Fig 2: Components of cell-surface binder display-based MitoCatch-C.a. Schematic diagram of a construct designed to display nanobodies on the surface of a cell (top) and super-resolution image of a HEK293T cell expressing the cell-surface anti-GFP nanobody (bottom). Nanobodies are identified by anti-OLLAS antibodies (magenta), and cell nuclei are labelled with Hoechst (blue). Igκ SP, immunoglobulin kappa light chain signal peptide; TM, transmembrane domain; 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Schematic diagram of a construct designed to target GFP to the outer membrane of mitochondria (top) and super-resolution image of a HEK293T cell expressing GFP at the outer membrane of mitochondria (bottom, green). Matrix of mitochondria is labelled with dsRed2 protein fused to COX8 signal peptide (red) and MitoTracker Deep Red (magenta). The construct was validated in at least three independent experiments. c. Western blotting on different subcellular fractions. Depletion of endoplasmic reticulum proteins in the isolated mitochondrial fraction was assessed with anti-GRP78, anti-PDIA3, and anti-KDELR1 antibodies. Enrichment of mitochondrial proteins in the mitochondrial fraction was assessed with anti-MCU and anti-TOMM20 antibodies in three independent mitochondrial preparations. For gel source data, see Supplementary Fig. 1. d. Western blotting on isolated mitochondria to assess the presence of mitochondrial proteins from matrix, intermembrane space, inner and outer membranes validated with an antibody cocktail consisting of anti-ATP5a, anti-UQCRC1 (Complex III Core Protein I), anti-VDAC1, anti-Cyclophilin D, and anti-Cytochrome c antibodies in three independent mitochondrial preparations. For gel source data, see Supplementary Fig. 1. e. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of isolated mitochondria from HEK293T cells. At least three independent mitochondrial preparations were used for integrity validations using the EM techniques. f. Oxygen consumption rate (OCR) of isolated mitochondria with and without substrates (10 mM succinate and 10 mM ADP). For all conditions, 2 µM rotenone is added to inhibit Complex I. Dashed line illustrates the time point when 4 µM Antimycin A is added. n = 3, Succinate + ADP vs. Succinate + ADP + Antimycin A: P = 0.0032, Succinate + ADP vs. No substrates: P = 0.014, Succinate + ADP + Antimycin A vs. No substrates: P = 0.85, Welch’s ANOVA test with two-sided Dunnett’s multiple comparisons test. g. Binding of anti-GFP and anti-mCherry nanobodies to GFP, mCherry, dsRed2, and tdTomato proteins assayed by enzyme-linked immunosorbent assay. OD490, optical density (OD) values measured at 490 nm. n = 3, each ‘n’ refers to a technical replicate. The assay was repeated two times independently. h. Histograms displaying the distribution of mean GFP fluorescence in nanobody-positive and nanobody-negative cells. Threshold for significant enrichment is shown with a black dashed line. Numbers indicate the percentage of cells that pass the threshold. P < 0.0001, Kolmogorov-Smirnov test. i. HEK293 cells expressing either a control nanobody (top) or the anti-GFP nanobody (bottom) on the cell surface after two hours of mito-GFP transplantation. Nanobody-positive cells are identified by anti-OLLAS antibodies (magenta). Transplanted mito-GFP are stained with anti-GFP antibodies (green). Nanobody-positive cells are outlined with grey dashed lines. j. Quantification of the efficacy of mito-GFP delivery in HEK293T cells two hours after transplantation. Percentage of GFP-positive cells among all nanobody-positive and the mean GFP fluorescence ratio between nanobody-positive cells and nanobody-negative cells. n = 7, P < 0.0001 (top) and P = 0.0014 (bottom), two-sided Welch’s t test. k. Co-localization of GFP from transplanted mito-GFP and TOMM20 among total mitochondria in HEK293T cells. Mito-GFP are stained with anti-GFP antibodies (green); anti-GFP nanobody is identified by anti-OLLAS antibodies (magenta); TOMM20 is identified by anti-TOMM20 antibodies (red); nuclei labelled with Hoechst (blue). Anti-GFP nanobody-positive cells are outlined with white dashed lines. l. Quantification of the co-localization of GFP and TOMM20 in (k). Donor mitochondria-positive pixels (GFP channel) were spatially randomized within a cell region to compute unspecific co-localization values. n = 10, P < 0.0001, two-sided paired t test. m. Histograms displaying the distribution of mean GFP fluorescence in tdTomato-positive and tdTomato-negative cells. Threshold for significant enrichment is shown with a black dashed line. Numbers indicate the percentage of cells that pass the threshold. P < 0.3766, two-sided Kolmogorov-Smirnov test. n. HEK293 cells expressing cytosolic tdTomato after two hours of mito-GFP transplantation. TdTomato-positive cells are identified by anti-RFP antibody immunostaining (red). Transplanted mito-GFP are stained with anti-GFP antibodies (green). Brightfield image is shown in grey. o. Quantification of the efficacy of mito-GFP delivery in (n). Percentage of GFP-positive cells among all tdTomato-positive cells (top, n = 6) and the mean GFP fluorescence ratio between tdTomato-positive cells and tdTomato-negative cells (bottom, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Data, mean ± s.e.m. Scale bars, 5 µm (a, i), 1 µm (b, e), 10 µm (k), 50 µm (n). Source data