methods.

  3. Fig. 4.

    Translocation of syntaxin17 and Atg9 to the autophagosome is SNARE-priming independent. (A, Left) HeLa cells were transfected with nontargeting (control) siRNA (non tgt) or αSNAP siRNA (siαSNAP) using DharmaFect 1 transfection reagent. After 24 h, the cells were transfected with FLAG-tagged syntaxin17 using jetPEI transfection reagent for an additional 24 h. Cells were then fixed with 100% methanol, stained with anti-LC3 and anti-FLAG antibodies, and analyzed by an Olympus confocal microscope. (Scale bar, 20 μm.) Right column shows large magnification of stained cells. (Right) At least three independent trials were quantified, using Imaris ×64 8.2.0 software, Bitplane. NS, not significant. Values are means ± SE. (B, Top Left) HeLa cells were transfected as in A. After 48 h, the cells were homogenized, floated upon a sucrose gradient as described in Materials and Methods, and quantified by Image Studio Lite software. (Top Right) Flotation assay scheme as detailed in Materials and Methods. (Bottom) Protein levels in the fractions were quantified using Image Studio Lite software. (C) HeLa cells were transfected as in A, fixed with 4% paraformaldehyde, immunostained with anti-Atg9, anti-p115, and anti-p62 antibodies, and analyzed by the Olympus confocal microscope. (Scale bar, 20 μm.) Large magnification of stained cells is presented in the Right column of each panel.

  4. Fig. 5.

    A low level of αSNAP is sufficient to partially promote autophagy under starvation conditions. (A, Left) HeLa cells were transfected with nontargeting (control) siRNA (non tgt) or αSNAP siRNA using DharmaFECT 1 transfection reagent. After 48 h, the cells were incubated for 4 h in normal growth medium or EBSS in the absence or presence of 0.1 μM BafA, lysed with RIPA extraction buffer, and then analyzed by Western blotting. (A, Right) At least four independent trials were quantified, using Image Studio Lite software normalized to the nontargeting control sample. Values are means ± SE. Starv, starvation conditions. (B, Left) HeLa cells were treated as in A, fixed with 100% methanol, immunostained with anti-LC3 antibodies, analyzed by a Leica confocal microscope using the same threshold for all samples and deconvolved using Huygens software. Z-stack projection was done using ImageJ software. (Scale bar, 10 μm.) Large magnification of stained cells is presented at Right. (Middle) Quantification of LC3 puncta using LAS X software, Leica Microsystems. n > 1,000, ***P < 0.0001. Values are means ± SE. (Right) HeLa cells stably expressing GFP-LC3 were treated as in A, then harvested by trypsin, fixed with 100% methanol, and stained with DAPI. Cells were then subjected to ImageStream analysis, as described in Materials and Methods. Four independent trials were analyzed and quantified by IDEAS v. 6.2 software. (C) HeLa cells were transfected as in A, homogenized, and fractionated as described in Materials and Methods. Total cell homogenates are presented on the Right.

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