Fig. 4: Initiation hubs coalesce at the vacuolar contact site to trigger phagophore initiation.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, Initial (top) and final (bottom) snapshots of a molecular simulation corresponding to a very-low-affinity, low-affinity and high-affinity interaction of Atg11–Atg19 subcomplexes (grey to green) with a cargo (blue). The clustering is shown by visualizing the number of neighbours of Atg11–Atg19 particles. Stronger colours indicate that more neighbours are in proximity and will result in a stronger coupling. b, GFP–Atg8 cells expressing mScarlet–Atg11, endogenous BFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO4. Images of one out of three biological replicates are shown. Scale bar, 2 µm. c, mScarlet–Atg8 atg19∆ cells expressing endogenous GFP–Ape1 and copper-inducible untagged Ape1 along with Atg19 or Atg19–GBP were grown to mid-log phase in medium containing CuSO4 and rapamycin to induce phagophore formation32. Scale bar, 2 µm. Quantification: elongated mScarlet–Atg8-positive structures on cargo. Data show the mean (n = 100 structures per condition and replicate, three biological replicates). Circles show the mean of each replicate, bars show the mean. Statistical analysis was conducted by a two-tailed unpaired t-test, P = 0.0061. d, Atg9–3×GFP mScarlet–Atg8 cells expressing endogenous BFP–Ape1 and copper-inducible untagged Ape1 grown and treated as in c. Scale bar, 2 µm. Quantification: cargo-associated Atg9 clusters. Data are mean values (n = 100 cells per condition and replicate, three biological replicates). Circles show the mean of each replicate, bars show the mean. Statistical analysis was conducted by a two-tailed unpaired t-test. P values: rich versus rapa(for 1–2 clusters), ***P = 0.0002; rich versus rapa(for >2 clusters), ***P = 0.0002. e, GFP–Atg19 cells overexpressing copper-inducible untagged Ape1 were grown to mid-log in medium containing CuSO4 and rapamycin (one out of three identified examples shown). (i) In situ cryo-electron tomographic slice. Orange arrowhead indicates phagophore; V, vacuole. (ii) Different slice (+16.6 nm from i). (iii) Segmentation and 3D rendering of the tomographic volume. Orange, phagophore; grey, vacuole membrane. Scale bar, 200 nm (i), 50 nm (ii,iii). Correlative fluorescence images are shown in Extended Data Fig. 5e. f, Indicated strains expressing endogenous GFP–Ape1 and copper-inducible untagged Ape1, Atg19–GFP–µNS or pp–GFP–µNS were grown to mid-log phase in the presence of 50 µM CuSO4. GFP cleavage was monitored by immunoblotting. pp, Ape11–45. One out of three biological replicates is shown. g, Cells as in f coexpressing mScarlet–Atg11 were monitored by fluorescence microscopy. Scale bar, 2 µm. Quantification: GFP clustering as the coefficient of variance (s.d./mean GFP intensity) in a box plot. Horizontal lines show the median, box shows 25th to 75th percentiles, whiskers indicate the 5th and 95th percentiles, circles show the mean value of each replicate, outliers are shown as black dots (n = 50 structures per condition and replicate, three biological replicates). Statistical analysis was conducted by a one-way ANOVA followed by a Dunnett’s post hoc test. P values: pp–GFP–µNS versus Atg19–GFP–µNS, ****P < 0.0001; pp–GFP–µNS versus GFP–Ape1, P = 0.9826. Source numerical data and unprocessed blots are available in Source data.
