Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy

Abstract

Autophagy is a catabolic process essential for cell homeostasis, at the core of which is the formation of double-membrane organelles called autophagosomes. Atg9 is the only known transmembrane protein required for autophagy and is proposed to deliver membrane to the preautophagosome structures and autophagosomes. We show here that mammalian Atg9 (mAtg9) is required for the formation of DFCP1-positive autophagosome precursors called phagophores. mAtg9 is recruited to phagophores independent of early autophagy proteins, such as ULK1 and WIPI2, but does not become a stable component of the autophagosome membrane. In fact, mAtg9-positive structures interact dynamically with phagophores and autophagosomes without being incorporated into them. The membrane compartment enriched in mAtg9 displays a unique sedimentation profile, which is unaltered upon starvation-induced autophagy. Correlative light electron microscopy reveals that mAtg9 is present on tubular-vesicular membranes emanating from vacuolar structures. We show that mAtg9 resides in a unique endosomal-like compartment and on endosomes, including recycling endosomes, where it interacts with the transferrin receptor. We propose that mAtg9 trafficking through multiple organelles, including recycling endosomes, is essential for the initiation and progression of autophagy; however, rather than acting as a structural component of the autophagosome, it is required for the expansion of the autophagosome precursor.

FIGURE 1:

mAtg9 affects autophagy at an early omegasome stage. (A) Domain structure of yeast and human Atg9. Red and blue boxes represent conserved transmembrane domains. (B) HEK293 cells were treated with either control siRNA (ctrl) or siRNA against mAtg9 (Atg9 KD) and incubated in full (F) or starvation medium (S) for 2 h. Cell lysates were analyzed by Western blot using antibodies against mAtg9, β-tubulin, and LC3. (C) Confocal microscopy of HEK293/GFP-LC3 and HEK293/GFP-DFCP1 cells treated with RISC-Free (ctrl) or mAtg9 (Atg9 KD) siRNA and starved for 2 h. ULK1, WIPI2, and Atg16 were detected by indirect immunofluorescence using anti-ULK1, -WIPI2, and -Atg16 antibodies. Bars, 10 μm. (D–H) Quantification of ULK1, GFP-DFCP1, WIPI2, Atg16, or GFP-LC3 spots from B expressed as spots/cell area (ULK1, GFP-DFCP1) or spots/cell (WIPI2, Atg16, GFP-LC3). Error bars, SEM; **p < 0.01 ***p < 0.001 (n = 3, two-tailed unpaired t test). (I) Colocalization of GFP-LC3 with WIPI2 and Atg16 in HEK293/GFP-LC3 cells treated with mAtg9 siRNA, starved, and detected in C. (J) Quantification of colocalization of WIPI2 and Atg16 with GFP-LC3 as shown in (I). Error bars, SEM, n = 3.

FIGURE 2:

mAtg9 colocalizes with both early and late autophagosome autophagy markers. (A) Confocal microscopy of mAtg9 (red) in HEK293/GFP-LC3 and HEK293/GFP-DFCP1 cells. GFP-DFCP1, ULK1, WIPI2, Atg16, or GFP-LC3 in green as indicated. ULK1, WIPI2, Atg16, and mAtg9 were detected using antibodies as in Figure 1. Arrows in merge show mAtg9 colocalization with markers. Bars, 10 μm. (B) Confocal microscopy of HEK293/GFP-LC3 cells, GFP-LC3 (green), ULK1 (blue), mAtg9 (red); HEK293/GFP-DFCP1 cells with mAtg9 (red), LC3 (blue); HEK293/GFP-LC3 cells, GFP-LC3 (green), Atg9 (red), and WIPI2 and Atg16 (blue). A low and partial colocalization of ULK1 and Atg9 is shown and is in contrast to the colocalization of ULK1 with LC3. LC3 emerges from the GFP-DFCP1 structure, and mAtg9 coincides partially with both structures. Bottom, WIPI2, Atg16, and GFP-LC3 signals overlap, whereas mAtg9 is found in close proximity with WIPI2, Atg16, and GFP-LC3. Quantification of the percentage of Atg9 found with ULK1, DFCP1, WIPI2, Atg16, and LC3 is shown in Table 1.

