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Figure S3. Analyses of mitochondrial contiguity in cells expressing high levels of MARCH5H43W. Interconnectivity of mitochondria was analyzed in MARCH5H43W-CFP and mito-YFP expressing cells with apparent rounding and perinuclear accumulation of these organelles. The round mitochondrion (region of interest; ROI 1; A) was bleached, followed by imaging of fluorescence recovery (B) that is predicted to occur if the bleached area is connected with the bulk mitochondrial network. Photobleach (indicated with �V�) and recovery cycles were repeated seven times. Images 1-5 correspond to the time points indicated on the recovery curves. Note efficient recovery of fluorescence in photobleached ROI 1 (B) over the time of experiment, and delayed response to photobleaching of the mitochondrion (ROI 2; C) adjacent to ROI 1 indicating an exchange of matrix content between these two mitochondria. Negligible changes in fluorescence intensity of ROI 3 (D) indicate low nonspecific photobleaching and validate fluorescence changes occurring in ROIs 1 and 2.