Extended Data Fig. 7: Autophagy initiation hubs are conserved in human cells.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, U2OS cells were grown in nutrient-rich medium. mCherry–p62 condensates were photobleached and recovery of the signal was followed. A representative image is shown from one out of three biological replicates. The white arrowhead indicates the photobleached area. Scale bar: 1 µm. Quantification: recovery of the mCherry signal. Data are mean values ± SEM (n = 28 structures across three biological replicates). b, U2OS wild-type cells stably expressing mito-mKeima and FRB-FIS93-152 and FKBP–GFP-ULK1 were grown in nutrient-rich medium and treated with Bafilomycin A1 (Baf) and rapalog as indicated. Gating for GFP-expressing cells was performed. The mito-mKeima ratio of lysosomal mitochondria (561 nm) to cytosolic mitochondria (488 nm) was analysed by flow cytometry and is shown as a ratio normalized to the Baf treatment. Data are mean values (n > 50,000 cells per condition and replicate, two biological replicates). Circles: mean values of each replicate, bars: mean. c, Further examples of Fig. 7f. Quantification: peak-to-peak signal distance in a box plot. Horizontal lines: median, box: 25th to 75th percentiles, whiskers: expand to 5th and 95th percentiles, outliers: black dots (n = 47 plot profiles across three biological replicates). Statistical analysis: one-way ANOVA followed by Sidak’s multiple comparison test. P values: p62-FIP200:p62-ER P < 0.0001, FIP200-ER:p62-ER P < 0.0001. d, Zoomed in 3D reconstruction of Fig. 7f. The 3D surface of BFP–Sec61β (blue), FKBP–GFP-FIP200 (green) and mCherry–p62 (magenta) was rendered with the IMARIS software using the machine learning tool for surface segmentation. A z-stack of 0.125 µm was taken to define the borders in z. e, U2OS cells stably expressing FRB-FIS193-152 and transfected with 2xFKBP-GFP-ULK1 were cultured in nutrient-rich medium and stained with MitoTracker DeepRed. Tethering of 2xFKBP-GFP-ULK1 to FRB-FIS93-152 was induced by adding rapalog for 2 h. Mitochondria-associated 2xFKBP-GFP-ULK1 clusters were photobleached, and the recovery of the signal was monitored. Scale bar: 1 µm. Quantification: recovery of the GFP signal. Data are mean ± SEM (n = 28 structures across three biological replicates). White arrowheads indicate the photobleached cluster. The quantification and the representative image are the same as shown in Fig. 8b. Source numerical data are available in source data, not significant (n.s.), arbitrary units (a.u.).
