Fig. 2: Receptor mobility enables the formation of initiation hubs.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, (i) atg19∆ cells expressing 2×GFP–Ede1 and mScarlet–Atg11 were grown to mid-log phase. (ii) 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. (iii) atg19∆ cells expressing GFP–Atg11 were grown to mid-log phase. Mitochondria were stained with MitoTracker Red and mitophagy was induced by starvation. Images of one out of three biological replicates are shown. Scale bar, 2 μm. b, Nup170–GFP, Vph1–GFP vac8∆ atg19∆ and GFP–Atg11-expressing cells (as in a(ii)) were grown to mid-log phase. 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 the 25th to 75th percentiles, whiskers show the 5th and 95th percentiles, circles show the mean value of each replicate, outliers are indicated by 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: GFP–Atg11 versus Vph1–GFP, ****P < 0.0001; GFP–Atg11 versus Nup170–GFP, *P = 0.0439. c, Atg9–3×GFP atg11∆ or Atg1–3×GFP 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. d, Cells expressing Ede1–BFP, mScarlet–Atg11 and either Atg1–mNeon or Atg9–mNeon were grown to mid-log phase. Images of one out of three biological replicates are shown. Scale bar, 2 μm. e, mScarlet–Atg11 atg19∆ cells coexpressing either Atg19, an empty control vector (−) 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. Scale bar, 2 µm. Quantification: mScarlet–Atg11 clustering as the coefficient of variance (s.d./mean mScarlet intensity) in a box plot. Horizontal lines show the median, box shows 25th to 75th percentiles, whiskers show the 5th and 95th percentiles, circles show the mean value of each replicate, outliers are indicated by black dots (n = 50 structures per condition and replicate, three biological replicates). Statistical analysis: two-tailed unpaired t-test. **P = 0.0012. f, atg19∆ cells expressing mScarlet–Atg11 and 2×GFP–Ede1 without (−) or with (+) 3×GBP under the control of a copper-inducible promoter were grown to mid-log phase. Scale bar, 5 µm. Data are mean values (n > 37 ENDs per condition and replicate, four biological replicates). Circles show mean values of each replicate, bars show the mean. Statistical analysis was carried out by two-tailed unpaired t-test. P value: 3×GBP(−) versus 3×GBP(+), ****P < 0.0001. Source numerical data are available in Source data.
