Extended Data Fig. 3: Initiation hubs on selective cargo are dynamic.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, Further examples of coalescence of in vitro formed GFP–Atg11 droplets as shown and quantified in Fig. 3e. Scale bar: 2 µm. b, Schematic illustration of Atg11 domains. c, atg19∆ vac8∆ or atg13∆ atg19∆ vac8∆ were transformed with Atg11–GFP (OE Atg11) or Atg111-454-GFP (OE Atg111-454) overexpressed under a GPD promoter or Atg11–GFP expressed under its native promoter (End. Atg11) and grown to mid-log phase. Quantification: cells with GFP puncta. Data are mean values (n = 100 cells per condition and replicate, three biological replicates). Circles: mean values of each replicate, bars: mean. Statistical analysis: two-way ANOVA followed by a Tukey post hoc test. P values: atg19∆vac8∆: OE Atg11:End. Atg11 P < 0.0001, OE Atg11:OE Atg111-454 P < 0.0001; atg13∆ atg19∆ vac8∆: OE Atg11:End. Atg11 P < 0.0001, OE Atg11:OE Atg111-454 P < 0.0001. d, atg11∆ atg19∆ cells expressing endogenous BFP–Ape1 and copper-inducible untagged Ape1 along with Atg111-873-GFP–Atg19, Atg111-607-GFP–Atg19, or Atg111-454-GFP–Atg19 were grown to mid-log phase in the presence of 50 µM CuSO4. Statistical analysis: one-way ANOVA followed by a Dunnett post hoc test. Scale bar: 2 µm. (n = 50 structures per condition and replicate, three biological replicates). P value: Atg111-873-GFP–Atg19:Atg111-454-GFP–Atg19 P < 0.0001, Atg111-873-GFP–Atg19: Atg111-607-GFP-Atg1 P = 0.451. e, Cells overexpressing Atg11–GFP under a GPD promoter and expressing Atg9-3xmCherry or 3xmCherry in atg19∆ or atg19∆ vac8∆ strains were grown to mid-log phase. Scale bar: 2 µm. Quantification: co-localization of Atg9-3xmCherry and Atg11–GFP puncta. Data are mean values (n > 148 punctae per condition and replicate, three biological replicates). Circles: mean values of each replicate, bars: mean. Statistical analysis: two-tailed unpaired t-tests. P values: WT P < 0.0001, vac8∆ P < 0.0001. f, GST–BFP or GST–BFP–Atg193D (a hosphor-mimetic mutant of Atg19 known to stably interact with Atg11, S390D, S391D, and S396D29) were expressed in E. coli and bound to Glutathione Sepharose (GSH) beads, incubated with Sf9 insect cell lysates containing overexpressed GFP–Atg11, and bound GFP–Atg11 was analysed. Scale bar: 20 µm. Quantification: ratio of bead-bound protein to soluble protein 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 > 18 beads per condition across replicates, three technical replicates). Statistical analysis: two-tailed unpaired t-test. P values: P = 0.2102. g, In vitro formed GFP–Atg11 droplets coupled to Atg193D containing beads were photobleached, and recovery of the signal was measured. Quantification: recovery of the GFP signal. Data are mean ± SEM (n = 10 structures across replicates, three technical replicates). Source numerical data are available in source data, not significant (n.s.), arbitrary units (a.u.).
