Fig. 6: FIP200 colocalizes with initiation hubs on mitochondria and p62 condensates.

a, Schematic of the synthetic tethering setup in U2OS cells. FRB is targeted to the outer mitochondrial membrane by its fusion with the tail anchor domain of the mitochondrial membrane protein FIS1 (FRB–FIS1^93–152, residues 93–152 of FIS1). Expression of an FKBP–GFP–ULK1 fusion construct allows its inducible tethering to mitochondrial FRB–FIS1^93–152 by rapalogue addition, resulting in mitophagy induction. b, U2OS cells stably expressing FRB–FIS1^93–152 and transfected with FKBP–GFP–ULK1 or FKBP–GFP were cultured in nutrient-rich medium and stained with MitoTracker DeepRed. Tethering was induced with rapalogue. Scale bar, 10 µm, scale bar inset, 1 µm. Quantification: GFP foci on mitochondria. Data are mean values (n > 80 cells per condition across replicates, three biological replicates). Circles show mean values of each replicate, bars show mean. Statistical analysis was carried out by a one-tailed unpaired t-test. ****P < 0.0001. c, U2OS cells stably expressing FRB–FIS1^93–152 and transfected with FKBP–GFP–ULK1 were cultured in nutrient-rich medium and stained with MitoTracker DeepRed. Tethering of FKBP–GFP–ULK1 to FRB–FIS^93–152 was induced with rapalogue. Scale bar, 10 µm; scale bar inset, 1 µm. Quantification: GFP foci on mitochondria. Data are mean values (n > 100 cells per condition across replicates, three biological replicates). Circles show mean 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: 0 h versus 1 h, ****P < 0.0001, 1 h versus 2 h, ****P < 0.0001. d, U2OS cells stably expressing FRB–FIS1^93–152, transfected with FKBP–GFP–ULK1 and mScarlet–FIP200 were cultured in nutrient-rich medium. Tethering of FKBP–GFP–ULK1 to FRB–FIS^93–152 was induced by adding rapalogue for 1 h. Scale bar, 10 µm, scale bar inset, 1 µm. Quantification: mScarlet–FIP200 foci co-localizing with FKBP–GFP–ULK1 foci. Data are mean values (n > 100 cells per condition across replicates, three biological replicates). Circles show mean values of each replicate, bars show mean. Statistical analysis was carried out by a one-tailed unpaired t-test. ****P < 0.0001. e, U2OS cells transfected with FKBP–GFP or FKBP–GFP–FIP200 and mCherry–p62 were cultured in nutrient-rich medium. Scale bar, 10 µm; scale bar inset, 1 µm. Quantification: cells with GFP foci on p62 condensates. Data are mean values (n > 100 cells per condition across replicates, three biological replicates). Circles show mean values of each replicate, bars show the mean. Statistical analysis was conducted by a one-tailed unpaired t-test. ****P < 0.0001. Source numerical data are available in Source data.