The biophysical properties of sphingolipids containing lignoceric (C24:0) or nervonic (C24:1) fatty acyl residues have been studied in multicomponent lipid bilayers containing cholesterol (Chol), by means of confocal microscopy, differential scanning calorimetry and atomic force microscopy. Lipid membranes composed of dioleoyl phosphatidylcholine and cholesterol were prepared, with the addition of different combinations of ceramides (C24:0 and/or C24:1) and sphingomyelins (C24:0 and/or C24:1). Results point to C24:0 sphingolipids, namely lignoceroyl sphingomyelin (lSM) and lignoceroyl ceramide (lCer), having higher membrane rigidifying properties than their C24:1 homologues (nervonoyl SM, nSM, or nervonoyl Cer, nCer), although with a similar strong capacity to induce segregated gel phases. In the case of the lSM-lCer multicomponent system, the segregated phases have a peculiar fibrillar or fern-like morphology. Moreover, the combination of C24:0 and C24:1 sphingolipids generates interesting events, such as a generalized bilayer dynamism/instability of supported planar bilayers. In some cases, these sphingolipids give rise to exothermic curves in thermograms. These peculiar features were not present in previous studies of C24:1 combined with C16:0 sphingolipids. Conclusions of our study point to nSM as a key factor governing the relative distribution of ceramides when both lCer and nCer are present. The data indicate that lCer could be easier to accommodate in multicomponent bilayers than its C16:0 counterpart. These results are relevant for events of membrane platform formation, in the context of sphingolipid-based signaling cascades. Sphingolipids are important structural membrane lipids, as well as second messengers in diverse cellular signaling cascades 1-4. Among the sphingolipid subfamilies, ceramides (Cer) have received the attention of investigators due to their particular biophysical properties and their role in apoptotic processes 5-8. Cer are also important in the stratum corneum of the skin 9. The biophysical properties of Cer cause dramatic changes in membranes, as this sphingolipid tends to induce the formation of segregated areas ('domains'), giving rise to bilayer lateral heterogeneity 10-13. In turn the presence of domains has an impact on the diffusivity of proteins and their capacity to cluster and exert their physiological effects 14-16. Mammals have 6 ceramide synthases, of which CerS5 predominantly produces C16:0 Cer, while CerS2 yields ceramides incorporating C22-C24 fatty acids 17. For long and very-long chain Cer (longer than 14 carbons in their N-acyl group), the effects on membranes may also include lipid trans-bilayer motion, or flip-flop 18 (therefore affecting bilayer asymmetry) and a notable increase in solute permeability across the membrane 19,20 , which may decisively distort cell homeostasis. Most of the experiments classically performed with model membranes have been focused on C16:0 ceramide (pCer), as it is a simple, saturated long-chain ceramide and, more importantly, C16:0 s...
