Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets

Abstract

Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723-1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.

FIGURE 1:

Atg2A and Atg2B are essential for autophagy in mammalian cells. (A) HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNAs twice. Cell lysates were analyzed by immunoblotting with anti-Atg2A, anti-Atg2B, and anti-Hsp90 (internal control) antibodies. (B) HeLa cells stably expressing GFP-LC3 were treated with siRNA directed against Atg2A and/or Atg2B twice for 5 d and then cultured in regular DMEM or starvation medium in the presence or absence of protease inhibitors (E64d and pepstatin A) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. (C) HeLa cells stably expressing GFP-LC3 were treated with control siRNA (a–c) and siRNAs against Atg2A (d–f), Atg2B (g–i), and both Atg2A and Atg2B (j–l) for 5 d and then cultured in regular DMEM (a, d, g, and j) or starvation medium for 1 h (b, e, h, and k) or 5 h (c, f, i, and l). GFP-LC3 signals were analyzed by fluorescence microscopy. Scale bar, 50 μm. (D) HeLa cells stably expressing GFP-LC3 were treated with control siRNA or siRNA against Atg2A and Atg2B and cultured as in C in the presence or absence of 0.2 μM wortmannin. Total cellular GFP-LC3 signals were analyzed by flow cytometry.

FIGURE 2:

Autophagy-related proteins accumulate in Atg2A/B-depleted cells. HeLa cells or HeLa cells stably expressing the indicated GFP-fused proteins were treated with control siRNA or siRNA against Atg2A and Atg2B. Cells cultured in regular medium were fixed and stained with the anti-Atg9A, anti-LC3 (CTB-LC3-2-1C; Cosmo Bio), and anti-GFP (A6455; Invitrogen) antibodies. Immunofluorescence images were obtained using a confocal microscope. Signal color is indicated by color of typeface. Scale bar, 5 μm.

FIGURE 3:

Unsealed autophagosomes accumulate in Atg2A/B-depleted cells. (A) Schematic drawing of autophagosome formation and protease protection assay. (B–E) HeLa cells expressing GFP-LC3 were treated with control siRNA (B, C) or a mixture of siRNAs against Atg2A and Atg2B (D, E), and cultured in regular DMEM (B, D) or starvation medium containing 0.2 μM bafilomycin A₁ (Baf) for 2 h (C, E). The PNS was separated into LSP, HSP, and HSS fractions and then analyzed by SDS–PAGE and immunoblotting using anti-GFP, anti-p62, and anti-Hsp90 antibodies. The subfractions were treated with proteinase K (ProK) with or without Triton X-100.

FIGURE 4:

Aberrant membrane structures accumulate in Atg2A/B-depleted cells. HeLa cells expressing GFP-LC3 were treated with control siRNA (A–D) or mixture of siRNAs against Atg2A and Atg2B (E–F) and cultured in regular DMEM (A, E), starvation medium (B, F), or starvation medium with bafilomycin A₁ (C, D). PNS fractions were layered at the bottom of the OptiPrep density gradient and fractionated by centrifugation as illustrated in G. Fractions in C were treated with proteinase K after fractionation (D). The fractions were analyzed by SDS–PAGE and immunoblotting using anti-GFP and anti-p62 antibodies. N and P, nonfloat and PNS fractions, respectively.

FIGURE 5:

Atg2A is present on both autophagic structures and lipid droplets. (A, B) HeLa cells stably expressing GFP-Atg2A were cultured in the presence or absence of oleic acid-BSA (OA) for 16 h and then incubated in regular DMEM or starvation medium without oleic acid for 2 h. Cells were incubated with BODIPY 558/568-C₁₂ during the last 1 h of culture. Cells were fixed, permeabilized, stained with anti-GFP and anti-LC3 antibodies, and examined by confocal microscopy (A). Cells were also directly observed by fluorescence microscopy without fixation and antibody staining (B). (C) HeLa cells were cultured in the presence of oleic acid-BSA for 24 h and then starved for 2 h. Cells were stained with BODIPY 558/568-C₁₂ as in A. Endogenous Atg2A and LC3 were detected with anti-Atg2A and anti-LC3 antibodies. Signal color is indicated by color of typeface. Scale bars, 5 μm, and 1 μm in inset (A–C).

FIGURE 6:

Atg2 is recruited to the autophagosome formation site later than Atg5 but before LC3. HeLa cells stably expressing GFP-Atg5 were cultured in starvation medium for 2 h. Cells were stained with anti-GFP, anti-Atg2A, and anti-LC3 antibodies (A). The ratios (%) of Atg2⁻Atg5⁻, Atg2⁺Atg5⁻, Atg2⁻Atg5⁺, and Atg2⁺Atg5⁺ populations to the total LC3-positive structures (B) and of Atg2⁻LC3⁻, Atg2⁺LC3⁻, Atg2⁻LC3⁺, and Atg2⁺LC3⁺ populations to the total Atg5-positive structures (C) were calculated from 10 cells. Data represent mean ± SE of three independent cultures. Signal color is indicated by color of typeface. Scale bars, 5 μm, and 1 μm in inset.

