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. 2018 Jul 23;6:114–127. doi: 10.1016/j.isci.2018.07.015

Figure 5.

Figure 5

Alterations of Mitochondrial Dynamics in the Soma after Focal mt-KR Activation

(A) Time-lapse imaging of mt-DS before and after photostimulation (left panel, dashed square indicates photobleached area). Fluorescence recovery in the stimulated region was monitored every 10 min for 30 min. mt-paGFP was photoactivated in the same region (middle panel), and colocalization with mt-DS was assessed every 10 min for 30 min in 3 zones: the stimulated region, the zone directly surrounding the stimulated region (zone 1), and the rest of the soma (zone 2, inset). The area of mt-paGFP fluorescence is displayed over time as binary images in the right panel.

(B–D) Quantification of mt-DS fluorescence recovery after mt-DS photostimulation in the stimulated region (B), mt-DS and mt-paGFP colocalization (C), and diffusion of mt-paGFP-positive mitochondria (D). Values represent the mt-DS fluorescence intensity in the ROI (B), the percentage of colocalization (red × green signal) in the total area corresponding to the ROI or surrounding zones (C), and the mt-paGFP-positive area as a percentage of the total area corresponding to the ROI or surrounding zones, indicating the spreading of the GFP signal (D). The dashed line indicates the time of mt-DS photostimulation.

(E) Time-lapse imaging of mt-KR before and after photostimulation (left panel, dashed square indicates photobleached area). Fluorescence recovery in the stimulated region was monitored every 10 min for 30 min. mt-paGFP was photoactivated in the same region (middle panel), and colocalization with mt-KR was assessed every 10 min for 30 min in 3 zones: the stimulated region, the zone directly surrounding the stimulated region (zone 1), and the rest of the soma (zone 2). The area of mt-paGFP fluorescence is displayed over time as binary images in the right panel.

(F–H) Quantification of fluorescence recovery after mt-KR photostimulation in the soma (in the stimulated region) (F), mt-KR and mt-paGFP colocalization (in the stimulated regions and surrounding zones) (G), and diffusion of mt-paGFP-positive mitochondria (H). Values represent the mt-KR fluorescence intensity in the ROI (F), the percentage of colocalization (red × green signal) in the total area corresponding to the ROI or surrounding zones (G), and the mt-paGFP-positive area as a percentage of the total area corresponding to the ROI or surrounding zones, indicating the spreading of the GFP signal (H). The dashed line indicates the time of mt-KR activation.

Values represent the mean ± SEM of 10–11 neurons from three independent hippocampal cultures. Two-way ANOVA (repeated measurements) and Dunnett's multiple comparison test versus T = 0 min, *p < 0.05; **p < 0.01; ****p < 0.0001. mt-KR: mito-KillerRed, mt-DS: mito-DsRed.