Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel-liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer.
Keywords: confocal laser scanning microscopy; giant unilamellar vesicles; milk fat globule membrane; phospholipid bilayer.