Formation Of Membrane Domains Research Articles

Protein Domain Formation in Lipid Membranes

Clustering of Gramicidin within a DMPC membrane has been studied with Small Angle Neutron Scattering (SANS). Hydrogen and Deuterium scatter neutrons very differently, thus deuteration allows protein scattering to be studied independent of lipid and solvent scattering (when the lipid and solvent are contrast matched). Different protein to lipid ratios were probed and a strict protocol was followed to ensure uniform vesicle size with limited polydispersity. The experiments were performed above the melting temperature of DMPC.A 100 nm deuterated lipid vesicle in deuterated solvent exhibits q-independent scattering showing that the two are truly contrast matched (see Figure 1). While the scattering obtained from lipid vesicles containing Gramicidin have significant q-dependent scattering. If the Gramicidin were uniformly distributed throughout the membrane the data should be well represented by vesicle scattering. However the vesicle fit of the data (also shown in Figure 1) clearly does not agree with the experimental scattering. Thus it is concluded that the protein is forming clusters within the lipid membrane giving rise to the difference in scattering. The system has been modeled and a 3D contrast map (inset Figure 1) has been generated. The map shows protein clustering within the membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein-Lipid Interaction and Domain Formation in Asymmetric Membranes

The lateral organization of lipids and receptors in cellular membranes is connected to a multitude of biological and pathological processes. The role of sphingolipid- and cholesterol-rich lipid “rafts” as membrane domains that control protein-protein interaction has recently attracted much attention. While the model membrane approach has yielded fundamental insights into cell membrane structure and function, the majority of these studies did not take into account the very important asymmetry between the composition of the inner and the outer leaflets of cell membranes. Therefore, certain key questions remain still unanswered: for example, how is the partition of membrane components in raft domains affected by the presence of non-raft lipids in the inner leaflet? To answer questions of this type we have developed a new method based upon cyclodextrin-induced lipid exchange to create giant unilamellar vesicles (GUVs) featuring the same type of asymmetry encountered in biological membranes. These vesicles can be produced with a very large yield, while avoiding use of organic solvents. The asymmetry of the bilayer can be confirmed by leaflet-targeted Fluorescence Correlation Spectroscopy (FCS). The use of asymmetric GUVs as novel model bilayers should allow the investigation of the principles underlying communication between raft-like domains in opposite leaflets of the bilayer, as well as investigation of the origin of lipid and membrane protein affinity for rafts.

Breaching the Membrane Barrier with Antimicrobial Agents that Cluster Anionic Lipids

The bacterial membrane plays an important role in the action of antimicrobial agents. The presence of a larger exposed fraction of negatively charged lipids in bacterial membranes contributes to the higher toxicity against bacteria. The mechanism of action of many antimicrobial agents is thought to be by damaging the bacterial membrane. Several mechanisms exist that result in such damage. The membrane also must be breached in order for the agent to reach an intracellular target. One recently recognized contribution to membrane damage by certain antimicrobial agents is their ability to cluster anionic lipids from zwitterionic lipids. This results in the formation of membrane domains enriched in the antimicrobial agent and the anionic lipid. Such lipid clustering has been demonstrated by DSC, FTIR, 31P-MAS/NMR, 2H-NMR, freeze fracture transmission electron microscopy and AFM combined with polarized fluorescence microscopy. In cases where this is the principal mechanism of membrane damage, it predicts that those species of bacteria whose membrane is composed largely of anionic lipids are more resistant to these agents, while other bacterial species that contain both anionic and zwitterionic lipids in their membrane exhibit greater susceptibility. The smallest active antimicrobial fragment of LL-37 (KRIVQRIKDFLR) is capable of inducing clustering of anionic lipids and is toxic against E. coli that has a high PE content but not against S. aureus that is composed largely of anionic lipids. The loss of both lipid clustering ability and antimicrobial action that occurs on removal of two cationic residues to make RI-10, gives further support to the role of lipid clustering in the antimicrobial activity. These predictions also hold well for certain antimicrobial oligo-acyl-lysines and also for the peptide PFWRIRIRR-amide and its analogs against several Gram positive bacterial strains having different membrane compositions.

