Cholesterol Displaces Ceramide from its Tight Packing with Sphingomyelin

Abstract

The present study deals with a combination of fluorescence spectroscopy, atomic force microscopy (AFM), differential scanning calorimetry (DSC) and confocal microscopy to study two different effects: i) lateral segregation in pSM/Chol binary mixtures, and ii) the effects of pCer incorporation into pSM/Chol mixtures. The data reveals the segregation of large cholesterol-enriched microdomains within the range XChol = 0-0.25 in the binary pSM/Chol mixtures. In comparison with the pSM/pCer mixture (Busto et al. 2009), sphingomyelin shows a higher preference for ceramide than for cholesterol. In ternary pSM/Chol/pCer mixtures, an immiscibility between cholesterol- (pSM/Chol) and ceramide-enriched (pSM/pCer) phases at high pSM/(Chol+pCer) ratio is observed, where no ceramide over cholesterol nor cholesterol over ceramide displacement is detected. Furthermore, the calorimetric and confocal microscopy data concur in showing an inability of pCer to displace cholesterol both at low and high cholesterol concentrations. Interestingly, an inverse cholesterol-mediated ceramide displacement from its tight packing with sphingomyelin is clearly observed. These observations in model membranes in the absence of the lipids commonly used to form a liquid-disordered (Lα) phase support the proposed role of raft-like domains (Silva et al, 2007) rather than ceramide, in regulating ceramide-induced platform formation within cell membranes.∗ Busto J.V. et al. (2009) Coexistence of immiscible mixtures of palmitoylsphingomyelin and palmitoylceramide in monolayers and bilayers. Biophys. J., in press.∗ Silva L.C. et al. (2007) Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid. Biophys. J., 92(2):502-516.