Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.

Influence of the length of the spacer on the partitioning properties of amphiphilic fluorescent membrane probes.

Four fluorescent diphenylhexatriene derivatives were considered as membrane probes, namely two ammonium compounds, 3-(diphenylhexatrienyl)propyltrimethylammonium (TMAP-DPH) and 22-(diphenylhexatrienyl)docosyltrimethylammonium (LcTMA-DPH), and two phospholipids, 1-palmitoyl-2-[3-(diphenylhexatrienyl)propanoyl]-sn-glyc ero-3-phosphocholine (DPHpPC) and 1-palmitoyl-2-[21-(diphenylhexatrienyl)henicosanoyl]-sn-phos phocholine (LcDPHpPC). For each pair, the molecules differ by the length of the polymethylenic spacer between the fluorescent moiety and the polar head, so one pair comprises two short chain molecules (C3 spacer) and the other two long chain molecules (C21 or C22 spacer). The partitioning of these probes between gel and liquid crystalline phases of multilamellar vesicles with binary composition (DEPC and DSPC) was measured by a method based on fluorescence anisotropy. The partitioning was shown to depend strongly on the length of the spacer. Short chain probes preferably partition into fluid phases (Kf/s = 1.7 +/- 0.3 for TMAP-DPH; 2.6 +/- 0.11 for DPHpPC), whereas long chain probes show a strong preferential partitioning for gel phases of the vesicles (Kf/s = 0.12 +/- 0.06 for LcTMA-DPH; 0.22 +/- 0.11 for LcDPHpPC). This strong partitioning may be explained by the interdigitation of the long polymethylenic chains across the mid-point of the lipid bilayer (I.E. Mehlhorn et al. (1988) Biochim. Biophys. Acta 939, 151-159), which is enhanced by the better packing provided by a gel phase.

New diphenylhexatriene derivatives as fluorescent membrane probes: Partitioning properties.

Three new diphenylhexatriene derivatives, two phospholipids and one single-chain amphiphilic molecule, have been synthesized and considered as probes for measuring membrane fluidity by fluorescence anisotropy. The possibility of using these probes to determine specifically fluidity of inner leaflets of cellular plasma membranes was inferred from their partitioning properties between gel and liquid crystalline phases of phospholipid vesicles of binary composition.