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Single cell analysis of TAX1BP1 translocation to mitochondria after CCCP- mediated depolarization. ( A ) Representative immunofluorescence images showing TAX1BP1 (yellow), parkin (magenta) and SSBP1 (cyan) after mitochondria depolarization in 5KO HeLa cells. Solid arrowheads indicate TAX1BP1 structures co-localizing with mitochondrial material. Arrows indicate TAX1BP1 structures not engaged with mitochondrial material. ROI 1 represents > 50 % of cell population while ROI 2 represents < 50% of cases. Scale bars: 10 µm. Zoom insets for WT (i), TAX1BP1 K549R (ii), TAX1BPΔCC3 (iii and iv) are shown. ( B ) Mean mitochondrial size of SSBP1 foci after 4h CCCP. ( C-D ) Normalized SSBP1 (C) area or (D) perimeter per cell. ( E ) Correlation analysis of the percentage of mitochondrial area recognized by TAX1BP1 (X axis) in relation to the percentage of total TAX1BP1 found on mitochondria (Y axis). Significance p-values are indicated in the graph for the indicated TAX1BP1 constructs. ( F ) Left panel: quantitative analysis of WT, TAX1BP1 K549R or TAX1BP1ΔCC3 localization after mitochondria depolarization in 5KO-PRKN cells after mitophagy induction (4h CCCP). TAX1BP1 localization is classified between cells where <t>COX4</t> clumps are fully recognized by TAX1BP1 (complete apposition) and cells where COX4 clumps recognition is limited (localized apposition). Bars: mean ± SEM. Right panel: representative immunofluorescence images stained for TAX1BP1 (yellow) and COX4 (magenta) are shown for each category.
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Single cell analysis of TAX1BP1 translocation to mitochondria after CCCP- mediated depolarization. ( A ) Representative immunofluorescence images showing TAX1BP1 (yellow), parkin (magenta) and SSBP1 (cyan) after mitochondria depolarization in 5KO HeLa cells. Solid arrowheads indicate TAX1BP1 structures co-localizing with mitochondrial material. Arrows indicate TAX1BP1 structures not engaged with mitochondrial material. ROI 1 represents > 50 % of cell population while ROI 2 represents < 50% of cases. Scale bars: 10 µm. Zoom insets for WT (i), TAX1BP1 K549R (ii), TAX1BPΔCC3 (iii and iv) are shown. ( B ) Mean mitochondrial size of SSBP1 foci after 4h CCCP. ( C-D ) Normalized SSBP1 (C) area or (D) perimeter per cell. ( E ) Correlation analysis of the percentage of mitochondrial area recognized by TAX1BP1 (X axis) in relation to the percentage of total TAX1BP1 found on mitochondria (Y axis). Significance p-values are indicated in the graph for the indicated TAX1BP1 constructs. ( F ) Left panel: quantitative analysis of WT, TAX1BP1 K549R or TAX1BP1ΔCC3 localization after mitochondria depolarization in 5KO-PRKN cells after mitophagy induction (4h CCCP). TAX1BP1 localization is classified between cells where COX4 clumps are fully recognized by TAX1BP1 (complete apposition) and cells where COX4 clumps recognition is limited (localized apposition). Bars: mean ± SEM. Right panel: representative immunofluorescence images stained for TAX1BP1 (yellow) and COX4 (magenta) are shown for each category.

Journal: bioRxiv

Article Title: Parkin-dependent ubiquitination of TAX1BP1 directs efficient autophagic removal of defective mitochondria

doi: 10.1101/2025.05.16.654474

Figure Lengend Snippet: Single cell analysis of TAX1BP1 translocation to mitochondria after CCCP- mediated depolarization. ( A ) Representative immunofluorescence images showing TAX1BP1 (yellow), parkin (magenta) and SSBP1 (cyan) after mitochondria depolarization in 5KO HeLa cells. Solid arrowheads indicate TAX1BP1 structures co-localizing with mitochondrial material. Arrows indicate TAX1BP1 structures not engaged with mitochondrial material. ROI 1 represents > 50 % of cell population while ROI 2 represents < 50% of cases. Scale bars: 10 µm. Zoom insets for WT (i), TAX1BP1 K549R (ii), TAX1BPΔCC3 (iii and iv) are shown. ( B ) Mean mitochondrial size of SSBP1 foci after 4h CCCP. ( C-D ) Normalized SSBP1 (C) area or (D) perimeter per cell. ( E ) Correlation analysis of the percentage of mitochondrial area recognized by TAX1BP1 (X axis) in relation to the percentage of total TAX1BP1 found on mitochondria (Y axis). Significance p-values are indicated in the graph for the indicated TAX1BP1 constructs. ( F ) Left panel: quantitative analysis of WT, TAX1BP1 K549R or TAX1BP1ΔCC3 localization after mitochondria depolarization in 5KO-PRKN cells after mitophagy induction (4h CCCP). TAX1BP1 localization is classified between cells where COX4 clumps are fully recognized by TAX1BP1 (complete apposition) and cells where COX4 clumps recognition is limited (localized apposition). Bars: mean ± SEM. Right panel: representative immunofluorescence images stained for TAX1BP1 (yellow) and COX4 (magenta) are shown for each category.

Article Snippet: The following primary antibodies were used: CD63(1:250, Antibodies online, ABIN94214), COX4 (1:10000, Cell signaling, 4850), HIS-tag (1:500, Addgene, 184180), HSP60 (1:1000, Novus Biologicals, NBP3-05536), LC3B (1:500, Cell signaling, 835065), Parkin(1:10000, Cell signaling, 4211S), RAB7 (1:200, Cell signaling, 95746), TAX1BP1 (1:500, Novus Biologicals, NBP1-86662), VPS35 (1:250, Novus Biologicals, NB100-1397).

Techniques: Single-cell Analysis, Translocation Assay, Immunofluorescence, Construct, Staining