Figure 3.

Mitochondrial PA is required for the subcellular location of N17-GFP. (A) The increase of mitochondrial PA by targeting optoPLD^WT onto mitochondria induces their clustering. optoPLD^WT or optoPLD^H170A were transfected into Cos-7 cells, and these cells were stained with mitotracker deep red prior to conversion. After 70 min of blue-light-induced mitochondrial targeting by pulse with 90 s intervals, the effect of optoPLD^WT on mitochondrial clustering was examined and quantified by the signal of mitotracker deep red. Scale bar, 10 μm. (B) Bar for inset image, 2 μm. The average mitochondrial area was quantified, compared to control cells, and shown in B. Each dot represents the average mitochondrial area of one cell. (C) optoPLD^WT enhances the mitochondrial localization of N17-GFP. After the co-transfection of N17-GFP and optoPLD^WT into Cos-7, the mitochondria were labeled with mitotracker deep red and subjected to blue-light conversion for 45 min. (D and E) Scale bar, 10 μm. The intensity of N17-GFP on mitochondria was monitored, magnified and shown (D), quantified and compared with optoPLD^H170A by ImageJ (E). Bar for heatmaps, 2 μm. To quantify the mitochondrial targeting efficiency, the total intensity of N17-GFP on mitochondria in a cell was calculated by ImageJ and divided by the total N17-GFP intensity of a cell. Each dot represents the ratio of one cell. (F) PLD6 depletion reduces the mitochondrial localization of N17-GFP. The proportion of N17-GFP co-localized with mitotracker was measured in HeLa cells depleted with PLD6. Scale bar, 10 μm. Bar for inset images, 2 μm. (G) The mitochondrial localization efficiency of N17-GFP was quantified. Data are expressed as mean ± SD of at least three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.