Fig. 3: Phase separation of Atg11 drives initiation hub formation.

a, GFP–Atg11 cells expressing endogenous BFP–Ape1 and copper-inducible untagged Ape1 or wild-type cells expressing GFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO₄. GFP–Atg11 and GFP–Ape1 structures were photobleached and recovery of the signal was monitored. White arrowheads show the photobleached area. Scale bar, 1 µm. Quantification: recovery of the GFP signal. Data show mean ± s.e.m. (n > 32 foci per condition across replicates, three biological replicates). b, 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 represented as kymographs. Images of one out of three biological replicates are shown. Scale bar, 2 µm. Kymograph scale bar, 1 µm. c, GFP–Atg11 was purified from Sf9 insect cells and droplet formation was monitored in vitro at different concentrations by fluorescence microscopy after 20 min incubation at room temperature in a buffer containing 150 mM NaCl. Images from one out of three biological replicates are shown. Scale bar, 5 µm. Quantification is shown in Extended Data Fig. 2e. d, In vitro formed GFP–Atg11 droplets were photobleached and recovery of the signal was measured. White arrowheads indicate the photobleached area. Scale bar, 1 µm. Quantification: recovery of the GFP signal. Data show mean ± s.e.m. (n = 30 structures per condition and replicate, three biological replicates). e, Coalescence of in vitro formed GFP–Atg11 droplets was monitored. Scale bar, 2 µm. Quantification: droplet size, represented in a scatter-plot. Statistical analysis was carried out by a two-tailed unpaired t-test. Circles show mean values of each replicate, horizontal lines show the median (n = 6 coalescence events, three biological replicates). *P = 0.026. Further examples are shown in Extended Data Fig. 3a. f, atg19∆, atg13∆ atg19∆ or atg13∆ atg19∆ vac8∆ cells were transformed with Atg11–GFP overexpressed under a GPD promoter. The formation of Atg11 condensates was monitored. Scale bar, 2 µm. The percentage of cells with Atg11–GFP foci was quantified, displayed in a bar graph. Data are mean values (n = 100 cells per condition and replicate, three biological replicates). Circles show mean values of each replicate, bars show mean. Statistical analysis was conducted by one-way ANOVA followed by a Dunnett’s post hoc test. P values: atg19∆ versus atg19∆ atg13∆, P = 0.9885; atg19∆ versus atg19∆ atg13∆ vac8∆, P = 0.688. g, Atg11–GFP was overexpressed in atg19∆ atg13∆ vac8∆ cells and cells were grown to the mid-log phase. GFP condensates were fully photobleached and their recovery was monitored. White arrowheads indicate the bleached area. Scale bar, 2 µm. Quantification: recovery of the GFP signal. Data show mean ± s.e.m. (n = 25 structures per condition across replicates, three biological replicates). Source numerical data are available in Source data.