Gangliosides and β1-Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin-mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo-glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol ('lipid rafts'), reduced caveolae and caveolin-1 at the plasma membrane by approximately 80%, and blunted activation of beta1-integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo-gangliosides or other sphingolipids) at 10 degrees C, suggesting that sialo-lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Igamma away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin-1 could be recapitulated by beta1-integrin knockdown. These results suggest that both gangliosides and beta1-integrin are required for maintenance of caveolae and plasma membrane domains.

Inhibiting Lateral Domain Formation in Lipid Bilayers: Simulations of Alternative Steroid Headgroup Chemistries

Cholesterol is an essential component of lateral domain formation in lipid bilayers. Experiments using structural analogues of cholesterol have revealed that slight changes to the steroid headgroup chemistry can lead to significantly different degrees of domain formation. For example, the seemingly slight modification to 4-cholesten-3beta-one changes the molecule from domain enhancing to domain inhibiting. To investigate the relationship between steroid headgroup structure and domain formation, we have performed a series of coarse-grain molecular dynamics simulations in which the hydrophobicity of the steroid headgroup is altered. We show that bilayers containing steroids with polar headgroups undergo lateral phase separation, with the steroids adopting the canonical, upright orientation. However, increasing the hydrophobicity of the steroid's headgroup dramatically changes the orientation of the molecule such that it lies at the center of the bilayer, perpendicular to the bilayer normal. As a fundamental consequence, we show that these steroids inhibit the formation of phase-separated domains in membranes.

Biophysical studies of the interactions between the phage ϕKZ gp144 lytic transglycosylase and model membranes

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.

Calcium-Dependent Lateral Organization in Phosphatidylinositol 4,5-Bisphosphate (PIP2)- and Cholesterol-Containing Monolayers

Biological membrane function, in part, depends upon the local regulation of lipid composition. The spatial heterogeneity of membrane lipids has been extensively explored in the context of cholesterol and phospholipid acyl-chain-dependent domain formation, but the effects of lipid head groups and soluble factors in lateral lipid organization are less clear. In this contribution, the effects of divalent calcium ions on domain formation in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2), a polyanionic, multifunctional lipid of the cytosolic leaflet of the plasma bilayer, are reported. In binary monolayers of PIP2 mixed with zwitterionic lipids, calcium induced a rapid, PIP2-dependent surface pressure drop, with the concomitant formation of laterally segregated, PIP2-rich domains. The effect was dependent upon head-group multivalency, because lowered pH suppressed the surface-pressure effect and domain formation. In accordance with previous observations, inclusion of cholesterol in lipid mixtures induced coexistence of two liquid phases. Phase separation strongly segregated PIP2 to the cholesterol-poor phase, suggesting a role for cholesterol-dependent lipid demixing in regulating PIP2 localization and local concentration. Similar to binary mixtures, subphase calcium induced contraction of ternary cholesterol-containing monolayers; however, in these mixtures, calcium induced an unexpected, PIP2- and multivalency-dependent decrease in the miscibility phase transition surface pressure, resulting in rapid dissolution of the domains. This result emphasizes the likely critical role of subphase factors and lipid head-group specificity in the formation and stability of cholesterol-dependent domains in cellular plasma membranes.

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.

A monolayer study on phase behavior and morphology of binary mixtures of sulfatides with DPPC and DPPE

Sulfatides are important constituents of brain myelin membranes and it is thought to be involved in lateral domain formation in biological membranes. In this work, the interaction of mixed systems of sulfatide with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1, 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine (DPPE), two of the major components in biological membranes, was investigated using the monolayer technique at the air–water interface. Based on the regular solution theory, the miscibility of the two binary systems in the mixed monolayer was evaluated in terms of mean surface area per molecule ( A m), excess molecular area (Δ A (ex)), surface excess Gibbs energy (Δ G (ex)), interaction parameter ( ω) as well as activity coefficients ( f 1 and f 2) of formed films. Thermodynamic analysis indicates in the two binary systems with negative deviations from the ideal behavior. Accordingly, the values of the Gibbs energy of mixing, sulfatide-DPPC form stable mixtures at X sul = 0.4 ( X sul is molar ratio of sulfatide in binary mixture) for all the selected pressures. As for sulfatide/DPPE system, at π = 5 and 30 mN m −1, the minimum for the Gibbs energy of mixing was found at X sul = 0.6 and 0.2 respectively. But the minimum appeared at X sul = 0.4 for other surface pressures. The activity coefficients ( f 1 and f 2) of mixed monolayers were evaluated which show a marked dependence on the mole faction of sulfatide X sul. AFM images could support the above findings as well as interpretation.

Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry

The solubilization and reconstitution of biological or liposomal membranes by detergents and biomolecules with detergent-like properties play a major role for technical applications (e.g., the isolation of membrane proteins) and biological phenomena (of, e.g., amphiphilic peptides). It is therefore important to know and understand the amounts of a given detergent required for the onset and completion of membrane solubilization and the detergent-lipid interactions in general. Lipid-detergent systems can form a variety of aggregate structures, which can be grouped into two pseudophases (lamellae and micelles) so that solubilization can be approximately described as a phase transition. Here we present a protocol for establishing the phase diagram and a detailed thermodynamic description of a lipid-detergent system based on isothermal titration calorimetry (ITC). The protocol can also be used to detect additive-induced membrane destabilization, permeabilization, domain formation and lipid-dependent transitions between rod-like and spherical micelles. A minimal protocol consisting of all sample preparation procedures and a single solubilization experiment can be accomplished within 2 days; a more extensive series comprising both solubilization and reconstitution experiments requires several days to a few weeks, depending on the number of titrations performed.

Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes

A methyl-beta-cyclodextrin-induced lipid exchange technique was devised to prepare small unilamellar vesicles with stable asymmetric lipid compositions. Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-beta-cyclodextrin exchange step. In asymmetric vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition. Analogous behavior was observed in vesicles containing cholesterol. This shows that an asymmetric lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asymmetric vesicles containing approximately 25 mol % cholesterol formed ordered domains more thermally stable than those in asymmetric vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Additional studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asymmetric vesicles represents an important improvement in model membrane studies and should find many applications in the future.

New strategies for novel antibiotics: peptides targeting bacterial cell membranes

Membranes are targets of host defence or antimicrobial peptides, effector molecules of innate immunity that evolved in nature to contend with invaders as an active system of defence. The different physicochemical properties of the lipids found in biological membranes allow antimicrobial peptides to discriminate between bacterial and mammalian cell membranes. Such cationic amphipathic peptides will interact predominantly with negatively charged lipids exposed on the outer leaflet of bacterial cell membranes. The molecular mechanism(s) of membrane rupture mutually depends on the nature of the peptide and membrane lipid composition. Biophysical studies demonstrated a complex behavior in terms of membrane perturbation, which can range from pore formation to micellization (carpet model). Peptides aligned parallel to the membrane surface can induce a quasi-interdigitated structure in the gel phase, while depending on the hydrophobic matching of the lipid bilayer core and the peptide membrane thinning or thickening can be observed in the fluid phase. As a consequence, besides of peptide-lipid pores, formation of peptide-enriched membrane domains and promotion of cubic structures can be observed, which adversely affect membrane integrity and function. A strategy using the membrane damaging properties of these peptides will form the basis for the development of such peptides as potential novel antibiotic drugs.

Phase behavior of multicomponent membranes: Experimental and computational techniques

Recent developments in biology seems to indicate that the Fluid Mosaic model of membrane proposed by Singer and Nicolson, with lipid bilayer functioning only as medium to support protein machinery, may be too simple to be realistic. Many protein functions are now known to depend on the composition of the membrane. Experiments indicate that biomembranes of eukaryotic cells may be laterally organized into small nanoscopic domains. This inplane organization is expected to play an important role in a variety of physiological functions such as signaling, recruitment of specific proteins and endocytosis. However, mainly because of their complexity, the precise in-plane organization of lipids and proteins and their stability in biological membranes remain difficult to elucidate. This has reiterated the importance of understanding the equilibrium phase behavior and the kinetics of fluid multicomponent lipid membranes. Current increase in interest in the domain formation in multicomponent membranes also stems from the experiments demonstrating liquid ordered-liquid disordered coexistence in mixtures of lipids and cholesterol and the success of several computational models in predicting their behavior. This review includes basic foundations on membrane model systems and experimental approaches applied in the membrane research area, stressing on recent advances in the experimental and computational techniques.

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.

