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. 2022 Aug 12;221(9):e202112107. doi: 10.1083/jcb.202112107

Figure 1.

Figure 1.

ORP5 localizes to MAM subdomains in contact with LD. (A) Representative confocal images showing single focal planes of HeLa cells expressing EGFP-ORP5A, EGFP-ORP5B, or EGFP-ORP5ΔPH (green) and treated with Mitotracker (red) and LTox Deep Red (LTox, purple) to label mitochondria and lipid droplets (LDs), respectively. Scale bar, 10 μm (entire cell), or 5 μm (zoom). (B) Quantification of the % of HeLa cells showing localization of EGFP-tagged ORP5A, ORP5B, and ORP5ΔPH to ER-LD contacts close to mitochondria in the absence of oleic acid (OA) or after 2 h of 300 μM OA loading. Data represent the mean ± standard error of the mean (SEM) of n = 25 cells. **P < 0.01, ***P < 0.001, unpaired two-tailed t test. (C) SIM micrographs of HeLa cells expressing EGFP-ORP5B or EGFP-ORP5ΔPH (green), treated with OA for 2 h, and stained with Mitotracker (red) and LTox Deep Red (purple). 3D-SIM images were obtained by segmentation using Software Imaris (v 9.3, Bitplaine). Scale bar, 5 μm (entire cell), or 2.5 μm (zoom). (D) Electron micrograph of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5ΔPH, treated with OA for 2 h, and immunogold stained with anti-EGFP (15 nm gold). Left: red arrows indicate EGFP-ORP5ΔPH localized to MAM. Right: blue arrows indicate ORP5ΔPH localized to ER–LD contacts and red arrows indicate ORP5ΔPH localized to MAM–LD contacts. Scale bar, 500 nm. (E) Quantification of the distribution of EGFP-ORP5B and EGFP-ORP5ΔPH immunogold particles (15 nm) to the different ER compartments: MAM, MAM–LDs, ER–LDs (associated to MAM–LDs), ER–LDs (isolated) and reticular ER, after 2 h of OA. Data are shown as % of mean ± SEM of cell profiles with n = 20 (360 and 470 gold particles analyzed in EGFP-ORP5B and EGFP-ORP5ΔPH overexpression, respectively). (F) Representative electron micrographs of HeLa cells co-overexpressing EGFP-ORP5A or EGFP-ORP5ΔPH and HRP-KDEL after 2 h OA treatment, and 3D reconstruction of 27 serial sections by 3Dmod. LD, lipid droplet; Mito, mitochondria; ER, endoplasmic reticulum. Scale bar 500 nm.