Phase Behavior of Lipids and Membrane Domain Formation

A large number of phase diagrams for binary mixtures combining cholesterol with different saturated and unsaturated phosphatidylcholines have been established. Cholesterol at different bilayer concentrations can promote or suppress lateral segregation of phospholipids of differing acyl chain length.

Addition of 50 mol% cholesterol to selectively deuterated DPPC bilayers leads to an elimination of the gel-toliquid crystal phase transition at 41°C. In contrast, cholesterol is also found to enhance the tendency of the PC components to exhibit lateral segregation. These seemingly contradictory effects of cholesterol can be readily explained in light of the cholesterol–phospholipid phase diagrams.

The effect of cholesterol on the thermotropic phase behavior of aqueous dispersions of different lipids has been extensively investigated by means of differential scanning calorimetry. The results show an inverse correlation between the strength of intermolecular phospholipid–phospholipid interactions, as manifested by the gel-to-liquid crystalline phase transition temperatures of the pure phospholipids, and the miscibility of cholesterol with the respective bilayer (particularly gel-state bilayers). The miscibility of cholesterol with lipids carrying identical fatty acyl chains decreases in the order: PC ∼ PG ∼ SM > PS > PE > diglucosyl - and monoglucosyl - diacylglycerol > GalCer. However, if the higher melting components are dispersed as minor components of total lipid in a host matrix consisting of, for example, 1-stearoyl-2-oleoyl-phosphatidylcholine and cholesterol, neither short-chain nor long-chain cerebrosides or sphingomyelins show phase separation in the physiological temperature range despite their high phase transition temperatures.

Mixtures of cholesterol and sphingolipids have recently attracted attention since spingolipid–cholesterol domain formation has been observed in mammalian cell membranes upon cooling to 4°C and extraction with Triton X-100. This phenomenon has also been termed “lipid raft” formation. Lipid rafts exhibit a high lateral packing density and are suggested to entail a sorting of GPI-anchored proteins. The bulky intrinsic proteins remain in the fluid phase. At room temperature, lipid rafts are no longer detectable. Nevertheless, they are assumed to prevail as microdomains at growth temperature and to be relevant for membrane trafficking and protein sorting in mammalian cells.

Although domain formation is now a common theme among biologists, an unambiguous physical–chemical characterization of domains under physiological conditions is still missing. On physical grounds, domain formation is most likely to occur in mixtures of lipids with widely different gel-to-liquid crystal phase transition temperatures. Phase separation will occur if the measuring temperature is below the phase transition temperature of one of the components of the mixture and if this component constitutes a major lipid fraction. Lipids exhibiting high phase transition temperatures generally have long saturated acyl chains and small headgroups, or headgroups that may interact via hydrogen bonding. Typical examples are sphingolipids, glycosphingolipids, or long-chain phosphatidylethanolamines.

As a further mechanism, electrostatic interactions of anionic lipids with cationic compounds may also induce domain formation. Due to the biochemical complexity of biological membranes, the molecular mechanisms responsible for phase separation are not easily distinguished experimentally.

The difficulty in understanding the diverging results arises, on the one hand, from the use of techniques differing in spatial (nanometers to micrometers) and temporal (nanoseconds to tens of seconds) resolution and, on the other hand, from the application of different experimental conditions. For technical reasons, domain formation was generally investigated at unsphyiological temperatures using lipids with bulky reporter groups. Both factors may affect the phase behavior of lipids. Further experiments are therefore required to test whether oganizational processes are induced by lipid domain formation under physiological conditions.