The mitophagy receptors BNIP3 and NIX mediate tight attachment and expansion of the isolation membrane to mitochondria

Abstract

BNIP3 and NIX are the main receptors for mitophagy, but their mechanisms of action remain elusive. Here, we used correlative light EM (CLEM) and electron tomography to reveal the tight attachment of isolation membranes (IMs) to mitochondrial protrusions, often connected with ER via thin tubular and/or linear structures. In BNIP3/NIX-double knockout (DKO) HeLa cells, the ULK1 complex and nascent IM formed on mitochondria, but the IM did not expand. Artificial tethering of LC3B to mitochondria induced mitophagy that was equally efficient in DKO cells and WT cells. BNIP3 and NIX accumulated at the segregated mitochondrial protrusions via binding with LC3 through their LIR motifs but did not require dimer formation. Finally, the average distance between the IM and the mitochondrial surface in receptor-mediated mitophagy was significantly smaller than that in ubiquitin-mediated mitophagy. Collectively, these results indicate that BNIP3 and NIX are required for the tight attachment and expansion of the IM along the mitochondrial surface during mitophagy.

Figure 1.

Morphology of receptor-mediated mitophagy revealed by CLEM. (A) Representative CLEM images (left) and enlarged EM images (right) of WT and DRP1 KO HeLa cells expressing mito-mCherry (Mt-mChe) and GFP-LC3B and treated with DFP for 16 h. Boxed areas in the main (upper) CLEM images are shown below as single-color images. Further enlargement of the boxed areas in the EM images are shown at the far right. Arrows, IM; asterisks, ER; mt, mitochondrion. Scale bars: 1 µm (CLEM), 500 nm (EM, middle), and 100 nm (EM, right). See Fig. S1 for an additional CLEM image of DRP1 KO HeLa cells. (B–F) Representative CLEM-FIB–scanning EM images of DRP1 KO HeLa cells expressing the same markers as in A. (B) Confocal image of a cell that was processed for FIB-scanning EM, with the boxed area enlarged and shown below. Arrows indicate the direction of milling. Scale bars: 10 μm (top) and 1 μm (bottom). (C) Serial EM images (intervals of 20 sections) taken from the bracketed region in B, represented in the x–z planes. An IM with high electron density (arrows) can be seen in section numbers 258, 278, and 298. Scale bar: 500 nm. (D) 3D image reconstruction from serial sections 209–408, illustrating a mitochondrion (mt, pink), ER (blue), and IM (green). (E) In views 1 and 2, the image from D is rotated as indicated by arrows, and the entire IM is shown with the ER (left) or IM-ER contact sites (right, red). See also Videos 1 and 2. (F) Two representative EM sections illustrating some of the contact sites in E (colored arrowheads). Scale bars: 500 nm.

Figure S1.

Morphology of receptor-mediated mitophagy revealed by CLEM (related to Fig. 1 A ). (A) Serial ultrathin sections of WT and DRP1 KO HeLa cells, with the grid and section numbers shown above each image. The framed images appear in Fig. 1 A. (B) CLEM (left) of another set of serial ultrathin sections of DRP1 KO HeLa cells showing co-localization of mito-mCherry (Mt-mChe, red) and GFP-LC3B (green). The boxed area is also displayed as single-color images for GFP-LC3B, Mt-mChe, and EM below the main image. Enlargements of the grid2 sec 3 EM image (also framed in the bottom panel) are shown in the middle and right panels. EM images of serial ultrathin sections are shown in the bottom row, with the grid and section numbers shown above each. Asterisks, ER; arrows, IM; mt, mitochondrion. Scale bars: 1 µm (CLEM) and 500 nm (EM).

Figure S2.

Analysis of the fine morphology of receptor-mediated mitophagy. Serial ultrathin sections of HeLa cells expressing mito-mCherry and GFP-LC3B, which were treated with DFP for 16 h and fixed with a mixture of aldehyde and osmium solutions. (A and B) Representative EM images of protruding (A) and slightly flattened (B) mitochondrial surfaces with tightly attached, electron-dense IMs (arrows). Red arrowheads indicate the frequently observed connections between ER (blue) and IM edges via thin linear/tubular structures. Boxed areas are magnified and shown in insets in A and below the main images in B. Section (sec) numbers are as indicated. Scale bars: 500 nm (lower magnification view) and 100 nm (higher magnification view).

Figure 2.

