Fig. 5: Dynamics of single mitochondrial matrix ROS release.
From: All-optical spatiotemporal mapping of ROS dynamics across mitochondrial microdomains in situ
a Fluorescent change over baseline of single mitochondria in HEK293T cells co-expressing various microdomain-targeted HyPer7 with matrix-KillerRed. Data taken from 12 frames (one minute) following the first 5 sec photostimulation pulse and displayed as a function of distance from the spot stimulation point. Data expressed as mean ± 95% CI, N = 192 matrix-HyPer7, 357 IMS-HyPer7, and 120 OMM-HyPer7 mitochondria, on average. b Average traces of fluorescent change over baseline of single mitochondria co-expressing microdomain-targeted HyPer7 and matrix-KillerRed following repeated photostimulation. Mitochondrial populations were separated based on distance to spot stimulation, where proximal and distal roughly approximates under 5 µm and over 15 µm, respectively. Data expressed as smoothed mean ± SEM. N = matrix-HyPer7: 39 proximal and 49 distal; IMS-HyPer7: 32 proximal and 34 distal; OMM-HyPer7: 53 proximal and 31 distal mitochondria, on average. c Fluorescent change over baseline of single mitochondria expressing OMM-HyPer7 and matrix-KillerRed following a single, longer duration (30 sec) photostimulation pulse of KillerRed. Mitochondrial populations were separated as proximal and distal, but also included a “spot” subpopulation of mitochondria within 1.5 µm of the stimulation point. Data expressed as smoothed mean ± SEM, N = 7, 27, and 52 individual mitochondria for spot, distal, and proximal mitochondrial populations per frame, on average. d Area under the curve analysis of C. Data expressed as mean ± SEM, one-way ANOVA with Tukey post-hoc correction, N = 4 independent experiments. e Representative images of mitochondria in a cell expressing OMM-HyPer7 (rainbow LUT applied) and matrix-KillerRed (grayscale) before and after photostimulation. Spot stimulation point depicted as a red circle, 5 µm surrounding area as a dashed white circle. Scalebar denotes 1 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Note: Matrix-HyPer7 data in b) taken from Fig. 1 and re-expressed as dF/F.
