Fig. 8: Receptor mobility is also a required feature for selective autophagy in human cells.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, U2OS cells stably expressing FRB–FIS193–152 and transfected with 2×FKBP–GFP–ULK1 were cultured in nutrient-rich medium. To induce the homo-oligomerization of 2×FKBP–GFP–ULK1 the cells were treated with the homodimerizer AP20187 for 24 h. Tethering of FKBP–GFP–ULK1 to FRB–FIS93-152 was induced by adding rapalogue for 2 h. Images of one out of three biological replicates are shown. Scale bar, 10 µm; scale bar inset, 1 µm. b, U2OS cells stably expressing FRB–FIS193–152 and transfected with 2×FKBP–GFP–ULK1 were cultured in nutrient-rich medium and stained with MitoTracker DeepRed. To induce the homo-oligomerization of 2×FKBP–GFP–ULK1 the cells were treated with AP20187 for 24 h. Tethering of 2×FKBP–GFP–ULK1 to FRB–FIS93–152 was induced by adding rapalogue for 2 h. 2×FKBP–GFP–ULK1 clusters were photobleached, recovery of the signal was monitored. White arrowheads indicate the bleached area. Scale bar, 1 µm. Quantification: recovery of the GFP signal. Data are mean values ± s.e.m. (n = 30 structures per condition across replicates, three biological replicates). c, U2OS wild-type cells stably expressing mito-mKeima and FRB–FIS93–152 and FKBP–GFP–ULK1 were grown in nutrient-rich medium and treated with Bafilomycin A1 (Baf), rapalogue and AP20187 (24 h) as indicated. Cytosolic and lysosomal mt-mKeima fluorescence signal was monitored using flow cytometry and gating for GFP-expressing cells was performed. Data are mean values (n > 50,000 cells per condition and replicate, three biological replicates). Circles show mean values of each replicate, bars show the mean. Statistical analysis was carried out by a one-way ANOVA followed by Sidak’s multiple comparison test. P values: 2 h, ****P = 0.0001; 4 h, ****P < 0.0001. d, U2OS WT cells were transfected with either GFP–p62–GFP alone or together with mCherry–3×GBP. GFP–p62–GFP clusters were photobleached and recovery of the signal was monitored. White arrowheads indicate the photobleached area. Scale bar, 1 µm. Quantification: recovery of the GFP signal. Data are mean values ± s.e.m. (n > 20 structures per condition across replicates, three biological replicates). e, WT and ATG13KO U2OS cells were transfected with either GFP–p62–GFP alone or together with mCherry–3×GBP. The cells were starved in EBSS medium. GFP cleavage was monitored by immunoblotting. RFP, red fluorescent protein. Quantification: ratio between free GFP and 2×GFP–p62. Data are mean values (n = 4 biological replicates). Circles show values of each replicate, bars show mean. Statistical analysis was carried out by a one-way ANOVA followed by Sidak’s multiple comparison test. P values: WT, ****P < 0.0001; ATG13KO, P = 0.9982. f, A universal model of selective autophagy (see text for details). Source numerical data and unprocessed blots are available in Source data.
