Lipid Dynamics and Domain Formation in Model Membranes Composed of Ternary Mixtures of Unsaturated and Saturated Phosphatidylcholines and Cholesterol

Abstract

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.

Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles

“…When cooling the GUVs from the miscibility temperature, the stripe domains appeared below the pretransition temperature of DPPC and became wider and more interconnected with further temperature decrease ( Figure 6). Independent FCS studies by Scherfeld et al (12) and Korlach et al (7) have showed that stripe domains in DOPC/DPPC and DLPC/DPPC GUVs have a diffusion rate close to pure DPPC below the transition temperature (35). Based on these previous studies and our observations, we propose that the stripe domains are in the gel phase.…”

Differential Effect of Plant Lipids on Membrane Organization

“…5B) and Bdp-cholesterol (Fig. 5C), classical markers of the L d (11) and sterol-enriched phases (82). Both fluorophores exhibited a spatial segregation in distinct domains (Fig.…”

“…5D). To discriminate between L o and L d phase, we considered a threshold limit of 1.2 for RGP because pure DOPC GUVs exist in a unique L d phase at room temperature (11,81) and consistently exhibit more than 95% of their RGP values greater than 1.2. In these conditions, the pattern obtained with di-4-ANEPPDHQ (Fig.…”

“…The DOPC (T m ϭ Ϫ20°C)/DPPC (T m ϭ 41°C) lipid mixture, which has been widely used in the literature to characterize the physical properties of the membrane model system (11,14,83,108,109), exhibits, at temperatures below 45°C, an L o and L d phase coexistence (83). Performing our experiments at 22°C and minimizing temperature fluctuations, we actually favored a long time equilibrium formation of L o and L d phase separation, allowing us to focus our study on the structuring effect of plant lipid on L o and L d phase partitioning.…”

A new detection algorithm for image analysis of single, fluorescence‐labeled proteins in living cells

“…Single-molecule microscopy and single-molecule tracking has revealed detailed motions of membrane proteins such as micro-scale diffusion on cell surfaces revealing inhomogeneities of lipid rafts (Dietrich et al, 2002;Scherfeld et al, 2003;Schutz et al, 2000;Simons and Ikonen, 1997;Simons and Tommre, 2000) and cytoskeletal corrals (Kusumi and Sako, 1996) or the entry and intracellular fate of single viruses in living cells (Lakadamyali et al, 2003;Seisenberger et al, 2001). The tracking and observation of colocalization of individual proteins on cell surfaces is not only important in revealing microdiffusion but represents a physiological strategy to complement protein overexpression models and avoid nonphysiological cross talk of multiple signals generated from a single receptor or ion channel (Sako and Yanagida, 2003;Sako et al, 2000;Yoshiharu and Yanagida, 2002).…”