ER–LD contact sites mediated by MOSPD2 depends on its CRAL-TRIO and TM domains. (A) Schematic representation of the different WT and mutant proteins used in the study. Two kinds of mutants were utilized: deletions of specific domains (ΔCRAL-TRIO, ΔMSP, ΔTM) and point mutation (RD/LD) impairing the MSP domain function. (B) Representative confocal images of the GFP-MOSPD2 WT and mutants (green) localization. Cells were treated with OA and LDs stained with Nile Red (magenta). (C) Quantification of cells presenting ring- and comma-shaped staining. Mean ± SD; n = 3 independent experiments (WT: 67 cells; ΔMSP: 138 cells; RD/LD: 152 cells; ΔCRAL-TRIO: 140 cells; ΔTM: 64 cells). (D) EM images of HeLa/GFP-MOSPD2, HeLa/GFP-MOSPD2 ΔMSP, HeLa/GFP-MOSPD2 RD/LD, and HeLa/GFP-MOSPD2 ΔCRAL-TRIO cells (top) and their interpretation scheme (bottom); the ER and LDs are in green and magenta, respectively. Mitochondria and endosomes are in light yellow and gray, respectively. Scale bars: 500 nm. (E) Left: schematic representation of the different chimeric constructs in which the MOSPD2 TM domain is replaced by the TM of SAC1 (purple). Right: localization of these chimeric proteins (green) and LDs stained with Nile Red (magenta) in HeLa cells treated with OA. In B and E, subpanels on the bottom are higher magnification images of the area outlined. The overlay panel shows merged channels. (B and E) Images were acquired on a confocal microscope (Leica SP8, 63× NA 1.4). Scale bars: 10 µm (insets, 2 µm).