Aritz B. García-Arribas

Lipid bilayers containing sphingomyelins and ceramides of varying N-acyl lengths: a glimpse into sphingolipid complexity.

The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain.

Lamellar Gel (Lβ) Phases of Ternary Lipid Composition Containing Ceramide and Cholesterol

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (Lβ) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide Lβ phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.

Atomic Force Microscopy Characterization of Palmitoylceramide and Cholesterol Effects on Phospholipid Bilayers: A Topographic and Nanomechanical Study

Supported planar bilayers (SPBs) on mica substrates have been studied at 23 °C under atomic force microscopy (AFM)-based surface topography and force spectroscopy with two main objectives: (i) to characterize palmitoylceramide (pCer)-induced gel (Lβ) domains in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin (pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC) and (ii) to evaluate effects of incorporating cholesterol (Chol) into the previous mixtures in terms of Cer and Chol cooperation for the generation of lamellar gel (Lβ) phases of ternary composition. Binary phospholipid/pCer mixtures at XpCer < 0.33 promote the generation of laterally segregated micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer ratios, by means of domain thickness, roughness, and mechanical resistance to tip piercing, reveals unvarying AFM-derived features over increasing pCer concentrations. These results suggest that the domains grow in size with increasing pCer concentrations while keeping a constant phospholipid/pCer stoichiometry. Moreover, the data show important differences between pCer interactions with pSM or DPPC. Gel domains generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore, a higher breakthrough force is observed for pSM/pCer as compared to DPPC/pCer domains, which can be associated with the preferential pCer interaction with its sphingolipid relative pSM. Cholesterol incorporation into both binary mixtures at a high Chol and pCer ratio abolishes any phospholipid/pCer binary domains. Bilayers with properties different from any of the pure or binary samples are observed instead. The data support no displacement of Chol by pCer or vice versa under these conditions, but rather a preferential interaction between the two hydrophobic lipids.

Ether- versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains

We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces Fb decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different Fb values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.

Ceramide increases free volume voids in DPPC membranes

Positron annihilation lifetime spectroscopy (PALS) can measure changes in local free volume voids in lipid bilayers. PALS has been applied, together with differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations, to study free volume voids in DPPC and DPPC : ceramide (85 : 15 mol : mol) model membranes in the 20–60 °C range. The free volume void average size clearly increases with the gel–fluid phase transition of the lipid, or lipid mixture. Ceramide increases void size at all temperatures, particularly in the range causing the gel–fluid transition of the mixture. A parallel study of PALS and calorimetric data indicates that, for the complex thermotropic transition of the DPPC–ceramide mixture, PALS is detecting the transition of the DPPC component, while calorimetry changes indicate mainly the melting of the ceramide-enriched domains. Molecular dynamics calculations provide a clear distinction between ceramide-rich and poor domains, and show that the voids are predominantly located near the membrane nodal plane. The ceramide-induced increase in void volume size occurs as well at temperatures when both phospholipid and ceramide are in the fluid state, indicating that the effect is not the result of phospholipid–ceramide domain coexistence. The above observations may be related to hitherto unexplained properties of ceramide, such as the increase in membrane permeability, and the induction of transmembrane (flip-flop) lipid motion.

Cholesterol–Ceramide Interactions in Phospholipid and Sphingolipid Bilayers As Observed by Positron Annihilation Lifetime Spectroscopy and Molecular Dynamics Simulations

Free volume voids in lipid bilayers can be measured by positron annihilation lifetime spectroscopy (PALS). This technique has been applied, together with differential scanning calorimetry and molecular dynamics (MD) simulations, to study the effects of cholesterol (Chol) and ceramide (Cer) on free volume voids in sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC) bilayers. Binary lipid samples with Chol were studied (DPPC:Chol 60:40, SM:Chol 60:40 mol ratio), and no phase transition was detected in the 20-60 °C range, in agreement with calorimetric data. Chol-driven liquid-ordered phase showed an intermediate free volume void size as compared to gel and fluid phases. For SM and SM:Cer (85:15 mol:mol) model membranes measured in the 20-60 °C range the gel-to-fluid phase transition could be observed with a related increase in free volume, which was more pronounced for the SM:Cer sample. MD simulations suggest a hitherto unsuspected lipid tilting in SM:Cer bilayers but not in pure SM. Ternary samples of DPPC:Cer:Chol (54:23:23) and SM:Cer:Chol (54:23:23) were measured, and a clear pattern of free volume increase was observed in the 20-60 °C because of the gel-to-fluid transition. Interestingly, MD simulations showed a tendency of Cer to change its distribution along the membrane to make room for Chol in ternary mixtures. The results suggest that the gel phase formed in these ternary mixtures is stabilized by Chol-Cer interactions.

Complex Effects of 24:1 Sphingolipids in Membranes Containing Dioleoylphosphatidylcholine and Cholesterol

The effects of C24:1 sphingolipids have been tested in phospholipid bilayers containing cholesterol. Confocal microscopy, differential scanning calorimetry, and atomic force microscopy imaging and force curves have been used. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide, nCer) were evaluated and compared to those of C16:0 ceramide (palmitoyl ceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcholine, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol. Combination of equimolecular amounts of C24:1 and C16:0 sphingolipids were also studied under the same conditions. Results show that both pCer and nCer are capable of forming segregated gel domains. Force spectroscopy data point to nCer having a lower stiffening effect than pCer, while the presence of nSM reduces the stiffness. DSC reveals Tm reduction by nSM in every case. Furthermore, pSM seems to better accommodate both ceramides in a single phase of intermediate properties, while nSM partial accommodation of ceramides generates different gel phases with higher stiffnesses caused by interceramide cooperation. If both pSM and nSM are present, a clear preference of both ceramides toward pSM is observed. These findings show the sharp increase in complexity when membranes exhibit different sphingolipids of varying N-acyl chains, which should be a common issue in an actual cell membrane environment.

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Coating Graphene Oxide with Lipid Bilayers Greatly Decreases Its Hemolytic Properties

Toxicity evaluation for the proper use of graphene oxide (GO) in biomedical applications involving intravenous injections is crucial, but the GO circulation time and blood interactions are largely unknown. It is thought that GO may cause physical disruption (hemolysis) of red blood cells. The aim of this work is to characterize the interaction of GO with model and cell membranes and use this knowledge to improve GO hemocompatibility. We have found that GO interacts with both neutral and negatively charged lipid membranes; binding is decreased beyond a certain concentration of negatively charged lipids and favored in high-salt buffers. After this binding occurs, some of the vesicles remain intact, while others are disrupted and spread over the GO surface. Neutral membrane vesicles tend to break down and extend over the GO, while vesicles with negatively charged membranes are mainly bound to the GO without disruption. GO also interacts with red blood cells and causes hemolysis; hemolysis is decreased when GO is previously coated with lipid membranes, particularly with pure phosphatidylcholine vesicles.