MICOS subcomplexes independently localize in proximity to ERMES-marked ER-mitochondria MCSs. (A) MICOS is organized into two subcomplexes that independently assemble when the holo-MICOS complex is disrupted. (B and C) Images of wild-type cells coexpressing Mic60-mCherry (B; magenta) or Mic27-mCherry (C; magenta), mito-TagBFP (blue), and GFP-HDEL (ER; green). (D and E) As in B and C for Δmic19 cells expressing Mic60-mCherry or Δmic60 cells expressing Mic27-mCherry, respectively. Arrows mark assemblies that localize in proximity to the ER. (F) Time-lapse images of a cell as in D highlighting a subcomplex that remains in proximity to the ER. White arrows indicate the original position and yellow arrows track its movement. See Video 1 and Video 2. (G) As in F for Δmic60 cells expressing Mic27-mCherry. See Video 3 and Video 4. (H) Images of Δmic19 cells expressing Mic60-mCherry (magenta), Mdm34-EGFP (green), and mito-TagBFP (blue). Arrows mark subcomplexes that colocalize with Mdm34. (I) As in H for Δmic60 cells expressing Mic27-mCherry. (J) Graph of colocalization between Mic60 and Mic27 assemblies and ERMES complexes from cells as in H and I. Data are the summation of the indicated number of foci from at least three independent experiments. See Fig. S1, A and B. (K) Images of Δmic19 ρ⁰ cells expressing Mic60-EGFP (green), Mdm34-HaloTag (magenta), and mito-dsRed (blue). Arrows mark Mic60 assemblies that localize in proximity to Mdm34. Cells are outlined with dashed lines. Scale bars = 2 µm.