FIGURE 3:

(A) Live-cell imaging of HEK293/GFP-DFCP1 cell transfected with mRFP-Atg9 using widefield fluorescence microscopy. (A′) Magnified frames from the boxed region. Frames are taken from Supplementary Video S1, which started after 5 min of incubation in starvation medium. Images were captured every 5 s, and every other frame is shown. White arrowhead, mRFP-Atg9 vesicle; empty arrowhead, GFP-DFCP1. Note how the two structures orbit each other without significant overlap except at +110 s, immediately after which they separate. (B) Live-cell imaging of HEK293/GFP-LC3/mRFP-Atg9 cells by widefield fluorescence microscopy. (B′) Magnified boxed regions. Images are extracted from Supplementary Video S2. Frames were taken every 5 s, and every other frame is shown. Note how the two structures orbit each other without significant overlap except at +60 s, immediately after which they separate. White arrowhead, mRFP-Atg9 vesicle.

FIGURE 4:

CLEM and cryoimmuno-EM demonstrates mAtg9 is present on tubulovesicular membranes surrounding autophagosomes. (A) HEK293/GFP-LC3/mRFP-Atg9 cells starved for 2 h were fixed, and GFP-LC3 (green) or mAtg9 (red) fluorescent structures were identified by confocal microscopy and subsequently in thin sections prepared for TEM. Low-magnification TEM of the whole cell is shown in Supplemental Figure S3A. (B, C) two 70- to 100-nm serial sections of the boxed area in Supplemental Figure S3A. The mRFP-Atg9–positive membranes (red arrows) corresponding to vesicular clusters and tubules emerging from multivesicular structures (red arrows). Bar, 500 nm. (D, E) Cryoimmuno-EM of HEK293/GFP-LC3/mRFP-Atg9 cells (D) or primary rat hepatocytes (E), transduced with mRFP-Atg9, starved for 2 h, and labeled for mAtg9 (10- or 5-nm gold, respectively). mAtg9 (arrowheads) is found on vesicles and tubules, often surrounding large endosomal-like structures. In E, note that mAtg9 is not found on the autolysosome membrane, although some signal is present inside that could represent trapped material. Bar, 500 nm. a, autolysosome; e, endosome; m, mitochondria. Bars, 500 nm (B, C), 1 μm (D, E).

FIGURE 5:

mAtg9 is on recycling endosomes. (A) Homogenates from HEK293 cells were separated on 1–22% Ficoll gradients. Gradient fractions were loaded from left (heavy) to right (light) and then analyzed by Western blot using antibodies against CI-MPR (marker for late endosomes/TGN), EEA1 (early endosomes), TGN46 (TGN), mAtg9, TfR (RE), and SOD1 (cytosol). (B) Distribution of marker proteins. The intensity of each band (in arbitrary units) is plotted on the y-axis and the fraction number on the x axis. mAtg9, black solid line; all other markers are dashed lines: red, CI-MPR; yellow, EEA1; green, TGN46; blue, TfR; gray, SOD1. (C) Lysates from HEK293 cells incubated in full medium (F) or starved for 2 h (S) were immunoprecipitated using beads alone (–), a nonrelevant antibody (NR) at 1× and 2× immunoglobulin G (IgG), and anti-mAtg9 IgG and then analyzed by Western blot using antibodies against mAtg9 and TfR. Input, 5% of total lysate before immunoprecipitates (IP). Data are representative of three independent experiments.

FIGURE 6:

mAtg9 colocalizes with endosomes and TGN. Confocal microscopy of HEK293/GFP-LC3 cells. mAtg9 (red) and (A) TfR, (B) Rab11, (C) EEA1, (D) TGN46, and (E) tubulin were detected by indirect immunofluorescence with antibodies to indicated proteins (green). TfR and TGN46 show the highest degree of partial colocalization with mAtg9. Insets in the merge panel are shown on the right. Quantification is shown in Table 2. Bar, 10 μm.

