Panel A shows time lapse confocal microscopy images of reversible and irreversible mitochondrial depolarization after laser injury. Panel B shows fluorescence intensity line graphs of irreversibly depolarized mitochondria after large laser wounding. Panel C shows fluorescence intensity line graphs of recovered mitochondria near the laser wound region. Panel D shows confocal microscopy images of mitochondrial membrane recovery and persistent mitochondrial damage after localized injury. Panel E shows fluorescence intensity line graphs of irreversibly damaged single mitochondria following laser induced injury. Panel F shows fluorescence intensity line graphs of recovered single mitochondria after transient membrane depolarization. Panel G shows confocal microscopy images of mitochondrial depolarization after FCCP treatment without MAI one recruitment.
MAI-1 distinguishes damage from reversible membrane depolarization. (A) Time-lapse images showing MAI-1::GFP and TMRE after large laser wounding. MAI-1::GFP only translocated to irreversibly depolarized mitochondria (a1) but not recovered mitochondria (a2). The yellow arrowhead (a1) indicates irreversibly depolarized mitochondria. The white arrowhead (a2) indicates recovered mitochondria. Strains: Pcol-19-MAI-1::GFP (zjuSi510). Scale bar, 10 and 5 μm (zoom in). UW, unwounded; W, wounded. (B) Line graph showing the intensity change (ΔFw/Fuw) of MAI-1::GFP and TMRE (yellow arrowhead in panel D) in the large laser-wounded site at different time points after wounding (a1). Multiple mitochondria in an ROI (13.8 μm × 13.8 μm) at the wound site were measured. n represents the number of ROIs. Data are mean ± SD, n = 3. (C) Line graph showing the intensity change (ΔFw/Fuw) of MAI-1::GFP and TMRE (white arrowhead in panel D) near the large laser-wounded site at different time points after wounding (a2). Data are mean ± SD, n = 3. (D) Representative confocal time-lapse images of translocation of MAI-1::GFP and recovery of TMRE at the small laser wound site in 150 s. The yellow arrowhead indicates irreversibly depolarized mitochondria, and the white arrowhead indicates recovered mitochondria. Strains: Pcol-19-MAI-1::GFP (zjuSi510). Scale bar, 5 μm. (E) Line graph showing the intensity change (ΔFw/Fuw) of MAI-1::GFP and TMRE (yellow arrowhead in panel D) at different time points after wounding. A single fragmented mitochondrion was measured. Data are mean ± SD, n = 5. (F) Line graph showing the intensity change (ΔFw/Fuw) of MAI-1::GFP and TMRE (white arrowhead in panel D) at different time points after wounding. A single fragmented mitochondrion was measured. Data are mean ± SD, n = 3. (G) Representative confocal images of MAI-1::GFP after FCCP treatment (15 μM, 2 h). TMRE was abolished, but MAI-1 remains cytosolic. Strains: Pcol-19-MAI-1::GFP (zjuSi510). Scale bar: 10 and 5 μm (zoom in).