REEP5-MFNs serve as molecular tethers at MERCs. (A) Formation of ER–mitochondria contact sites under hypotonic conditions (also see Videos 1 and 2). COS-7 cells were co-transfected with HaloTag-MFN1/2 and mEmerald-REEP5. White arrowheads, contact sites. Bars: 1 µm. (B) ER–mitochondria LICV contact sites before and after photobleaching (also see Video 3). MFN1&2 DKO MEFs were transfected with mEmerald-REEP5/Sec61β and HaloTag-MFN2/HaloTag. Mitochondria were labeled with mito-RFP. Dash-line boxes, bleached regions. White arrowheads, contact sites. Bars: 2 µm. A total of six cells from three independent experiments were analyzed for each group. (C) Confocal images of MFN1&2 DKO MEFs co-transfected with mEmerald-REEP5 C-terminus (CT) and mito-HaloTag or HaloTag-MFN2 (also see Video 4). White arrowheads, REEP5 CT recruitment onto MFN2-mitochondria. Bars: 5 µm (whole cell view, left); 2 µm (zoomed-in images). (D) Confocal images of U2OS cells co-transfected with HaloTag-MFN2 and mGFP tagged LAMP1 wt or chimera (LAMP1 NT-3XREEP5 CT). White arrowheads, co-movement events (also see Video 5). Bars: 5 µm. (E) Comparison of LAMP1–mitochondria co-movement cases within 50 s between LAMP1 wt or LAMP1 chimeric mutant expressing cells. Values shown are means ± SEM (n = 15).