Figure 8.
BNIP3/NIX assemble at mitophagosome formation sites in an LIR-dependent manner. (A) Schematic representation of BNIP3/NIX mutant constructs, showing the LIR and TM domains in the WT control, LIRm, and dimerization-deficient OMP25C mutant proteins. (B and C) Immunofluorescence images of mCherry-BNIP3 variants (B, red) and mCherry-NIX variants (C, red), along with GFP-LC3B (green) and Tom20 (blue) in HeLa cells cultured in DFP-containing medium for 12 h, followed by treatment with 100 nM bafilomycin A1 and DFP for an additional 12 h. Mitophagosomes and tubular mitochondria are indicated by arrows and arrowheads, respectively. Scale bars: 10 μm (top) and 2 μm (bottom). (D and E) Quantification of the relative fluorescence intensity of mCherry-BNIP3 or mCherry-NIX variants within mitophagosomes, represented as the mean ratio to that on tubular mitochondria ± SEM (n = 10 cells). More than 10 mitophagosomes were analyzed in each cell. (F) Serine residues (arrows) located in the vicinity of the LIRs (underlined) in BNIP3/NIX. (G and H) Quantification of the relative fluorescence intensity of mCherry-BNIP3 or mCherry-NIX serine mutants within mitophagosomes, as described above. Data are represented as the mean ± SEM (n = 6 cells). More than 10 mitophagosomes were analyzed in each cell. *P < 0.05; ***P < 0.001; NS, not significant by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (D, E, G, and H).

BNIP3/NIX assemble at mitophagosome formation sites in an LIR-dependent manner. (A) Schematic representation of BNIP3/NIX mutant constructs, showing the LIR and TM domains in the WT control, LIRm, and dimerization-deficient OMP25C mutant proteins. (B and C) Immunofluorescence images of mCherry-BNIP3 variants (B, red) and mCherry-NIX variants (C, red), along with GFP-LC3B (green) and Tom20 (blue) in HeLa cells cultured in DFP-containing medium for 12 h, followed by treatment with 100 nM bafilomycin A1 and DFP for an additional 12 h. Mitophagosomes and tubular mitochondria are indicated by arrows and arrowheads, respectively. Scale bars: 10 μm (top) and 2 μm (bottom). (D and E) Quantification of the relative fluorescence intensity of mCherry-BNIP3 or mCherry-NIX variants within mitophagosomes, represented as the mean ratio to that on tubular mitochondria ± SEM (n = 10 cells). More than 10 mitophagosomes were analyzed in each cell. (F) Serine residues (arrows) located in the vicinity of the LIRs (underlined) in BNIP3/NIX. (G and H) Quantification of the relative fluorescence intensity of mCherry-BNIP3 or mCherry-NIX serine mutants within mitophagosomes, as described above. Data are represented as the mean ± SEM (n = 6 cells). More than 10 mitophagosomes were analyzed in each cell. *P < 0.05; ***P < 0.001; NS, not significant by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (D, E, G, and H).

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