Co-Existing Gel and Fluid Phases in Bilayers Containing Ceramide and Cholesterol

Abstract

In previous studies, we have described the homogeneous gel phase formed by phospholipid:Chol:Cer at 54:23:23 mol ratios, where phospholipid was either SM or dipalmitoylphosphatidylcholine (DPPC). We now provide new data, based on trans-parinaric acid and diphenylhexatriene fluorescence, supporting that the gel phase includes all three components in a single bilayer. The main question addressed in this paper is the stability of the ternary gel phase when bilayer composition is changed, specifically when the SM proportion is varied. To this aim, we have prepared bilayers of composition phospholipid:Chol:Cer at X:Y:Y ratios, in which phospholipid increased between 54 and 70 mol %. The N-palmitoyl derivatives of SM (pSM) and Cer (pCer) have been used. We observe that for X = 54 or 60 mol %, a gel phase is clearly predominant. However, when the proportion of phospholipid increases beyond 60 mol %, i.e., in 66:17:17 or 70:15:15 mixtures, a lateral phase separation occurs at the micrometer scale. These data can be interpreted in terms of a pCer:Chol interaction, that would predominate at the lower phospholipid concentrations. The putative pCer:Chol complexes (or nanodomains) would mix well with the phospholipid. At the higher SM concentrations pSM:pCer and pSM:Chol interactions would become more important, giving rise to the coexisting gel and liquid-ordered phases respectively. Heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicating a higher affinity of SM over DPPC for Chol or Cer. The observation that heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicates a higher affinity of SM over DPPC for Chol or Cer, and can be related to cell regulation through the sphingolipid signaling pathway.