Figure 3: ORP1L and VAP-A form cholesterol-dependent ER-AV contact sites
(a) MelJuSo cells expressing GFP or GFP-tagged ORP1L mutants were fixed and stained for LC3 and Calnexin. Y477 and D478 in the FFAT motif were mutated to alanines (A) in the ORP1L ydaa mutant. Scale bar, 10 μm. Right: co-immunoprecipitation for ORP1L (mutants) with VAP-A. GFP-ORP1L mutants or GFP were isolated from lysates of HEK293T cells co-overexpressing HA-VAPA using GFP-Trap beads. Western blot filters were probed for isolated GFP-tagged proteins, the associated HA-VAP-A and the input HA-VAP-A, as indicated. (b) Cryo-immuno-EM on HeLa cells expressing HA-LC3 and GFP-ORP1LΔORD, as detected by HA10 nm and GFP15 nm gold antibodies. Insets show ORP1L labelling in the membrane contact site between ER and autophagosome. The membranes of the ER are depicted in the bottom inset. Scale bar, 50 nm. (c) Three-colour super-resolution image of an autophagosomal vesicle labelled by LC3 (green), ORP1L (blue) and the ER protein VAP-A (red). Scale bar, 500 nm (d) MelJuSo cells cultured either in lipid depleted serum or control medium were fixed and stained for LC3 and ER marker Calnexin. Scale bar, 10 μm. Right panel: Manders coefficient for LC3 localization to the ER was calculated on at least 10 cells over three independent experiments. Bars indicate mean+s.d. Student’s t-test statistical analysis (****P<0.0001).