Thin tubular/linear structures connect the IM and ER in the late phase of receptor-mediated mitophagy. DRP1 KO HeLa cells were treated with DFP for 16 h and fixed with a mixture of aldehyde and osmium solutions for electron tomography. EM images of 102 serial sections (∼6-nm thickness) were reconstructed from two resin-embedded sections (300-nm thick each). (A–C) Representative EM image of section 79 (A) and its color-segmented 3D version (B) with ∼180° rotation (C), as indicated by the arrow. See also Video 3. (D and F) Rotated images of the boxed areas in B and C, respectively. (E and G) Representative EM images of the areas corresponding to D and F, respectively, in the indicated section(s) numbers. See also Videos 4 and 5. mt, mitochondria (pink); IM (green); TL, tubular/linear structures (cyan); ER (blue). Arrows in A, E, and G indicate IM. Colored arrowheads in D and F indicate tubular/linear structures, which correspond to the same-colored arrowheads in E and G, respectively. Scale bars: 200 nm (A) and 100 nm (D–G).

Figure 3.

Short linear structures connect the IM and ER in the early phase of receptor-mediated mitophagy. DRP1 KO HeLa cells were treated with DFP for 16 h and fixed with a mixture of aldehyde and osmium solutions for electron tomography. EM images of 115 serial sections (∼6.5-nm thickness) were reconstructed from three resin-embedded sections (300-nm thick each). (A–D) Representative EM image of section 51 (A) and its color-segmented 3D version (B), rotated ∼100° vertically, as indicated by the arrow (C and D). See also Video 6. (E–G) Enlargements of the boxed and labeled areas in D, captured from slightly different angles. (H and I) Serial images of two representative sites indicated in the boxed areas (left), arranged from left to right and labeled with the section(s) numbers. See also Videos 7 and 8. mt (pink), mitochondria; IM (green); L, linear structure (cyan); ER (blue); Go, Golgi complex (pale yellow). Arrows in A, H, and I indicate the IM. Colored arrowheads in E–G indicate linear structures, which correspond to the same-colored arrowheads in H and I. Scale bars: 200 nm (A) and 100 nm (E–I).

Figure S3.

Mitolysosome formation and recruitment of ULK1 and IM in BNIP3/NIX DKO cells. (A) Representative images of mt-Keima in WT, BNIP3/NIX (B/N) DKO, and FIP200 KO HeLa cells, with or without DFP treatment. In the merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (B) Quantification of the mitolysosomes in the cells shown in A. (C) Immunoblot analyses of BNIP3, NIX, BCL2L13, FKBP8, and FUNDC1 in WT and B/N DKO cells under DFP treatment. (D) Quantification of mitolysosomes in B/N DKO cells with re-expression of BNIP3 or NIX, or the empty vector control, with or without DFP induction. Data in B and D expressed as the means ± SEM of three independent experiments involving analysis of >200 cells per experiment; ***P < 0.001 and not significant (NS), determined by one-way ANOVA followed by a Tukey–Kramer post hoc test. (E) Serial ultrathin sections of WT and B/N DKO HeLa cells. Section (s) numbers are indicated at the top right corner of each image. The framed images are also displayed in Fig. 5 A. Scale bars: 500 nm. Source data are available for this figure: SourceData FS3.

Figure 4.

BNIP3/NIX are required for IM elongation and mitophagosome formation. (A) Immunofluorescence staining of Tom20 in GFP-ULK1–expressing WT, B/N DKO, and FIP200 KO HeLa cells following culture in medium containing DFP (DFP⁺) for 12 h. GFP-ULK1 puncta on mitochondria are indicated by arrows. Boxed areas are enlarged and shown below each main image. Scale bars: 10 μm (top) and 5 μm (bottom). (B) Quantification of the GFP-ULK1 puncta shown in A. Data are represented as the mean ± SEM (n = 3 biological replicates). More than 100 cells were analyzed in each replicate. (C and D) Time-lapse imaging of mito-mCherry (Mt-mChe) and GFP-LC3B in WT (C) and B/N DKO (D) cells cultured in DFP-containing medium. Nascent IM, elongated IM, and mitophagosomes are indicated by allows. Scale bars: 2 μm. (E) Quantification of the ratio of GFP-LC3B signals undergoing mitophagosome formation to the total signals shown in C and D. Data are represented as the mean ± SEM. More than 500 GFP-LC3B puncta in each cell type were analyzed. (F) Quantification of GFP-LC3B puncta on mitochondria shown in C and D for a duration of 30 min. Data are represented as the mean ± SEM (n = 30 cells). ***P < 0.001; NS, not significant by one-way ANOVA followed by Tukey–Kramer post hoc test (B) or Mann–Whitney U test (E and F).

