Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains

Abstract

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.Cholesterol (Chol) 3 is the most abundant sterol in mammalian plasma membrane and has unique biophysical properties (1, 2). Chol interacts with the high melting temperature (T m ) sphingolipids (SL) in the membrane, leading to the formation of SL/Chol-enriched microdomains (so-called lipid rafts). These domains are in a more ordered state (usually referred to as liquid-ordered (l o ) phase) than the bulk membrane (liquid-disordered phase (l d )) (3, 4). Ceramide (Cer) is an SL formed in stress situations either from sphingomyelin (SM) in rafts or synthesized de novo by serine palmitoyltransferase and ceramide synthase. Both of these processes can be induced by diverse stimuli (5). Cer-induced membrane alterations (e.g. raft fusion into large signaling platforms (6)) were proposed to be the mechanism by which this lipid mediates diverse cellular processes, namely apoptosis (7-10). Cer presents an unusually small polar headgroup and in general very high gel-fluid T m (e.g. for palmitoyl-Cer (PCer) it is ϳ90°C) (11)....

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae

“…Thus, although ergosterol has some ability to solubilize phytoceramide-enriched gel domains, the results suggest that at least a small fraction of those domains should persist at physiological concentrations of those lipids. Similarly, in the ternary mixture of mammalian lipids POPC/ceramide/cholesterol, for 17 mol % of ceramide the gel phase was completely solubilized for ϳ30 mol % cholesterol (21). Therefore, the ability of the main mammalian sterol to abolish gel phases was similar to that of ergosterol.…”

“…Liposome Preparation-Lipid stock solutions were made in chloroform except for phytoceramide, which was dissolved in chloroform/methanol (2:1, v/v). Multilamellar vesicles (MLV) of binary and ternary mixtures of POPC with ergosterol and/or phytoceramide or of lipid extracts from plasma membrane fractions and whole cells were prepared in buffer A with either t-PnA or DPH, as described previously (15,21). Total lipid concentration in MLV suspensions was 0.2 mM in the binary mixtures to avoid incomplete hydration of ceramide and 0.4 mM for the ternary mixtures.…”

“…In a pure gel, a pure liquid ordered or a pure liquid disordered phase, both probes have similar fluorescence anisotropy values (21,36). In the case of liquid disordered/liquid ordered domains, the anisotropy of t-PnA is only slightly larger than that of DPH because the quantum yield of t-PnA is moderately higher in this phase.…”

“…4). Although the fluorescence decay of t-PnA has been previously studied in synthetic saturated phosphatidylcholine and in mammalian-sphingolipid-containing membranes (17,21), no studies have been performed in the presence of fungal sphingolipids. In Fig.…”

“…4A), average lifetime, and anisotropy (data not shown) attains the highest value at ϳ20 mol % phytoceramide, but still the system is close enough to the gel/fluid coexistence boundary so that the fluorescence of t-PnA will be sensitive to even a partial solubilizing effect of ergosterol. In addition, studying this system allows us to compare its behavior with an analogous system composed of the mammalian lipids POPC/ ceramide/cholesterol previously studied by us (21) and to assess which sterol has a stronger ability to solubilize a gel phase. From Fig.…”