Journal: Nature
Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration
doi: 10.1038/s41586-026-10391-0
Figure Lengend Snippet: a. Top, schematic diagram of the construct used for directing a nanobody to the outer membrane of mitochondria. Bottom, a super-resolution image of an HEK293T cell with mitochondria-targeted nanobody detected by anti-alpacaV H H antibodies (magenta). Cell nuclei are labelled with Hoechst (blue). Mitochondria matrix is labelled with dsRed2 (cyan). 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Top, schematic diagram of the construct used for directing GFP to the cell surface. Bottom, a super-resolution image of an HEK293T cell with cell surface-targeted GFP detected by anti-GFP antibodies (green). Cell nuclei are labelled with Hoechst (blue). The construct was validated in at least three independent experiments. c. HEK239T cells transplanted with donor mitochondria displaying the anti-GFP nanobody, two hours after transplantation. HEK293T cells are transfected with cell-surface mCherry (cyan) or GFP (green). Nanobodies are detected by anti-alpacaV H H antibodies (magenta). d. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria two hours after transplantation. n = 6, P < 0.0001 (top) and P = 0.0012 (bottom), two-sided Welch’s t test. e. HEK239T cells transplanted with donor mitochondria displaying the anti-mCherry nanobody, two hours after transplantation. HEK293T cells were transfected with cell-surface GFP (green) or mCherry (cyan). Nanobodies detected by anti-alpacaV H H antibodies (magenta). f. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria, two hours after transplantation. n = 8, P = 0.0003 (top) and P = 0.0012 (bottom), two-sided Welch’s test. g. Live-imaged endothelial cells expressing cell surface GFP and endosome-targeted RAB5A-TagRFP with (top) and without (bottom) transplanted with mito-mTagBFP2 (cyan) displaying anti-GFP nanobody, six hours after mitochondrial transplantation. White arrows, endosome-free donor mitochondria (confirmed in at least three independent experiments). For an example of mito-mTagBP2 in endothelial cells transplanted without the anti-GFP nanobody, see Supplementary Fig. . h. Donor mito-mTagBFP2 (cyan) inside endocytic vesicles (red arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. i. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. j. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta), 24 h after mitochondrial transplantation. Outlined region, mito-mTagBFP2 free from endocytic vesicles and lysosomes (yellow). Lysosomes are stained with LysoTracker Deep Red dye. The experiment was repeated at least three times with similar results. k. Quantification of proportion of endosome-free mitochondria by pixel-based co-localization analysis. Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P = 0.0756, two-sided Mann-Whitney U test. l. Quantification of abundance of endosome-free mitochondria by pixel-based co-localization analysis. The values were normalized to cell size (µm 2 of donor mitochondria area per 1 µm 2 cell area). Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P < 0.0001, two-sided Mann-Whitney U test. m. Endothelial cells stained with pH-dependent lysosome staining dye pHLys Red (yellow). Cells expressed cell surface GFP and were transplanted with mito-mTagBFP2 displaying anti-GFP nanobody or no binder. In addition, Bafilomycin A1 was used as a positive control for pH acidification change in lysosomes. For mitochondria transplanted conditions, images of cells positive for mito-mTagBFP2 are shown (Supplementary Fig. ). n. Quantification of pH changes in lysosomes relative to untreated condition. Untreated: n = 6; Bafilomycin A1: n = 4; mito-mTagBFP2: n = 4; mito-mTagBFP2 + anti-GFP nanobody: n = 6, Untreated vs. Bafilomycin A1: P = 0.0036, Untreated vs. mito-mTagBFP2: P = 0.8235, Untreated vs. mito-mTagBFP2 + anti-GFP nanobody: P = 0.9648, Welch’s ANOVA test corrected with two-sided Dunnett’s test for multiple comparisons. o. Live-imaged endothelial cell expressing cell surface GFP (green), and transplanted with mito-dsRed2 (cyan) displaying anti-GFP nanobody, four days after mitochondrial transplantation. The cell is outlined with a grey dashed line. The zoomed-in region is outlined with a white dashed square. Two timeframes are shown on the right. The tracked mitochondrion is indicated with a red arrow. The experiment was repeated at least three times with similar results. p. Live-imaged endothelial cell expressing cell surface GFP and transplanted with mito-dsRed2 (cyan) displaying outer membrane anti-GFP nanobody, four days after mitochondrial transplantation. Mitochondria are labelled with 50 nM MitoTracker Deep Red (magenta). The zoomed-in region is outlined with a white dashed square and the tracked mitochondrion is indicated with a white arrow. The experiment was repeated at least three times with similar results. q. Labelling of donor and native mitochondria with MitoTracker Deep Red dye in live-recorded endothelial cells. At the used concentration, the dye stained both native (black) and donor mitochondria (cyan) with stronger enrichment in the native mitochondria. Donor mitochondria positive for matrix-labelled dsRed2 and MitoTracker Deep Red are indicated with red arrows. NS not significant, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m and median for k, l. Scale bars, 2.5 µm (a, b), 25 µm (c, e), 5 µm (g, h, i, o, p, q), 10 µm (j), 20 µm (m).
Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.
Techniques: Construct, Membrane, Microscopy, Transplantation Assay, Transfection, Expressing, Staining, MANN-WHITNEY, Positive Control, Concentration Assay
