Fig 2: TAX1BP1 is dispensable for ferritin–NCOA4 condensate formation but required for its recognition for macroautophagy and endosomal microautophagy. (A) Colocalization of TAX1BP1 with ferritin condensates. WT and FIP200 KO HeLa cells expressing mGFP-TAX1BP1 and mRuby3-FTH1 were grown in DMEM and observed by fluorescence microscopy. Scale bars, 10 µm (main) and 1 µm (inset). (B) TAX1BP1 is dispensable for condensate formation. TAX1BP1 KO and FIP200 TAX1BP1 DKO HeLa cells expressing mGFP-FTH1 were observed as in A. Scale bars, 10 µm (main) and 1 µm (inset). (C) Ferritin–NCOA4 condensates are recognized by autophagosomes in a TAX1BP1-dependent manner. WT and TAX1BP1 KO HeLa cells expressing mGFP-FTH1 (green) and Halo-LC3 (red) were treated with 100 µg/ml FAC for 24 h followed by 50 µM DFO for 5.5 h and then observed by fluorescence microscopy. Scale bars, 5 µm (main) and 1 µm (inset). (D) The colocalization rate of mGFP-FTH1 puncta with Halo-LC3 in C was quantified (n = 1,130–2,716). Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed by Welch’s t test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (E) Ferritin–NCOA4 condensates are incorporated into endosomes in a TAX1BP1-dependent manner. mRuby3-RAB5Q79L was expressed by treatment with 2 µg/ml doxycycline for 48 h in WT and TAX1BP1 KO HeLa cells expressing mGFP-FTH1. Scale bars, 10 µm. (F) The rate of the endosomes containing mGFP-FTH1 puncta in E was quantified (n = 359–436). Solid bars indicate the means, and dots indicate the data from three independent experiments. Differences were statistically analyzed by Welch’s t test (two-tailed test). Data distribution was assumed to be normal, which was not formally tested. (G) A model of NCOA4-dependent formation of ferritin condensates and TAX1BP1-dependent recognition of condensates by macroautophagy and endosomal microautophagy.

Fig 3: Ferritin particles assemble to form liquid-like condensates. (A and B) Fluorescent images of the ferritin subunits FTH1 and FTL. WT and macroautophagy-deficient FIP200 KO HeLa cells stably expressing mGFP-FTH1 or mGFP-FTL were grown in DMEM (A) or treated with 10 µg/ml ferric ammonium citrate (FAC) for 24 h (B) and observed by fluorescence microscopy. Scale bars, 10 µm (main) and 1 µm (inset). (C) Immunoblots showing the expression levels of mGFP-FTH1 and mGFP-FTL in cells used in A. (D) TEM images of the ferritin condensates in FIP200 KO HeLa cells expressing GFP-FTH1. Scale bars, 500 nm (main) and 100 nm (magnified). (E) FRAP analyses of the ferritin condensates. WT HeLa cells expressing mGFP-FTH1 were treated with 100 µg/ml FAC for 24 h followed by 50 µM deferoxamine (DFO) for 5 h, and subjected to FRAP analyses. Scale bars, 10 µm (main) and 1 µm (magnified). (F) Quantification of E. Fluorescence intensities before photobleaching were set to 1. Representative results from two independent experiments are presented as means ± SEM (n = 5). (G) Coalescence of the ferritin condensates. WT HeLa cells expressing GFP-FTH1 were grown in DMEM and observed by time-lapse fluorescence microscopy at ~11-s intervals. Scale bars, 10 µm (main) and 1.5 µm (magnified). Source data are available for this figure: SourceData F1.

Fig 4: The ferritin–NCOA4 condensates are targeted by both macroautophagy and endosomal microautophagy. (A) Ferritin–NCOA4 condensates are engulfed by autophagosomes. WT HeLa cells expressing mGFP-FTH1 (green) and Halo-LC3 (red) were treated with 50 µg/ml FAC for 24 h followed by 50 µM DFO for 5 h and then observed by time-lapse fluorescence microscopy at 2-min intervals (see also Video 1). Arrows indicate part of ferritin condensates engulfed by an autophagosome. Scale bars, 5 µm (main) and 1 µm (magnified). (B) 3D-CLEM of cells treated as in A. Arrowheads indicate the surface of a ferritin–NCOA4 condensate exposed to the cytosol, and arrows indicate an elongating autophagosomal membrane. Scale bar, 500 nm. (C) The ferritin–NCOA4 condensates are incorporated into endosomes. WT HeLa cells expressing GFP-FTH1 or mGFP-NCOA4 were treated with 2 µg/ml doxycycline for 48 h to induce mRuby3-RAB5Q79L expression and then observed by time-lapse fluorescence microscopy at 4-s intervals under normal growing conditions (see also Videos 2, 3, and 4). Arrowheads indicate ferritin–NCOA4 condensates in the cytosol, and arrows indicate the ferritin–NCOA4 condensates in enlarged endosomes. Scale bars, 5 µm. (D and E) 3D-CLEM of WT HeLa cells expressing mGFP-FTH1 treated as in C. Correlative scanning electron microscopy (SEM) and fluorescent images are shown. An enlarged endosome containing mGFP-FTH1 puncta is magnified. Scale bars, 5 µm (main) and 1 µm (magnified; D). TEM images of the enlarged endosome indicated by arrows in SEM and fluorescent images are shown. Scale bars, 5 µm (SEM and fluorescent images), 1 µm (TEM), and 100 nm (magnified TEM; E). (F) NCOA4 KO HeLa cells expressing mGFP-FTH1 were examined as in C. Scale bar, 10 µm.

Fig 5: Ferritin forms liquid-like condensates with NCOA4. (A and D) WT HeLa cells expressing mGFP-FTH1 (A) or Halo-NCOA4 (D) were grown in DMEM, treated with 100 nM HaloTag SaraFluor 650T ligand (D), and then subjected to immunostaining with anti-FTL monoclonal antibody. Scale bars, 10 µm (main) and 1 µm (inset). (B and C) FRAP analyses of ferritin condensates. WT HeLa cells expressing mGFP-FTH1 (B) or mGFP-FTL (C) were treated with 100 µg/ml FAC for 24 h followed by 50 µM DFO for 2 h, and subjected to FRAP analyses (4-s intervals for 128 s). Three individual mGFP-FTL puncta are shown in C. Scale bars, 2 µm. (E) WT, NCOA4 KO, TAX1BP1 KO, FIP200 KO, FIP200 NCOA4 DKO, and FIP200 TAX1BP1 DKO HeLa cells were grown in DMEM. Whole-cell lysates were analyzed by immunoblotting with antibodies against NCOA4, TAX1BP1, FIP200, and HSP90. (F) WT, NCOA4 KO, and TAX1BP1 KO cells were grown in DMEM and treated with 10 µg/ml FAC for 24 h followed by 50 µM DFO for the indicated hours. Whole-cell lysates were analyzed by immunoblotting with antibodies against FTH1 and HSP90. Source data are available for this figure: SourceData FS1.