Figure 1.

ULK1, Atg14, WIPI-1, Atg16L1 and LC3 co-localize to the same compartment, which is in close proximity to the DFCP1 structure. (A–H) Mouse embryonic fibroblasts (MEFs) stably expressing GFP-ULK1 (A), GFP-ULK1 and HA-Atg14 (B), HA-Atg14 and GFP-WIPI-1 (C), HA-WIPI-1 (D), GFP-DFCP1 (E and F), GFP-DFCP1 and HA-Atg14 (G) and GFP-DFCP1 and HA-WIPI-1 (H) were cultured in starvation medium for 1 hour. Cells were then fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-HA (B, C, G and H), anti-Atg16L1 (A, D and E) and anti-LC3 antibodies (F). Due to low expression, GFP-ULK1 and GFP-DFCP1 were stained with anti-GFP antibodies. Nearly complete co-localization is indicated by arrowheads and adjacent co-localization is indicated by arrows. The structures indicated with broken lines were subjected to linescan analysis (shown in J, K and Suppl. Fig. 3). Signal color is indicated by color of typeface. St. M., starvation medium. Scale bars, 10 µm (white) and 1 µm (yellow). (I) Quantification of complete and adjacent co-localization between indicated Atg protein pairs after 1-hour starvation. Data represent mean ± SE of ten images. (J–K) Linescans were obtained from representative punctate structures showing co-localization between GFP-WIPI-1 and Atg16L1 (J) and between HA-WIPI-1 and GFP-DFCP1 (K). Original structures are shown in (D and H) (indicated with dashed lines). (L) 3D reconstruction of Atg puntate structures. 1-hour starved cells were observed by confocal laser microscopy and then 3D images were reconstituted. Lateral images reconstituted from Z-sectioning are also shown. Scale bars, 1 µm.