C24:0 and C24:1 sphingolipids in cholesterol-containing, five- and six-component lipid membranes

Abstract

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...

Phase behaviour of C18-N-acyl sphingolipids, the prevalent species in human brain

“…This may be an indication that the observed endotherm arises from highly stable SM:Cer complexes. This behavior is observed as a rule in all our Cer-containing samples (see below), and it has been repeatedly observed in our previous studies [13,19,20,41].…”

“…4C, D, F, G). Domains are expected to be enriched in Cer, as repeatedly observed in other samples [13,19,20,40,41]. Domain boundaries, as detected by AFM, tend to be more convoluted in the C18:0 (Fig.…”

“…Al Sazzad et al (2017) described how the long-chain sphingoid base of ceramides determined their propensity for lateral segregation. In the recent years, systems of increasing complexity, including glycerophospholipids, sphingolipids and Chol have been examined in this laboratory, integrating a variety of biophysical techniques, in particular DSC, confocal microscopy of GUV, and correlative AFM and fluorescence microscopy of SLB [17][18][19][20], as well as a detailed 2 H-NMR study with selectively deuterated SM and Cer [21].…”

Lipid Self-Assemblies under the Atomic Force Microscope

“…Regarding non-cytoskeleton supported SPB, a recent publication from our lab [ 34 ] also demonstrated, using AFM, that domains might be unstable in SPB for some lipid mixtures (related to the combined presence of C24:0 and C24:1 sphingolipids), to the point of completely disappearing over time, while non-supported liposomes of the same composition were perfectly stable. This would point to a support-driven effect that reduces domain size over time until they become undetectable by AFM imaging (the possibility of nanodomains under the AFM resolution limit cannot be discarded).…”

“…This makes them extremely impactful for the biophysical properties of membranes [ 18 ]. Ceramides, due to their high hydrophobicity as well as their high T m values, tend to form gel phase platforms [ 29 , 30 , 31 , 32 ] depending on their carbon chain length [ 33 , 34 , 35 ], while also inducing membrane permeability [ 36 , 37 , 38 , 39 , 40 ] and lipid flip-flop (loss of bilayer asymmetry) [ 41 ]. Sphingomyelins (which are non-bioactive per se ) are considered to be an integral part of the liquid-ordered domains as they have a strong affinity for cholesterol [ 42 ], but they are also reservoirs for quick ceramide generation via sphingomyelinases [ 43 ].…”