Panel A shows confocal microscopy images of mitochondrial membrane damage after laser wounding. Panel B shows time lapse confocal microscopy images of TOMM twenty redistribution following injury. Panel C shows confocal microscopy images and fluorescence quantification after mitochondrial matrix leakage. Panel D shows diffuse GFP microscopy images with fluorescence intensity measurements after wounding. Panel E shows time lapse confocal microscopy images of ATP three redistribution after injury. Panel F shows spinning disk confocal microscopy images of disrupted mitochondrial membrane protein organization. Panel G shows confocal microscopy images of mitochondrial clearance at progressive post injury stages.
Laser wounding triggers mitochondrial membrane damage and clearances. (A) Representative confocal image showing mito::GFP, TMRE, TOMM-20::GFP, and ATP-3::GFP before and after wounding. Wounding triggers loss of mitochondrial matrix and membrane potential (ΔΨm). Pcol-19-mito::GFP (zjuSi197), Pcol-19-TOMM-20::GFP (zjuSi42), and ATP-3::GFP (KI, zju133) animals were used for imaging and wounding. Scale bar, 10 μm. (B) Representative time-lapse confocal images of TOMM-20::GFP before and after laser wounding. Strain: Pcol-19-TOMM-20::GFP(zjuSi42). Scale bar, 10 µm. (C) Left, representative confocal image of mito::GFP before and 50 ms after wounding. Strains: Pcol-19-mito::GFP (zjuSi197). Scale bar, 10 μm. Right, quantification of relative cytosolic fluorescence intensity before and after wounding. Fluorescence was measured from ten cytosolic ROIs (0.476 μm × 0.476 μm) surrounding mitochondria at the wound site and at distal regions per animal. Statistical analysis was performed using the Mann–Whitney test. Data are mean ± SD (n = 7 animals per group); ***, P < 0.001. (D) Left, representative confocal image showing diffuse GFP before and 200 ms after wounding. Strains: Pcol-19::GFP (zjuSi60). Scale bar, 10 μm. Right, quantification of relative GFP intensity before and after wounding. Fluorescence was measured from 10 cytosolic ROI (0.476 μm × 0.476 μm) surrounding mitochondria at the wound site and at distal regions per animal. Statistical analysis was performed using the Mann–Whitney test. Data are mean ± SD (n = 6 animals per group); ***, P < 0.001. (E) Representative time-lapse confocal images of ATP-3::GFP before and after laser wounding. Strain: ATP-3::GFP (KI, zju133). Scale bar, 10 µm. (F) Representative spinning-disk confocal images of ATP-3::GFP and TOMM-20::mYonghong before and after laser wounding. Strains: ATP-3::GFP(zju133); Pcol-19-TOMM-20::mYonghong (zjuSi577). Scale bar, 10 and 5 µm (zoom in). (G) Representative confocal images of TOMM-20::mKate before and at different time points after laser wounding. Strain: Phyp-7-TOMM-20::mKate2 (zjuSi126). Scale bar, 10 µm. (A–G) The white dashed circle indicates the wound site. UW, unwounded; W, wounded.