Figure 5.
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.

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.

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