Fig. 3: Miro1 is enriched at EMCS and spatially associated with mtDNA.

a A multicolor SIM image of a Cos-7 cell expressing mEmerald-Sec61β and mito-DsRed, with Miro1 labeled with α-Miro1 antibody. b Higher magnifications of the boxes in a showing three examples of Miro1 enriched at EMCS. White arrowheads indicate Miro1 punctae. Graphs to the right show relative pixel intensities of mito-DsRed, anti-Miro1, and mEmerald-Sec61β from a linescan drawn on each corresponding image (dashed line). The white arrowheads correspond to the black arrows on the linescans. c The percentage of Miro1 punctae that colocalized with the ER–mitochondria contacts in fixed Cos-7 cells. Data are shown as mean ± SEM (n = 10 cells examined over three independent experiments). d Cos-7 cells were transfected with mito-YFP, and immunostained by anti-Miro1 and anti-mtDNA. Arrowheads show mtDNA foci with adjacent Miro1 foci. e Spatial cross-correlation analysis of mtDNA and Miro1 fluorescence intensity along linescans of mitochondria (n = 91 mtDNA from 17 images) in fixed Cos-7 cells. Blue dotted lines indicate 95% confidence interval cutoffs, corresponding to the position of 224 nm. f Percentage of mtDNA that colocalized with Miro1 in Cos-7 cells (distance < 224 nm), corresponding to the 95% confidence interval marked by the blue dotted lines in e. Significantly more mtDNA is spatially (74%) associated with Miro1 than that expected by a randomly scrambled distribution (19%; n = 91 mtDNA from 17 images examined over three independent experiments, P < 0.0001, ***P < 0.001, two-tailed, unpaired Student’s t-test). Scale bars: a, 5 µm; b and d, 2 µm. Source data are provided as a Source data file.