FIGURE 7:

CLEM of transferrin-positive compartments and mAtg9 compartments. HEK293/mRFP-Atg9 cells were incubated for 2 h with anti-TfR antibody conjugated to 10-nm gold and Alexa 647–conjugated Tfn. Cells were fixed, and (A) Tfn-Alexa 647 (blue) or mRFP-Atg9 (red) fluorescent structures were identified by confocal microscopy and subsequently in thin sections prepared for EM. (B) Low-magnification TEM of the top of the cell from A. (C, D) two sets of 70- to 100-nm serial sections from the boxed area in B. TfR-gold–positive vacuoles were identified in TEM (white arrowheads) near the Atg9 compartment: mRFP-Atg9–positive membranes (red arrowheads) corresponding to vesicular clusters and tubules, and vacuoles, some of which contain small amounts of TfR-gold. White arrows show phagophore and autophagosome membranes. Bar (A), 10 μM.

FIGURE 8:

Regulation of mAtg9 traffic by ULK1 and WIPI2. (A) Confocal microscopy of HEK293/GFP-DFCP1 cells treated with siRNAs against RISC-free (ctrl), ULK1, WIPI2, or WIPI2 and ULK1. Cells were incubated in full medium or starved for 2 h before fixation. mAtg9 was detected by indirect immunofluorescence. Arrows indicate areas of colocalization between GFP-DFCP1 and mAtg9. In WIPI2 KD virtually every DFCP1 spot colocalizes with mAtg9. (B) The efficiency of the knockdowns in A was confirmed by Western blot using antibodies against ULK1, WIPI2, and β-tubulin. One representative experiment of three is shown.

FIGURE 9:

The mAtg9 compartment is unaltered after starvation. (A) HEK293 cells were incubated in full (F) or starvation medium for 2 h (S) before homogenization and separation on a 1–22% Ficoll gradient as in Figure 6A. Fractions were loaded from left (heavy) to right (light) and analyzed by Western blot using an antibody against LC3. Membrane-associated LC3-II is found in fractions 13–21. (B) HEK293 cells were treated with RISC-Free (ctrl) siRNA or siRNA against WIPI2 or ULK1 and then incubated in full medium (F) or starved (S) for 2 h before homogenization and fractionation as in A, followed by Western blot for endogenous mAtg9. (C) The efficiency of the knockdown in B was evaluated using antibodies against ULK1, WIPI2, and β-tubulin and confirmed by inhibition of LC3 lipidation after both WIPI2 and ULK1 KD. (D) The intensity of mAtg9 signal was quantified (in arbitrary units) and plotted against the refractive index of each fraction (see example in Supplemental Figure S5D and Materials and Methods). To allow comparison of experiments, a Gaussian curve was fitted for each profile. For each experiment the values were normalized to the median value of the ctrl-F sample, and a Gaussian curve was fitted for each value. No significant shift from the median value was detected. Representative experiments are shown. Ctrl-F and ctrl-S, n = 4; WIPI2 and ULK1, n = 2.

FIGURE 10:

CLEM analysis of mRFP-Atg9 in WIPI2-depleted GFP-DFCP1 cells. (A) CLEM of WIPI2-depleted starved or (B) RF starved cells. (A) Left, GFP-DFCP1/mRFP-Atg9–expressing cell. Right, boxed area in low-magnification image and two serial sections from this region. Red arrowheads indicate tubular and vacuolar Atg9-positive membranes. Black arrowhead indicates double-membrane phagophore. (B) Top left, confocal microscopy of mRFP-Atg9 and GFP-DFCP1–positive structures (boxes 1–3). These structures were then identified in low-magnification TEM and then in higher-magnification images, and sections from three different areas (1–3) are shown. Black arrowhead indicates double-membrane phagophores. Asterisk indicates autophagosome. Bars, 50 μm (A, B), 1 μm (A, middle panel).