Cholesterol Displaces Palmitoylceramide from Its Tight Packing with Palmitoylsphingomyelin in the Absence of a Liquid-Disordered Phase

Abstract

A set of different biophysical approaches has been used to explore the phase behavior of palmitoylsphingomyelin (pSM)/cholesterol (Chol) model membranes in the presence and absence of palmitoylceramide (pCer). Fluorescence spectroscopy of di-4-ANEPPDHQ-stained pSM/Chol vesicles and atomic force microscopy of supported planar bilayers show gel L(beta)/liquid-ordered (L(o)) phase coexistence within the range X(Chol) = 0-0.25 at 22 degrees C. At the latter compositional point and beyond, a single L(o) pSM/Chol phase is detected. In ternary pSM/Chol/pCer mixtures, differential scanning calorimetry of multilamellar vesicles and confocal fluorescence microscopy of giant unilamellar vesicles concur in showing immiscibility, but no displacement, between L(o) cholesterol-enriched (pSM/Chol) and gel-like ceramide-enriched (pSM/pCer) phases at high pSM/(Chol + pCer) ratios. At higher cholesterol content, pCer is unable to displace cholesterol at any extent, even at X(Chol) < 0.25. It is interesting that an opposite strong cholesterol-mediated pCer displacement from its tight packing with pSM is clearly detected, completely abolishing the pCer ability to generate large microdomains and giving rise instead to a single ternary phase. These observations in model membranes in the absence of the lipids commonly used to form a liquid-disordered phase support the role of cholesterol as the key determinant in controlling its own displacement from L(o) domains by ceramide upon sphingomyelinase activity.

Insights into Sphingolipid Miscibility: Separate Observation of Sphingomyelin and Ceramide N-Acyl Chain Melting

“…However, Cer-enriched gel phase formation associated with the displacement of cholesterol by Cer appears to depend on lipid composition. Adding cholesterol to 70:30 PSM:PCer causes a homogeneous phase to form at room temperature (30), indicating that Cer, SM, and cholesterol can all interact closely. This result highlights the importance of understanding the interaction energies between pairs of lipids: a possible explanation for cholesterol's unpredictability is that Cer can only displace cholesterol in an Biophysical Journal 103(12) 2465-2474 environment that includes a cholesterol sink such as that provided by an unsaturated PC.…”

“…This result highlights the importance of understanding the interaction energies between pairs of lipids: a possible explanation for cholesterol's unpredictability is that Cer can only displace cholesterol in an Biophysical Journal 103(12) 2465-2474 environment that includes a cholesterol sink such as that provided by an unsaturated PC. Busto et al (30) provided, as an alternative explanation, the stabilization of SM:Cer: Chol ternary phases, with no cholesterol displacement. In principle, it would also be possible that Cer displaces cholesterol to form cholesterol-dense regions, likely leading to crystalline cholesterol monohydrate.…”

“…A more detailed understanding of lipid-lipid interactions in the plasma membrane would require, in addition to SM and Cer, the presence of at least cholesterol and unsaturated PC. However this quaternary mixture is hardly amenable to analysis by contemporary techniques; even the ternary mixtures of SM:PC:cholesterol (17,28,29) are difficult to study, and the mixture SM:Cer:cholesterol is known for its unusual behavior (30)(31)(32). Thus, we start with SM:Cer, a decision supported by the fact that these two lipids give rise to complexes that are stable even in the presence of PC or cholesterol under certain conditions (31).…”

The Long-Chain Sphingoid Base of Ceramides Determines Their Propensity for Lateral Segregation

“…In bilayers mimicking cellular plasma membranes, ceramide has been shown to displace cholesterol and prevent it from interacting with saturated phospholipids such as dipalmitoyl phosphatidylcholine (19) and palmitoyl SM (PSM) (20). The cholesterol-displacing effect is dependent on the ceramide acyl chain length (12,13) and on the balance of cholesterol and ceramide in the bilayer (21,22). The propensity for lateral segregation of ceramides in fluid phosphatidylcholine bilayers is affected by the length of the long-chain base, by the nature of the N-linked acyl chain, and by the hydrogen-bonding properties of the ceramides (23).…”

The many faces (and phases) of ceramide and sphingomyelin II – binary mixtures

“…The interaction of some species of SM and ceramide (Cer) has been studied in monolayers (Busto et al 2009) and bilayers (Busto et al 2009;Westerlund et al 2010). 16:0 Cer forms condensed domains segregated from more liquid-like phases enriched in SM (Busto et al 2009) and unsaturated PCs Pinto et al 2011) even in the presence of low amounts of cholesterol Ale et al 2012), leading to the idea that Cer and cholesterol can compete in ordered domains with SM (Staneva et al 2008;Busto et al 2010). Mixtures of Cer and SM N-acylated with palmitic acid (16:0) in monolayers show a thermodynamically stable point at X 16:0 Cer = 0.4 with molecular area condensation, whereas in bilayer systems, complex differential scanning calorimetry (DSC) thermograms occur (Busto et al 2009) with increasing melting temperatures at increasing ceramide content.…”