Fig. 3: Functional membrane asymmetry demonstrated with protein-lipid interactions.
From: Engineering synthetic cells with intramembrane domains possessing distinct bilayer asymmetries
a Confocal images of asymmetric GUVs (99.5 mol% POPC and 0.5 mol% DiD (red color) in the inner leaflet and 99.5 mol% POPC and 0.5 mol% Biotinyl Cap PE in the outer leaflet) with unbound alexa 488-tagged streptavidin (green color) in the lumen of the GUVs because the biotinylated lipids are present only in the outer leaflet, negative control. b Confocal images of the same GUVs as presented in (a) after the addition of alexa 488-tagged streptavidin (green color) in the outer solution. c Confocal images of His-tagged GFP in the outer solution of vesicles with 99.5 mol% POPC and 0.5 mol% DiD (red color) in the outer leaflet and 0.5 mol% DGS Ni-NTA in the inner leaflet, negative control. d Confocal images of His-tagged GFP (green color) in the outer solution of vesicles with 99.5 mol% POPC and 0.5 mol% DiD (red color) in the inner leaflet and 0.5 mol% DGS Ni-NTA in the outer leaflet. e Confocal images of GUVs with streptavidin (green color) (inside) and cholera toxin subunit-B tagged with AlexaFluor® 594 (CT-B Al-594) (red color) (outside) binding to biotinylated lipids present in the inner leaflet and to GM1 present in the outer leaflet of GUVs respectively (99.5 mol% POPC and 0.2 mol% biotinyl Cap PE in the inner leaflet and 1 mol% GM1 in the outer leaflet). f Confocal images of GUVs (negative control) with streptavidin (green color) (inside) and CT-B Al-594 (red color) (outside) not binding to GM1 present in the inner leaflet and biotinylated lipids present in the outer leaflet of GUVs respectively (99.5 mol% POPC and 0.2 mol% biotinyl Cap PE in the outer leaflet and 1 mol% GM1 in the inner leaflet). Corresponding schematics are shown to the left of each image. n ≥ 30, data is collected from three or more independent experiments. Scale bars correspond to 20 µm.