Figure 5.

BNIP3/NIX are required for tight attachment of the IM to the mitochondrial surface. (A) CLEM images of MitoTracker Deep Red (Mito) and GFP-ULK1 fluorescence (left panels) and EM (right panels) of WT and B/N DKO cells expressing GFP-ULK1 and treated with DFP for 16 h. Boxed areas of EM images are enlarged and shown at the right. (B) Colored circles at the upper right corner outside each image correspond to the colors in the graph in B. mt, mitochondria; yellow arrow, IM; green arrowhead, IMAT; red arrowhead, IM-like structure; orange arrowhead, ER structure. Scale bars: 500 and 100 nm (EM, rightmost). (B) Distance between the OMM and IMs or nascent IMs in 10 profiles of WT and B/N DKO cells. EM profiles are classified into three patterns: IM attached to the mitochondrial surface (IM on mito, blue); IM not covering the mitochondrial surface (IM not covering mito, white); and IM-like structures or ER attached to the mitochondrial surface (nascent IM on mito, orange). Horizontal bars indicate the median; **P < 0.005 determined by Mann–Whitney U test. IMAT, IM-associated tubules.

Figure 6.

BNIP3/NIX are required for IM tethering but not for ULK1 recruitment. (A) Schematic representation of artificial tethering of ULK1 or LC3B to mitochondria in a chemically induced dimerization system involving treatment of cells with 500 nM rapalog for 24 h. (B and D) Immunofluorescence images of Atg13 and LC3 in WT and B/N DKO cells with or without artificial tethering of ULK1 (B) or LC3B (D) to mitochondria. Scale bars: 10 μm. (C and E) Quantification of LC3 puncta on mitochondria in the cells shown in B and D. More than 100 cells were analyzed in each group. (F and H) Representative images of mt-Keima in WT and B/N DKO cells with or without artificial tethering of ULK1 (F) or LC3B (H). In merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (G and I) Quantification of the mitolysosomes in the cells shown in F and H. Data are the averages of three independent experiments. More than 200 cells were analyzed in each experiment. Data are represented as the mean ± SEM. ***P < 0.001; NS, not significant by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (C and E) or one-way ANOVA followed by a Tukey–Kramer post hoc test (G and I).

Figure S4.

Effects of artificial tethering of the FKBP domain, interaction analysis of BNIP3/NIX with other autophagy factors, and serial ultrathin sections related to Fig. 7 G . (A and B) Representative immunofluorescence images of Atg13 and LC3 (A) and mt-Keima (B) in WT and B/N DKO cells, with or without artificial tethering of the FKBP domain to mitochondria. In merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (C) Quantification of the mitolysosomes in the cells shown in B. More than 200 cells were analyzed in each experiment. Data expressed as the means ± SEM of three independent experiments; ***P < 0.001 and not significant (NS), determined by one-way ANOVA followed by a Tukey–Kramer post hoc test. (D) Immunoprecipitation (IP) analysis of mCherry-BNIP3 or mCherry-NIX expressed in B/N DKO cells cultured with DFP for 12 h and then treated for an additional 12 h with 100 nM bafilomycin A1 and DFP. The cells were lysed, and IP was performed using RFP-trap magnetic agarose. Input is 10% of the IP fraction. (E) Serial ultrathin sections of the mitophagy profile. The framed image appears in Fig. 7 G. Section (s) numbers are indicated at the top right corner of each EM image. mt, mitochondrion; arrows, IM. Scale bars: 500 nm. Source data are available for this figure: SourceData FS4.

Figure 7.

