The images are projections of Z-stacks of the lower hemispheres of GUVs consisting of POPC SM Ch (1 1 1) labeled with 0.5 mol% Rho-DPPE, encapsulating 200 mM sucrose, diluted in deionized water at 25°C (n = 5).
We examined GUVs of various compositions by confocal imaging.
We investigated the selectivity of the interaction between caspase-8 and GUVs of different lipid compositions.
Also needed was a method for preparing GUVs of well-defined size from lipids doped with transporter, at high ionic strength and with a cholesterol rich lipid mixture.
Instead of quantifying transport into a population of vesicles with a distribution of sizes, we can now analyze the transport into individual GUVs of which we can verify the lamellarity and measure the size.
For GUVs of ternary lipid mixtures of unsaturated DOPC, saturated SM and cholesterol, the spectral GP image analysis reflected the expected phase separation into Ld and Lo domains on a single GUV, with the GP values of the Lo and Ld environments depending on the ratio of the lipid mixture.
This experiment was a modification of the original method of Kwok and Evans and of Longo et al. as described by Sun et al. Giant unilamellar vesicles (GUVs) of a chosen lipid composition were produced by the electroformation method in a solution containing 199 mM sucrose for the purpose of controlling the osmolality and 1 mM Tris (pH 7).
This would further enhance the tendency for small domains in GUVs of this BSM/Chol/POPC mixture, in agreement with the fluorescence microscopy results (26), and is perhaps one of the reasons there is some discrepancy in observations of large phase separation or not when these mixtures are investigated in different vesicle systems.
(C D ) The process in (A B ) exemplified by wide-field fluorescence (C ) and deconvolved (D ) images of a solution of GUVs consisting of POPC SM Ch (1 1 1) labeled with 0.5 mol% Rho-DPPE at 25°C containing 200 mM sucrose concentration, osmotically balanced by 200 mM glucose in (C ), and under an osmotic differential of ∼200 mM in (D ) at 25°C.
The membrane interactions of HAMLET were further investigated by confocal microscopy imaging of GUVs composed of EYPC or soybean lipids at pH 7. LUVs were not suitable for confocal imaging, as they do not form stable unilamellar vesicles under these conditions.
