ALCAT1 Overexpression Affects Supercomplex Formation and Increases ROS in Respiring Mitochondria

Figure 1

Overexpressed ALCAT1 is localized in the ER and mitochondria. Fluorescence microscopic analysis of transiently transfected cells expressing CoxIV-sEcGFP, ALCAT1-mCherry stained with an antibody against ER marker KDEL. (a) Low ALCAT1-mCherry expression. (b) High ALCAT1-mCherry expression. Arrowheads in zoom image are pointing to ALCAT1-mCherry localized between ER and mitochondria, in other words MAMs. (c) Merged two-color images of ROIs shown in (a). (d) Merged two-color zoom images of ROI shown in (b). (e, f) Colocalization analysis showing scatterplots after setting thresholds in both color channels. (e) Colocalization analysis of the region shown in (a+c), (f) Colocalization analysis of the area shown in (b+d). Upper left quadrant = Ch2 above threshold; Ch1 below threshold; lower left quadrant = Ch1 below threshold; Ch2 below threshold; lower right quadrant = Ch1 above threshold; Ch2 below threshold; upper right quadrant = Ch1 above threshold; Ch2 above threshold; with linear regression; channel 1 (Ch1) = -axis, channel 2 (Ch2) = -axis. Thresholds for scatterplots were automatically determined for each channel (ImageJ plugin “colocalization threshold”), while presented images were manually processed with ImageJ to enhance the contrast. Scale bars: 10 μM (a, b).

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