mice  (Addgene inc)

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: Donor mito-mTagBFP2 can be free from endocytic vesicles. HUVECs expressing cell surface GFP and endosome-targeted Rab5a-TagRFP (magenta) transplanted with mito-mTagBFP2 displaying anti-GFP nanobody (cyan). The videos were recorded 6 h and 1 day after mitochondrial transplantation, respectively. Two different cells are shown. Scale bar, 5 mm.

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:

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

Journal: Cell Reports Methods

Article Title: MitoTracker transfers from astrocytes to neurons independently of mitochondria

doi: 10.1016/j.crmeth.2026.101338

Figure Lengend Snippet: Image acquisition immediately after astrocytes expressing GFP-OMM (green) and labeled with MitoTracker dye (magenta) are co-cultured with neurons expressing mito-mTagBFP2 (blue).

Article Snippet: Sequences can be found in Methods S1. pAAV2/1 was a gift from James M. Wilson (Addgene plasmid #112862; http://n2t.net/addgene:112862; RRID:Addgene_112862). pCAG mito-mTagBFP2 was a gift from Franck Polleux (Addgene plasmid #105011; http://n2t.net/addgene:105011; RRID:Addgene_105011).

Techniques:

Journal: Cell Reports Methods

Article Title: MitoTracker transfers from astrocytes to neurons independently of mitochondria

doi: 10.1016/j.crmeth.2026.101338

Figure Lengend Snippet: Neurons expressing mTagBFP2 (blue) are incubated with ACM from astrocytes expressing GFP-OMM (green) and labeled with MitoTracker dye (magenta).

Article Snippet: Sequences can be found in Methods S1. pAAV2/1 was a gift from James M. Wilson (Addgene plasmid #112862; http://n2t.net/addgene:112862; RRID:Addgene_112862). pCAG mito-mTagBFP2 was a gift from Franck Polleux (Addgene plasmid #105011; http://n2t.net/addgene:105011; RRID:Addgene_105011).

Techniques:

Journal: Cell Reports Methods

Article Title: MitoTracker transfers from astrocytes to neurons independently of mitochondria

doi: 10.1016/j.crmeth.2026.101338

Figure Lengend Snippet: MitoTracker, but not mitochondria, transfers rapidly to neurons from astrocytes (A) Mitochondria are dual labeled with outer mitochondrial membrane targeted GFP (GFP-OMM) and the MitoTracker dye. (B) Schematic diagram outlining the protocol for live co-culture experiments. Astrocytes are additionally labeled with CTDR (CellTracker Deep red) and neurons/neuronal mitochondria are identified by mTagBFP2 and mito-mTagBFP2 expression, respectively. (C) Timelapse images of a dual-labeled astrocyte (central) immediately following co-culture with neurons. ( n = 3 biological repeats, 4–5 positions of interest per repeat). (D) An orthogonal view of co-cultures following timelapse acquisition (>30 min). (E) Representative image of dual-labeled astrocytes co-cultured with mito-mTagBFP2 expressing neurons for >30 min. The yellow dashed line represents the astrocyte boundary. Insert highlights that MitoTracker but not GFP-OMM labels neuronal mitochondria. See also .

Article Snippet: Sequences can be found in Methods S1. pAAV2/1 was a gift from James M. Wilson (Addgene plasmid #112862; http://n2t.net/addgene:112862; RRID:Addgene_112862). pCAG mito-mTagBFP2 was a gift from Franck Polleux (Addgene plasmid #105011; http://n2t.net/addgene:105011; RRID:Addgene_105011).

Techniques: Labeling, Membrane, Co-Culture Assay, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Reliable quantification of multiplexed genetically encoded biosensors responsiveness in plant tissues

doi: 10.64898/2026.03.13.711581

Figure Lengend Snippet: mTagBFP2, mTurquoise2, Venus, mKO2, mCherry, mKate2, and mCardinal exhibit approximately comparable brightness across different excitation conditions. All fluorescent proteins were fused to a nuclear localisation signal. (a) Cumulative fluorescence intensity (RFU) of transiently expressed proteins in N. benthamiana, measured using standard laser lines on the Leica Stellaris 5 microscope. (b) The same measurement performed using the 405 nm or a white light laser on Stellaris 8, with excitation wavelengths set just below each protein’s emission peak. Fluorescent proteins are indicated below each bar chart, while laser wavelengths and power settings are shown above. Detector gain was kept at the minimum setting for all fluorescent proteins. For each condition, at least two independent images were analysed. Mean fluorescence intensity per nucleus was plotted as bar charts with overlaid points (each dot represents one nucleus). (c, e) Expected fluorescence intensity based on FPbase.org data (excitation × brightness), shown for (c) Stellaris 5 and (e) Stellaris 8, calculated for (a) and (d) standard laser lines, and (b) and (f) white light laser excitation. Among the tested fluorescent proteins, Venus, mKO2 and mKate2 were selected for stable transformation in potato (highlighted in yellow across all charts). (d, f) Brightness of fluorescent proteins measured in S. tuberosum (potato) leaves (d) using standard laser lines or (f) the white light laser, as described in panels (a‒b). Brightness is shown for one transgenic line per fluorescent protein. Analysis of a second transgenic line gave similar results (results available on Zenodo: 10.5281/zenodo.17600476).

Article Snippet: N7 localization signal together with mTurquoise2, Venus, and mKate2 (plasmids pHG128, pHG132, and pHG154, respectively) were kindly provided by Hassan Ghareeb ( ). mTagBFP2 (plasmid pJL1-mTagBFP2; Addgene #102638; ( )), mKO2 (plasmid pTU-A-005; Addgene #124412; ( )), mCardinal (plasmid mCardinal-pBAD; Addgene #54800; ( )), and miRFP713 (plasmid pmiRFP713-N1; Addgene #136559; ( )) were obtained from Addgene.

Techniques: Fluorescence, Microscopy, Transformation Assay, Transgenic Assay