Fig. 3: Membrane unsaturation promotes BAX pore-forming activity.
From: Lipid unsaturation promotes BAX and BAK pore activity during apoptosis
A and (B) Kinetics of BAX-induced LUVs permeabilization measured as % carboxyfluorescein release over time. LUVs treated with 10 nM cBID and increasing concentrations of BAX as indicated. LUVs composed of POPC:CL (16:0) (A) or PAPC:CL (16:0) (B) in 8:2 ratio. Data are presented as means ± S.D.; (POPC, n = 4; PAPC, n = 5) from independent experiment. C Dose-response curve of % carboxyfluorescein release as a function of BAX concentration after 1 h incubation (data from A, B) for the indicated lipid compositions. Data are presented as means ± S.D.; n = 4 (DPPC and POPC); n = 5 (PAPC) from independent experiment. D Representative images of GUV assay of membrane permeabilization. Internalization of Atto488 dye (green) in the external medium into the lumen of GUVs (membrane shown in grey), initially devoid of dye, indicate vesicle permeabilization. GUV composition was POPC:heart CL (8:2) or PC:CL(16:0) (8:2). Scale bar, 75 µm. E % of permeabilized GUVs of the indicated lipid composition at different time points after addition of 100 nM BAX and 20 nM cBID. Data are presented as means ± S.E.; n = 3; from independent experiments. F Distribution of degree of filling for the individual GUVs after 60 min incubation with cBID/BAX. Data are presented as means ± S.E.; n = 3; from independent experiments. G Pore stability assay showing the filling degree of individual GUVs with dye 1 (Atto488, blue lines) and dye 2 (Atto655, red lines) after 2 h incubation with cBID/BAX (n = 3; from independent experiments). H Representative images of the GUV assay to estimate pore size. GUVs (gray) in a solution of cyt c-488 (12 kDa, blue) and APC (104 kDa, red) were incubated for 1 h in absence or presence of 20 nm cBID and 100 nm BAX. cyt c-488 and/or APC internalization into the lumen of GUVs indicates vesicle permeabilization. Scale bar, 20 µm. I % GUVs permeabilized to cyt c-488 (blue bars) and APC (red bars), (J) Distribution of the degree of filling of individual GUVs composed of POPC:CL (16:0) with cyt c-488 (blue lines) and APC (red lines) after treatment with cBID/BAX. K Distribution of the degree of filling of individual GUVs composed of POPC:hCL with cyt c-488 (blue lines) and APC (red lines) after treatment with cBID/BAX. Data are presented as means ± S.E.; n = 3 from independent experiments. Source data are provided as a Source Data file.
