Panel A shows sequence alignment of mitochondrial proteins from nematodes and humans. Panel B shows time lapse confocal microscopy images of mitochondrial apoptosis inducing factor localization after injury. Panel C shows merged confocal microscopy images of mitochondrial apoptosis inducing factor and tetramethylrhodamine ethyl ester signals. Panel D shows fluorescence microscopy images with quantification of mitochondrial apoptosis inducing factor intensity changes. Panel E shows time lapse confocal microscopy images of mitochondrial apoptosis inducing factor and mitochondrial membrane proteins. Panel F shows merged microscopy images with Pearson correlation coefficient analysis of mitochondrial protein colocalization. Panel G shows merged microscopy images and Pearson correlation coefficient analysis of mitochondrial membrane association.
Discovery of a genetically encoded mitochondrial damage reporter. (A) Amino acid sequences alignment of C. elegans MAI-1, MAI-2, and human ATP5IF1. (B) Representative time-lapse confocal images of MAI-1::GFP before and at different time points (1–60 min) after laser wounding. Strain: Pcol-19-MAI-1::GFP(zjuSi510). Scale bar, 10 µm. (C) Representative time-lapse confocal images of MAI-1::GFP;TMRE before and after laser wounding. Strains: Pcol-19-MAI-1::GFP(zjuSi510). Scale bar, 10 and 5 μm (zoom in). (D) Left, representative confocal images of MAI-1::GFP before and 200 ms after laser wounding (first frame taken after laser wounding). Frame rate: 200 ms; exposure time: 100 ms. Strain: Pcol-19-MAI-1::GFP(zjuSi510). Scale bar, 10 µm. Right, quantitation of absolute MAI-1::GFP intensity before and after wounding to the left images. Multiple mitochondria within an ROI (6.9 μm × 6.9 μm) at the wound site were measured. n represents the number of ROI analyzed. Statistical analysis was performed using the Mann–Whitney test. Data are mean ± SD (n = 45 and 45, respectively); ***, P < 0.001. (E) Representative time-lapse confocal images of MAI-1::GFP; TOMM-20::mKate before and at different time points after laser wounding. Strains: Phyp-7-TOMM-20::mKate2(zjuSi126); Pcol-19-MAI-1::GFP(zjuSi510). Scale bar, 10 µm. (B–E) The white dashed circle indicates the wound site. UW, unwounded; W, wounded. (F) Left, representative confocal images of MAI-1::GFP and TOMM-20::mKate2 before and after laser wounding at the wound site. Strains: Phyp-7-TOMM-20::mKate2 (zjuSi126); Pcol-19-MAI-1::GFP (zjuSi510). Scale bar, 10 and 5 μm (zoom in). Right, Pearson’s colocalization analysis of MAI-1::GFP and TOMM-20::mKate2 before and after wounding to the left images. Multiple mitochondria within an ROI (13.8 μm × 13.8 μm) at the wound site were measured. n represents the number of ROI analyzed. Statistical analysis was performed using the Mann–Whitney test. Data are mean ± SD (n = 31 and 32, respectively); ***, P < 0.001. (G) Representative confocal images of ATP-3::GFP and MAI-1::mKate2 before and after laser wounding at the wound site. Strains: ATP-3::GFP (zju133); Pcol-19-MAI-1::mKate2 (zjuSi510). Scale bar, 10 and 5 μm (zoom in). Right, Pearson’s colocalization analysis of MAI-1::mKate2 and ATP-3::GFP before and after wounding to the left images. Multiple mitochondria in an ROI (13.8 μm × 13.8 μm) at the wound site were measured. n represents the number of ROI analyzed. Statistical analysis was performed using the Mann–Whitney test. Data are mean ± SD (n = 37 and 38, respectively); ***, P < 0.001.