FIGURE 7:

Targeting of Atg2A to autophagic structures depends on PI3K activity but not on Atg5, and that to lipid droplets depends on neither PI3K activity nor Atg5. Atg5 KO MEFs stably expressing GFP-Atg2A and HA-ULK1 were cultured in starvation medium containing BODIPY 558/568-C₁₂ with or without 0.2 μM wortmannin for 1 h. Cells were subjected to immunofluorescence microscopy using anti-GFP and anti-HA antibodies. Arrows indicate ULK1-positive structures, and arrowheads indicate lipid droplets. Signal color is indicated by color of typeface (A). Atg2A-positive ratio of total ULK1 structures (B) and that of total lipid droplets (C) were quantified from 10 cells. Data represent mean ± SE of three independent experiments. Scale bars, 5 μm and 1 μm in inset.

FIGURE 8:

Amino acids 1723–1829 of Atg2A are essential for localization to both lipid droplets and autophagosomes. (A) GFP-tagged Atg2A fragments were expressed in HeLa cells, and their colocalization with lipid droplets was examined. Representative images are shown in Supplemental Figure S4. (B) Amino acid alignment of human Atg2A (NP_055919) with human Atg2B (NP_060506), Drosophila melanogaster Atg2 (NP_647748), Schizosaccharomyces pombe Atg2 (XP_001713120), and S. cerevisiae Atg2 (NP_014157). The alignment was generated using CLUSTAL W. Identical residues are indicated with filled boxes. The black line above the human Atg2A sequence shows the minimal region required for lipid droplet binding (1723–1829). (C) HeLa cells stably expressing wild-type GFP-Atg2A or its mutant (GFP-Atg2A Δ1723–1829) were cultured in regular medium containing oleic acid-BSA for 24 h and then in starvation medium containing BODIPY 558/568-C₁₂) for 1 h. Cells were fixed, stained with anti-GFP and anti-LC3 antibodies, and subjected to confocal microscopy. Signal color is indicated by color of typeface. Scale bars 5 μm, and 2 μm in inset.

FIGURE 9:

Amino acids 1723–1829 of Atg2A are required for autophagy. (A) HeLa cells stably expressing either siRNA-resistant wild-type GFP-Atg2A or GFP-Atg2A Δ1723–1829 were treated with control siRNA or a mixture of siRNAs against Atg2A and Atg2B. Cells grown in regular medium were fixed and subjected to immunofluorescence microscopy using anti-LC3 and anti-GFP antibodies. Scale bar, 5 μm. (B) Ratio of cells containing large LC3 punctate structures was quantified. Data represent mean ± SE of three independent treatments with siRNA. (C) HeLa cells used in A were cultured in regular DMEM or starvation medium in the presence or absence of 20 μM of chloroquine for 2 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.

FIGURE 10:

Silencing of Atg2A/B causes aggregation of enlarged lipid droplets. (A–D) HeLa cells were treated with the indicated siRNAs for 5 d and cultured without (0 h) or with oleic acid (16 h). Cells were fixed, stained with Sudan III (2 mg/ml), and immediately analyzed by fluorescence microscopy (B–D), or stained with anti-LC3 antibody and Sudan III and analyzed by confocal microscopy (A). Total lipid droplet pixel area (B) and the number of lipid droplets (C) were quantified in 20 randomly selected cells. Histogram analysis of size variation of 1000 lipid droplets in each group is shown (D). Data represent mean ± SE of three independent treatments with siRNA and oleic acid. Statistical difference determined by one-way analysis of variance (ANOVA) with Bonferroni–Dunn posttest (*p < 0.05, **p < 0.01). Scale bar, 5 μm (A). (E) Quantification of cluster formation of lipid droplets. Cells used in A–C were cultured without (0 h) or with oleic acid (16 h). After additional 1 h incubation with BODIPY 558/568-C₁₂, the BODIPY signals were directly captured by confocal microscopy without fixation to avoid artificial disruption of lipid droplet clusters during the fixation processes. Ratio of cells having lipid droplet clusters was quantified from 50 randomly selected cells as described in Materials and Methods. Data represent mean ± SE of three independent treatments with siRNA and oleic acid. Statistical difference determined by one-way ANOVA with Bonferroni–Dunn posttest (**p < 0.01). Scale bar, 5 μm.