CHO/LY-B cell growth under limiting sphingolipid supply: Correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes.

Sphingolipids (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 linein whichserine 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 this 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%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. 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 only to a sixfold 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. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions.

Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients.

This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar-nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.

Lipid Self-Assemblies under the Atomic Force Microscope.

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid-lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Homogeneous and Heterogeneous Bilayers of Ternary Lipid Compositions Containing Equimolar Ceramide and Cholesterol.

Cell membranes have been proposed to be laterally inhomogeneous, particularly in the case of mammalian cells, due to the presence of "domains" enriched in sphingolipids and cholesterol (Chol). Among membrane sphingolipids, sphingomyelin (SM) in the cell plasma membrane is known to be degraded to ceramide (Cer) by acid sphingomyelinases under stress conditions. Since cholesterol (Chol) is abundant in the plasma membrane, the study of ternary mixtures SM:Chol:Cer is interesting from the point of view of membrane biophysics, and it might be physiologically relevant. In previous studies, we have described the homogeneous gel phase formed by phospholipid:Chol:Cer at 54:23:23 mol ratios, where phospholipid was either SM or dipalmitoylphosphatidylcholine (DPPC). We now provide new data, based on trans-parinaric acid and diphenylhexatriene fluorescence, supporting that the gel phase includes all three components in a single bilayer. The main question addressed in this paper is the stability of the ternary gel phase when bilayer composition is changed, specifically when the SM proportion is varied. To this aim, we have prepared bilayers of composition phospholipid:Chol:Cer at X:Y:Y ratios, in which phospholipid increased between 54 and 70 mol %. The N-palmitoyl derivatives of SM (pSM) and Cer (pCer) have been used. We observe that for X = 54 or 60 mol %, a gel phase is clearly predominant. However, when the proportion of phospholipid increases beyond 60 mol %, i.e., in 66:17:17 or 70:15:15 mixtures, a lateral phase separation occurs at the micrometer scale. These data can be interpreted in terms of a pCer:Chol interaction, that would predominate at the lower phospholipid concentrations. The putative pCer:Chol complexes (or nanodomains) would mix well with the phospholipid. At the higher SM concentrations pSM:pCer and pSM:Chol interactions would become more important, giving rise to the coexisting gel and liquid-ordered phases respectively. Heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicating a higher affinity of SM over DPPC for Chol or Cer. The observation that heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicates a higher affinity of SM over DPPC for Chol or Cer, and can be related to cell regulation through the sphingolipid signaling pathway.

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.

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.

Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts.

The effects of increasing amounts of palmitoylceramide (pCer) on human red blood cell lipid membranes have been studied using atomic force microscopy of supported lipid bilayers, in both imaging (bilayer thickness) and force-spectroscopy (nanomechanical resistance) modes. Membranes appeared homogeneous with pCer concentrations up to 10 mol % because of the high concentration of cholesterol (Chol) present in the membrane (∼45 mol %). However, the presence of pCer at 30 mol % gave rise to a clearly distinguishable segregated phase with a nanomechanical resistance 7-fold higher than the continuous phase. These experiments were validated using differential scanning calorimetry. Furthermore, Chol depletion of the bilayers caused lipid domain generation in the originally homogeneous samples, and Chol-depleted domain stiffness significantly increased with higher amounts of pCer. These results point to the possibility of different kinds of transient and noncompositionally constant, complex gel-like phases present in RBC lipid membranes rich in both pCer and Chol, in contrast to the widespread opinion about the displacements between pCer-enriched "gel-like" domains and liquid-ordered "raft-like" Chol-enriched phases. Changes in the biophysical properties of these complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine-tuning the properties of membranes as required.

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.

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.

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 (Lß) 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 (Lß) 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.

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.

Lamellar gel (lß) 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 (Lß) 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 Lß 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.

Plasma membrane effects of sphingolipid-synthesis inhibition by myriocin in CHO cells: a biophysical and lipidomic study.

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.

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

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.

ß-Amyloid (1-42) peptide adsorbs but does not insert into ganglioside-containing phospholipid membranes in the liquid-disordered state: modelling and experimental studies.

ß-Amyloid (Aß) is a 39-43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aß deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aß42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1-5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aß42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aß binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aß plaque formation.

Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough "holes" so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

The interaction of lipid-liganded gold clusters (Aurora ™) with lipid bilayers.

Lipid bilayers of different phospholipid compositions have been prepared, in the form of vesicles, or of supported lipid bilayers, and doped with Aurora™ at 0.1 mol%. Aurora™ consists of an Au55 gold nanoparticle (about 1.4 nm in diameter) capped with triphenylphosphine ligands and a single diglyceride (distearoyl glycerol) ligand. Gold nanoparticles have been incorporated in the past inside liposomes, or grafted onto their surfaces, with diagnostic or therapeutic aims. Including the gold nanoparticles in a stable form within the lipid bilayers has serious technical difficulties. We have tested the hypothesis that, because of the diglyceride ligand, Aurora™ would allow the easy incorporation of gold nanoclusters into cell membranes or lipid bilayers. Our results show that Aurora™ readily incorporates into lipid bilayers, particularly when they are in the fluid phase, i.e. the state in which cell membranes exist. Calorimetric, fluorescence polarization or fluorescence confocal microscopy concur in showing that bilayer-embedded Aurora™hardly changes the physical properties of the bilayers, nor does it perturb the phase equilibrium in lipid mixtures giving rise to lateral phase separation in the plane of the membrane. Atomic force microscopy shows, in fluid bilayers, well-resolved particles, 1.2-2.9 nm in height, that are interpreted as single Aurora™conjugates. Cryo-transmission electron microscopy allows the clear observation of lipid bilayers with an enhanced contrast due to the Aurora™ gold nanoparticles; the single particles can be resolved at high magnification. Our studies support the applicability of Aurora™ as a membrane-friendly form of nano-gold particles for biological research or clinical applications.

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.