Fig. 4: Mic60 links mtDNA to Miro1 and contributes to EMCS stability.

a Co-immunoprecipitation of Mic60 with endogenous TFAM and Miro1 in HEK293T cells, and the immunoprecipitates were blotted as indicated. Input: 2%. b Cos-7 cells immunostained by anti-mtDNA and anti-Mic60. Arrowheads show mtDNA foci with adjacent Mic60 foci. (Right) the graph is linescan drawn through the mitochondria, and show the relative fluorescence intensity of mtDNA (green) and Mic60 (purple) along their length. White arrow positions correspond to black arrows on the linescan. c As in b for cells immunostained by anti-Miro1 (green on linescan) and anti-Mic60 (purple on linescan). d Dual-color confocal image of a Cos-7 cell expressing mito-DsRed and mEmerald-Sec61β (left), and the corresponding mitochondrial perimeter profile (right). Mitochondrial perimeter profiles are presented by red pixels, and the contacts between the mitochondrial perimeter and the ER are presented by yellow pixels. e ER–mitochondria colocalization index (R), calculated as the ratio between the perimeter pixels of mitochondria colocalized with the ER and the total perimeter pixels of mitochondria for the control and Mic60 RNAi cells (n = 10 cells were used per condition from three independent experiments). f Fluorescence image (modified as described in “Methods” section) of a Mic60 RNAi cell expressing mito-DsRed and mEmerald-Sec61β. The boxed region is enlarged and shows time-lapse images. White arrowheads indicate unstable ER–mitochondria interactions. g Representative kinetics of the ratio (R) on the entire Cos-7 cell shown in f. ΔR represents the difference between the R of one image (frame) and that of the previous one. h Mean ΔR/R0 values (where R0 is the R of the first frame of each pair; n = 110 frames from ten control and ten Mic60 RNAi cells examined over three independent experiments). Scale bars: b, c, inset in d, and inset in f, 2 μm; d and f, 10 μm. Data are presented as mean ± SEM. P value from top to bottom: p = 0.4981, p = 0.0089. NS no significant difference; **p < 0.01, two-tailed, unpaired Student’s t-test. Source data are provided as a Source data file.