 Lipidomic data of Cer and SM species in 35 mammalian tissues/cells are provided.  The sphingoid base was predominantly d18-1 sphingosine in all cases.  The most abundant Cer species were those containing C24:0 and C24:1 acyl chains.  The main SM species was C16:0.  Brain was an exception, with Cer and SM C18:0 as the main species.

Ceramide is a major actor in the sphingolipid signaling pathway elicited by various kinds of cell stress. Under those conditions ceramide (Cer) is produced in the plasma membrane as a product of sphingomyelin (SM) hydrolysis, and this may lead to apoptosis. Thus, SM and Cer coexist in the membrane for some time, and they are known to separate laterally from the (more abundant) glycerolipids, giving rise to highly rigid domains or platforms. The properties of these domains/platforms are rather well understood, but the underlying SM:Cer molecular interactions have not been explored in detail. Infrared (IR) spectroscopy is a powerful analytical technique that provides information on all the chemical groupings in a molecule, and that can be applied to membranes and lipid bilayers in aqueous media. IR spectra can be conveniently retrieved as a function of temperature, thus revealing the thermotropic transitions of SM and its mixtures with Cer. Four regions of the IR spectrum of these sphingolipids have been examined, two of them dominated by the hydrophobic regions in the molecules, namely the C–H stretching vibrations (2800–3000 cm−1), and the CH2 scissoring vibrations (1455–1485 cm−1), and two others arising from chemical groups at the lipid-water interface, the sphingolipid amide I band (1600–1680 cm−1), and the phosphate vibrations in the 1000–1110 cm−1 region. The latter two regions have been rarely studied in the past. The IR data from the hydrophobic components show a gel (or ripple)-fluid transition of SM at 40 °C, that is shifted up to about 70 °C when Cer is added to the bilayers, in agreement with previous studies using a variety of techniques. IR information concerning the polar parts is more interesting. The amide I (carbonyl) band of pure SM exhibits a maximum at 1638 cm−1 at room temperature, and its position is shifted by about 10 cm−1 in the presence of Cer. Cer causes also a change in the overall band shape, but no signs of band splitting are seen, suggesting that SM and Cer carbonyl groups are interacting tightly, presumably through H-bonds. The 1086 cm−1 band, corresponding to PO2− vibrations, appears more stable in SM than in DPPC, and it is further stabilized by Cer, again suggesting an important role of H-bonds in the formation of SM:Cer clusters. Thus, SM and Cer can interact through their polar headgroups, in a way that is not accessible to other lipid classes.

ABSTRACTSphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of control LY-B cell growth rates. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led to a 6-fold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Concomitant changes were detected in glycerophospholipids under SL-restriction conditions.

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Biomembranes are assumed to contain lateral heterogeneities, usually called domains. When they are less than ∼100 nm in diameter, they are referred to as nanodomains. Biological nanodomains have been properly identified only in the twenty first century, largely through the advent of super‐resolution microscopy techniques. Size, irrespective of lifetime, is used as a defining parameter for nanodomains. The main methods used in these studies span from the arguably earliest atomic force microscopy detection of nanodomains in a lipid bilayer, in year 2000, to the most recent observations in living cells of synapsis, receptors, exocytosis or plant membranes, based on advanced fluorescence microscopy techniques. Key Concepts Nanodomains are lateral inhomogeneities in lipid bilayers or in cell membranes, of varying lifetimes, and diameters usually below ≈200 nm. Nanodomains are stabilised by low line tensions and high dipole–dipole interactions and crowding pressures. Observation of nanodomains requires the use of atomic force microscopy and super‐resolution fluorescence microscopy as the main tools.

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