Asymmetry determines the effects of natural ceramides on model membranes

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides,with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.