Extended Data Fig. 2: Atg11 forms initiation hubs on selective cargo.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, mScarlet–Atg11 atg19∆ cells coexpressing either Atg19 or Atg19–GBP and endogenous GFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO4. Quantification: mean mScarlet–Atg11 signal intensity in a box plot. Horizontal lines: median, box: 25th to 75th percentiles, whiskers: expand to 5th and 95th percentiles, circles: mean value of each replicate, outliers: black dots (n = 50 GFP–Ape1 positive particles per condition and replicate, three biological replicates). Statistical analysis: two-tailed unpaired t-test. b, Atg1-3xmCherry atg19∆ cells expressing endogenous GFP–Ape1 and copper-inducible untagged Ape1 together with an empty control vector (-), Atg19 or Atg19–GBP were grown to mid-log phase in the presence of 50 µM CuSO4. Scale bar: 2 µm. Quantification: Atg1-3xmCherry positive GFP–Ape1 structures. Data are mean values (n = 100 structures per condition and replicate, three biological replicates). Circles: mean values of each replicate, bars: mean. Statistical analysis: Dunnett post hoc test. P values: –:Atg19 P < 0.0001, Atg19:Atg19–GBP P < 0.0001. c, Indicated cells expressing GFP–Atg11, endogenous BFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO4.Scale bar: 2 µm. Quantification: GFP clustering as the coefficient of variance (SD/mean GFP intensity) in a box plot. Horizontal lines: median, box: 25th to 75th percentiles, whiskers: expand to 5th and 95th percentiles, circles: mean value of each replicate, outliers: black dots (n = 50 structures per condition and replicate, three biological replicates). Statistical analysis: one-way ANOVA followed by Dunnett post hoc test. P values: wild-type:atg9∆ P = 0.0498, wild-type:vac8∆ P = 0.9036, wild-type:atg9∆vac8∆ P = 0.9893. d, Vac8-mCherry and GFP–Atg11 cells expressing endogenous BFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO4. The dynamics of GFP–Atg11 foci were monitored and the fluorescence profile of GFP was measured along the Ape1 surface. Images of one out of three independent experiments are shown. Scale bar: 2 µm, scale bar inset: 1 µm. Time-lapse series are shown as Supplementary Videos 1–12. e, Quantification of GFP–Atg11 droplet size of Fig. 3c in a box plot. Horizontal lines: median, box: 25th to 75th percentiles, whiskers: expand to 5th and 95th percentiles, circles: mean value of each replicate, outliers: black dots (n = 30 structures per condition and replicate, three biological replicates). Statistical analysis: one-way ANOVA followed by a Dunnett post hoc test. P values: 0.005:2 P < 0.0001, 0.05:2 P < 0.0001, 0.25:2 P < 0.0001, 0.5:2 P < 0.0001, 1:2 P < 0.0001. Source numerical data are available in source data, not significant (n.s.), arbitrary units (a.u.).
