Fig 2: Autophagic membranes and LDs.a Distribution and effects of CCTβ3(1-206)-mCherry expression in MEFs starved for 8 h. (a) Distribution of CCTβ3(1-206)-mCherry (magenta) and LDs (green). Bar, 10 μm. (b) Western blotting of LC3. MEFs overexpressing CCTβ3 or CCTβ3(1-206) were compared with untransfected MEFs. A representative result of three independent experiments. b Distribution of GFP-DFCP1 (green) and LDs (magenta) in MEFs starved for 8 h (left) and in MEFs cultured in OA-containing regular medium for 1 day (right). GFP-DFCP1 puncta in starved MEFs are marked by arrows. Bar, 5 μm. c Expansion of the omegasome (green, GFP-DFCP1) around LDs (magenta) in MEFs starved for 8 h. Selected images from Supplementary Movie 1. Bar, 2 μm. d Distribution of GFP-LC3 (green) and LDs (magenta) in MEFs starved for 8 h. Bar, 10 μm. e Emergence of isolation membranes/autophagosomes (green, GFP-LC3; arrows) around LDs (magenta) in MEFs starved for 36 h. Selected images from Supplementary Movie 2. Bar, 5 μm. f EM of MEFs overexpressing CCTβ3 starved for 8 h. The isolation membrane (arrows) adheres to the LD surface (arrowheads). Bar, 0.5 μm. Source data are provided as a Source data file.

Fig 3: CCT isoforms in MEFs starved for 1 h.a Distribution of mCherry-tagged CCT isoforms in MEF. (a) Regular medium. (b) Starvation for 1 h. Bar, 10 μm. b Distribution of CCTβ3-mCherry (magenta) and Atg proteins (green) in MEFs starved for 1 h. Bar, 5 μm. The colocalization ratios of Atg proteins and CCTβ3-mCherry in the original picture (gray bar) and in the picture after 90° clockwise rotation of the green image (white bar) are shown. Pooled data obtained in three independent experiments (n = 60); *p = 0.0319, ****p < 0.0001 (two-tailed Mann–Whitney test). c PC synthetic activity. 3H-choline was added to medium 30 min before harvest. (a) MEFs with or without CCT overexpression cultured in regular medium or starvation medium for 1 h. (b) The result in starvation medium is normalized by the expression level of CCT isoforms, estimated by the CCT-mCherry band intensity in western blotting (c). A representative result of three independent experiments. d The effect of CCT overexpression on GFP-DFCP1 in MEFs starved for 1 h. Bar, 10 μm. (i) The punctum size. The number of puncta counted: 578 (WT), 486 (CCTα), 481 (CCTβ2), and 2320 (CCTβ3). (ii) The total area in a cell. The number of cells counted: 90. Pooled data from three independent experiments. ****p < 0.0001 (two-tailed Mann–Whitney test). e The effect of CCTβ3 overexpression on the autophagic flux estimated by western blotting of LC3. MEFs were cultured for 1 h in starvation medium, with or without the addition of 20 μM chloroquine (CQ) for the last 30 min. A representative result of three independent experiments. In box plots, the center line indicates the median, box boundaries indicate the 25th and 75th percentiles, whiskers are Tukey-type, and the average is marked as +. Source data are provided as a Source data file.

Fig 4: De novo-synthesized PC and autophagic membranes.a Labeling of de novo-synthesized PC. MEFs were cultured for 1 h with 0.25 mM propargylcholine in regular medium (a) or in starvation medium (b), fixed, and reacted with Cy3-azide for fluorescence microscopy. Bar, 10 μm. b De novo-synthesized PC and GFP-tagged Atg proteins in MEFs starved for 1 h. PC (magenta) and Atg proteins (green): GFP-ULK1, GFP-DFCP1, GFP-WIPI1, and GFP-LC3. Endogenous EEA1 is labeled as a control. Bar, 10 μm. The box plot shows the ratio of Atg protein (and EEA1) pixels colocalizing with PC puncta. Pooled data from three independent experiments (n > 130); *p = 0.0042, ****p < 0.0001 (two-tailed Mann–Whitney test). c Double labeling of de novo-synthesized PC (magenta) and endogenous LC3 (green) in MEFs starved for 1 h. Bar, 10 μm. d Labeling of existing PC. MEFs were cultured for 1 h with 0.25 mM propargylcholine in regular medium and then for the next 1 h with 5 mM choline in starvation medium. Bar, 10 μm. The total area and the number of PC puncta in a cell were measured. Pooled data from three independent experiments (n = 40); *p = 0.0367, ***p = 0.0002 (two-tailed Mann–Whitney test). In box plots, the center line indicates the median, box boundaries indicate the 25th and 75th percentiles, whiskers are Tukey-type, and the average is marked as +. Source data are provided as a Source data file.

Fig 5: DFCP1 mutants show delayed omegasome constriction.a 3D reconstruction of omegasomes in maturation and constriction phases. U2OS DFCP1 KO lines stably expressing mNG-DFCP1 WT were starved in EBSS, fixed, stained for p62, LC3B and mNG and SIM imaging was performed. Shown are examples from three different stages of omegasome formation, and their reconstruction from different angles. Scale bar: 5 µm, insets: 0.5 µm. b U2OS DFCP1 KO lines stably expressing mNG-DFCP1 WT and mCh-p62. Cells were starved in EBSS and SoRa live-cell super-resolution imaging was used to capture omegasome formation. Omegasome formation was divided into three phases as indicated. The scheme illustrates how DFCP1 and p62 co-localize throughout an omegasomes’ life cycle. Quantification of events in (d–g). Scale bar: 0.5 µm. c U2OS DFCP1 KO lines stably expressing mNG-DFCP1 WT or -mutant and mCh-p62. Cells were starved with EBSS and imaged live to capture omegasome formation. Shown are representative stills of omegasomes analyzed in (d–g). Scale bar 0.5 µm. d Quantification of the total omegasome lifetime, with a DFCP1 spot as beginning and end point. Each plotted point represents the mean value of one experiment. Bars: mean ± SD, 3 independent experiments, analyzing in total 58 (WT), 16 (K193A) and 49 (T189A) omegasomes. One-way ANOVA, Dunnett’s post hoc test, comparing against WT. e Duration of the individual phases of omegasome formation, dataset from (d). Each plotted point represents the mean value of one experiment. Bars: mean ± SD, 3 experiments, Ordinary One-way ANOVA, Dunnett’s multiple comparisons test, comparing against WT. 3 independent experiments; ns: not significant. Analyzed omegasomes: Initiation: WT (76), K193A (54), T189A (81); Maturation: WT (99), K193A (81), T189A (117); Constriction: WT (78), K193A (47), T189A (99). f Same dataset as in (d). The signal of DFCP1 was segmented, and mNG-DFCP1 diameter was measured over time. 3 independent experiments, total number of tracks: WT (329), K193A (133), T189A (266). Mean ± 95% CI. g Same dataset as in (f). Individual omegasomes have been classified by their maximum diameter and their diameter was plotted over time. The vertical line indicates complete disappearance of WT omegasomes, the horizontal line indicates successful constriction (to a diameter smaller than 0.5 µm). Shown is the mean, bands indicate ± 95% CI. Source data are provided as a Source data file.