Mitochondrial morphology is disrupted by reduced or enhanced REEP5-MFN1/2 interactions. (A) ER morphology in REEP5 depleted cells. Endogenous mitochondria and ER were immunostained in U2OS cells. Bars: 20 µm (whole cell view); 5 µm (zoomed-in images). (B) Schematic diagram of REEP chimeric mutants. Residue numbers are shown as indicated. NT, N-terminus; CT, C-terminus; TM, transmembrane; cytL, cytosolic loop. (C) Co-IP analysis of Myc-tagged REEP chimera with Flag-MFN2 from HEK293T cells. (D) Alignment of sequences from REEP1-5 and REEP5 mutants. Positions of the mutations were indicated in red and residue numbers were shown as indicated. (E) Co-IP analysis of Myc-tagged REEP5 mutants with Flag-MFN2 from HEK293T cells. (F) Confocal images of mEmerald-REEP5 mutants and mCherry-KDEL expressing U2OS cells. Bar: 5 µm. (G) Co-IP analysis of endogenous MFN2 with REEP5 from HEK293T cells expressing mEmerald tagged REEP5 CT. (H) Time-lapse images of ER–mitochondria dynamics in U2OS cells with rapamycin-induced REEP5–MFN1/2 binding (also see Videos 13 and 14). 20 nM rapamycin was added at the time point indicated. Bars: 10 µm (whole cell view); 5 µm (zoomed-in images). Source data are available for this figure: SourceData FS2.