Lipid Domains In Giant Vesicles Composed Of Ternary Lipid Mixtures Containing Cholesterol And Their Relationship With Thermodynamic Phases

Abstract

Fluorescence microscopy related techniques provide a powerful tool for direct observation of lipid domains in giant unilamellar vesicles (GUVs) [1]. Using these techniques it was reported that liquid-ordered (lo) - liquid-disordered (ld) phase coexistence can be observed in GUVs composed of cholesterol containing ternary lipid mixtures [1,2]. However, still it is not rigorously established if the lipid domains observed in these GUVs correspond to real thermodynamic phases. Recently we introduced a new method to measure the area fractions of the coexisting lipid domains in GUVs [3]. This novel procedure that involves deconvolution and segmentation of the individual GUV's fluorescence image stacks (including fitting with 3D surface models), allows reconstruction of GUVs 3D structure, permitting to retrieve, at the level of single vesicles, the area fractions of the coexisting lipid domains. The last procedure allowed us to demonstrate quantitatively the accomplishment of the lever rule in GUVs composed of binary phospholipid mixtures displaying solid ordered/liquid disordered domains [3].In this work we measured the relative areas of the two observed distinct regions (lipid domains) at the reported lo/ld coexisting region in GUVs composed of POPC/DPPC/cholesterol mixtures (approximately 20 different compositions). We explore subsequently if the relationship between the measured areas are consistent with that expected for coexistence of real thermodynamic phases. In particular our method provides a mean of characterizing the tie lines in the lo/ld coexistence region, providing evidences of a connection between what is observed in GUVs and what is predicted from the 3-component phase diagram of the lipid mixture.1) Bagatolli, L.A, 2006, Biochim Biophys Acta 1758:1541-1556.2) Veatch, S.L and Keller S.L, 2005, Biochim Biophys Acta. 1746:172-85.3) M. Fidorra et al., 1198-Pos Biophys. J. 2008 94:1198.