ATG9A-deficient cells show structures similar to those observed in FIP200-deficient cells. (A) ATG9A-knockout (KO) MEFs with or without stable expression of the indicated constructs were cultured in starvation medium for 1–2 h and subjected to fluorescence microscopy. Endogenous ATG16L1, LC3 and p62 were stained. Scale bars: 10 µm, 1 µm (inset). (B–E) ATG9A-knockout MEFs (B), HeLa cells treated with siRNA against ATG9A (C,D) and wild-type (WT) (Ea) and ATG9A-knockout (Eb,c) MEFs expressing GFP–ULK1 were cultured in starvation medium for 1 h and subjected to conventional electron microscopy (B,C) and immunoelectron microscopy using anti-p62 (D) and anti-GFP (E) antibodies. Arrowheads, clusters of high-density particles; arrows, ER surrounding the ribosome-free area; double-headed arrows, vesicles inside and at the periphery of the ribosome-free area. Scale bars: 500 nm (Ea,b), 200 nm (B–D,Ec).

The high-density particle clustered at the autophagosome formation site is ferritin. (A,B) Wild-type (WT), FIP200-knockout (KO) and ATG9A-knockout MEFs (A) and HeLa cells treated with oligos against ATG9A or control oligos (luciferase, siLuc) (B) were cultured in regular medium. Cells were stained with anti-ferritin (green) and anti-p62 (red) antibodies. Scale bars: 10 µm (white), 1 µm (yellow). (C) ATG9A-knockout MEFs (a,b) and HeLa cells treated with siRNA against ATG9A (c,d) were cultured in starvation medium for 1 h and subjected to immunoelectron microscopy using an anti-ferritin antibody. Panels b and d show magnified images of the indicated areas in panels a and c, respectively. Scale bars: 500 nm (a,c), 100 nm (b,d). Arrowheads, ferritin-positive high-density particle clusters. (D) ATG9A-knockout MEFs stably expressing GFP–ULK1 cultured in regular medium were analyzed by immunocytochemistry using anti-GFP (green) and anti-ferritin (red) antibodies. Scale bars: 10 µm (white), 1 µm (yellow).

Accumulation of small isolation-membrane-like structures in VMP1-silenced cells. (A) HeLa cells treated with siRNA against VMP1 with or without stable expression of the indicated constructs were cultured in starvation medium for 1–2 h and subjected to fluorescence microscopy. Endogenous ATG9A, LC3 and p62 (red) were stained. Scale bars: 10 µm (white); 1 µm (yellow). (B–F) HeLa cells treated with siRNA against VMP1 with (E) or without (B–D,F) stable expression of GFP–DFCP1 were cultured in regular (B) or starvation (St) medium (B–F) for 1 h and subjected to conventional electron microscopy (B) and immunoelectron microscopy using anti-p62 (C), anti-LC3 (D), anti-GFP (E) and anti-ATG9A antibodies (F). Arrowheads, ferritin clusters; arrows, ER surrounding the ribosome-free area; double-headed arrows, vesicles inside and at the periphery of the ribosome-free area; asterisks, small isolation membrane (IM)-like structures. The graph in B indicates the number of isolation-membrane-like structures (longer than 200 nm) per p62-positive region. Data are presented as the mean±s.e.m. of 37 [growth, St (-)] and 57 [starvation, St 1 h] p62-positive regions. Scale bars: 500 nm (B), 200 nm (C–F), 50 nm (D,F, inset).

Accumulation of small autophagosome-like structures in ATG2A/B-silenced cells. (A) HeLa cells treated with siRNA against ATG2A and ATG2B were cultured in regular or starvation (St) medium for 1 h and subjected to conventional electron microscopy. The graph indicates the number of small autophagosome-like structures (asterisks) per p62-positive region. Data are presented as the mean±s.e.m. of 11 [growth, St (-)] and 46 [starvation, St 1 h] different p62-positive regions. Arrowheads, ferritin clusters; arrows, ER surrounding the ribosome-free area; double-headed arrows, vesicles inside and at the periphery of the ribosome-free area; asterisks, small autophagosome (AP)-like structures. Scale bars: 500 nm. (B) HeLa cells treated with siRNA against luciferase (data shown in Fig. 3B), VMP1 or ATG2A/B were cultured in regular medium and subjected to fluorescence microscopy. Endogenous ferritin (green) and p62 (red) were stained. Scale bars: 10 µm (white), 1 µm (yellow). (C–E) HeLa cells treated with siRNA against ATG2A/B were starved and analyzed by immunoelectron microscopy using anti-p62 (C), anti-LC3 (D) and anti-ATG9A antibodies (E). Arrowheads, ferritin clusters. Scale bars: 200 nm (C,Da,E), 50 nm (Db,E inset).

Isolation membranes formed in ATG5-deficient cells. (A) ATG5-knockout (KO) MEFs with or without stable expression of the indicated constructs were cultured in starvation medium for 1 h and subjected to fluorescence microscopy. Endogenous ATG16L1, ATG9A and p62 were stained. Scale bars: 10 µm. (B–D) Wild-type (WT) and ATG5-knockout MEFs (B) and HeLa cells treated with siRNA against ATG5 (C,D) were cultured in starvation medium for 1 h and subjected to conventional electron microscopy (B,C) and immunoelectron microscopy using an anti-ferritin antibody (D). Arrowheads, ferritin clusters; arrows, ER surrounding the ribosome-free area; double-headed arrows, vesicles inside and at the periphery of the ribosome-free area. Scale bars: 200 nm (B,C), 500 nm (D), 100 nm (D, inset).

Ferritin is selectively incorporated in autophagosomes and delivered to lysosomes. (A) MEFs stably expressing GFP–ULK1 or GFP–LC3 were cultured in starvation (St) medium with or without 100 nM bafilomycin A₁ (Baf A) for 1 h. Cells were fixed and stained with an anti-ferritin antibody and analyzed by fluorescence microscopy. The outlined areas in each image are shown at higher magnification to the right. Arrows, colocalization of GFP and ferritin signals. Scale bars: 20 µm (left), 4 µm (right). Quantification of the ferritin-positive ratio (%) of total GFP–ULK1 or GFP–LC3 punctate structures is shown in the graph (circles). Triangles represent the random overlapping ratio (%) of the colocalization (ferritin-positive ratio) (see Methods). (B) Wild-type (WT) MEFs cultured in regular medium were analyzed by immunoelectron microscopy using an anti-ferritin antibody. Arrowheads (light blue), autophagosomes. Scale bars: 500 nm, 100 nm (inset). (C) Wild-type, ATG9A-knockout (KO) and FIP200-knockout MEFs were cultured in regular medium in the presence or absence of 50 µM pepstatin (pep) A and 50 µM E64d for 24 h. Cells were fixed and stained with anti-Lamp1 and anti-ferritin antibodies, and analyzed by fluorescence microscopy. Arrows, colocalization of Lamp1 and ferritin signals. Scale bars: 20 µm (left), 4 µm (right). Quantification of the ferritin-positive ratio (%) of total Lamp1 punctate structures is shown as in A. For graphs in A and C, data represent the mean±s.e.m. (seven randomly selected cells); *P<0.05, **P<0.01; unpaired Student's t-test.