BNIP3/NIX assemble at mitophagosome formation sites. (A and B) Immunofluorescence images of Tom20 (green) in WT HeLa cells expressing mCherry-BNIP3 (A, red) or mCherry-NIX (B, red). Scale bars: 10 μm. (C and D) Time-lapse imaging of mCherry-BNIP3 (C, red) or mCherry-NIX (D, red), GFP-LC3B (green), and MitoTracker Deep Red (blue) in HeLa cells. The cells were stained with MitoTracker Deep Red prior to culturing in DFP-containing DMEM. Mitophagosome formation sites labeled by GFP-LC3B are indicated by arrows. Bars, 2 μm. (E and F) Quantification of fluorescence intensity of mCherry-BNIP3 (E) or mCherry-NIX (F) at mitophagosome formation sites shown in C and D. Data are represented as the mean ratio of mCherry intensity at mitophagosome formation sites to that on tubular mitochondria (which are not targeted for mitophagy) ± SEM at each time point (n = 3 independent events). *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA followed by Dunnett’s post hoc test. (G) Representative CLEM (left) and EM (right) images of HeLa cells stably expressing mCherry-BNIP3 (mCh-BNIP3, red) and GFP-LC3B (G-LC3B, green) and cultured in DFP-containing medium for 16 h followed by staining with MitoTracker Deep Red (Mito, cyan) for 15 min. Single or overlapping images for mCh-BNIP3, G-LC3B, and mito are shown as indicated. The EM image is enlarged, with the boxed area magnified and shown as an inset. Arrowheads indicate a protruded mitochondrial region covered with IM (arrows). Fluorescence intensities of three signals along the white line in the CLEM images are plotted in the graph shown at the right. Scale bars, 500 and 100 nm (inset).

Figure S5.

BNIP3/NIX are accumulated in mitophagosome in an LIR-dependent manner (related to Fig. 8 ). (A and B) Immunofluorescence images of Tom20 (blue) and BNIP3 (A, red) or NIX (B, red) in GFP-LC3B (green)–expressing WT HeLa cells cultured in medium containing DFP for 12 h, followed by treatment with 100 nM bafilomycin A1 and DFP for an additional 12 h. Mitophagosomes (dots with positivity for both GFP-LC3B and Tom20) and tubular mitochondria are indicated by arrows and arrowheads, respectively. Scale bars: 10 μm (top) and 2 μm (bottom). (C) Quantification of the relative fluorescence intensities of BNIP3, NIX, and Tom20 within mitophagosomes, expressed as the mean ratio to the intensity on tubular mitochondria ± SEM (n = 10 cells, with >10 mitophagosomes analyzed per cell); ***P < 0.001, determined by Student’s t test. (D and E) Immunoblot analysis of WT or OMP25C hybrids of BNIP3 (D) and NIX (E) expressed in B/N DKO cells. (F) Quantification of the mitolysosomes in the B/N DKO cells expressing BNIP3/NIX variants. (G) Quantification of mitolysosomes in the B/N DKO cells expressing BNIP3/NIX serine mutants. Data in F and G represent the averages of three independent experiments involving analysis of >200 cells per experiment; ***P < 0.001, determined by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (F), or one-way ANOVA followed by Tukey–Kramer post hoc test (G). (H and I) Immunofluorescence images of serine mutants of mCherry-BNIP3 serine variants (H, red) or mCherry-NIX (I, red), GFP-LC3B (green), and Tom20 (blue), related to Fig. 8, G and H. Images were analyzed as in Fig. 8 A. Scale bars: 10 μm (top) and 2 μm (bottom). EV, empty vector. Source data are available for this figure: SourceData FS5.

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).

Figure 9.

Tight attachment of the IM to the mitochondrial surface. (A) EM images of CCCP-treated HeLa cells expressing mCherry-Parkin (mCh-Parkin HeLa) and WT HEK293 cells (HEK), DFP-treated WT and DRP1 KO HeLa cells, and rapalog-treated B/N DKO HeLa cells expressing FKBP-LC3B (FKBP-LC3B B/N DKO). Boxed areas are enlarged and shown below. The arrows indicate OMM and IM. mt, mitochondria. Scale bars: 200 nm (upper row) and 100 nm (lower row). (B) Box and whisker plot of the mean distance (open circles) between the OMM and the IM under CCCP-induced mitophagy (mCherry-Parkin HeLa, n = 16; HEK, n = 11), DFP-induced mitophagy (WT HeLa, n = 6; DRP1 KO HeLa, n = 10), and rapalog-induced mitophagy (FKBP-LC3B B/N DKO, n = 11); *P < 0.05, **P < 0.01, ****P < 0.0001, determined by a Tukey–Kramer test. (C) Model of receptor-mediated mitophagy. At the initiation of mitophagy, ULK1 complex and nascent IM are recruited to the mitochondrial surface independently of BNIP3/NIX (B/N). The IM is tethered to the mitochondrial surface, where it elongates and ultimately forms a mitophagosome, depending on B/N. During elongation of the IM, B/N accumulate, promoting tight attachment of the IM to the mitochondria, while ER contacts the IM rim through linear structures.