Nano-meter-sized domain formation in lipid membranes observed by small angle neutron scattering

Using a contrast matching technique of small angle neutron scattering (SANS), we have investigated a phase separation to liquid-disordered and liquid-ordered phases on ternary small unilamellar vesicles (SUVs) composed of deuterated-saturated, hydrogenated-unsaturated phosphatidylcholine lipids and cholesterol, where the equilibrium size of these domains is constrained to less than 10nm by the system size. Below a miscibility temperature, we observed characteristic scattering profiles with a maximum, indicating the formation of nano-meter-sized domains on the SUVs. The observed profiles can be described by a multi-domain model rather than a mono-domain model. The nano-meter-sized domain is agitated by thermal fluctuations and eventually ruptured, which may result in the multi-domain state. The kinetically trapped nano-meter-sized domains grow to a mono-domain state by decreasing temperature. Furthermore, between the miscibility and disorder-order transition temperature of saturated lipid, the integrated SANS intensity increased slightly, indicating the formation of nano-meter-sized heterogeneity prior to the domain nucleation.

Behavior of sulfatide/cholesterol mixed monolayers at the air/water interface

The monolayer properties of sulfatide and cholesterol binary system have been investigated with surface pressure–mean molecular area isotherms measurements and atomic force microscopy (AFM). The thermodynamic analysis indicates that the obtained negative deviation of the excess molecular area (Δ A (ex)) and surface excess Gibbs energy (Δ G (ex)) from the ideal behavior at various molar ratios, suggesting an attractive interaction between sulfatide and cholesterol in the monolayers as compared with the pure components monolayers. Meanwhile, the compression modulus ( C s − 1 ) vs. surface pressure ( π) and activity coefficients ( f 1 and f 2) of mixed films dependencies for mixed monolayers are drawn at different mole fractions. The AFM images for the mixed sulfatide/cholesterol monolayers deposited on the mica at 15 and 30 mN m −1 show the stronger molecular attractive force to form condensed structure. The behavior of sulfatide is thought to be involved in lateral domain formation in biological membranes. Therefore, the interaction between sulfatide and cholesterol becomes more important in mimicking “lipid rafts” domains.

Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15-32%. When the same quenchers have access to both leaflets, they quench 70-80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60-70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes approximately 40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.

FRET between non-substrate probes detects lateral lipid domain formation during phospholipase A 2 interfacial catalysis

The probes C 12-NBD-FA (12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)] dodecanoic acid) and C 18-R (octadecyl rhodamine B chloride) have been used as donor and acceptor, respectively, in FRET studies on liposomes subjected to pancreatic PLA 2 action. Neither of these fluorophores is a substrate for the enzyme but one of them, C 12-NBD-FA, is an analog of the fatty acid reaction product. The fluorophores were incorporated into 1,2-dipalmitoyl- sn-glycero-3-phosphatidylcholine (DPPC) liposomes and FRET was studied following the fluorescence of the donor, C 12-NBD-FA. Working with a molar ratio of acceptor to donor (A/D) of 1, we have found that FRET efficiency (E) decreases during DPPC hydrolysis. After 60 min, the decrease is equivalent to a reduction of more than five times in the effective A/D ratio, as estimated by interpolation in an efficiency vs. A/D reference curve. Using a more complete, empirical approach, the efficiency data, calculated from experiments at variable A/D proportions and constant donor concentration, were fitted by a rectangular hyperbolic function. The parameter K of this function, representing the A/D ratio at half-maximum transfer efficiency, increases more that five times after 60 min hydrolysis. This agrees with the reduction of the effective acceptor density sensed by the donor after hydrolysis, detected by the interpolation procedure. The heterogeneous distribution of acceptor and donor induced by hydrolysis can be attributed to the formation of product domains in the phospholipid membranes and is consistent with the preferential segregation of the donor, which is an analog of the fatty acid reaction product, in those domains. In conclusion, FRET between non-substrate probes detects the heterogeneities generated in phospholipid membranes by PLA 2 action.

Mass Spectral Imaging of Glycophospholipids, Cholesterol, and Glycophorin A in Model Cell Membranes

Time of flight secondary ion mass spectrometry (ToF-SIMS) and the Langmuir-Blodgett (LB) technique have been used to create and analyze reproducible membrane mimics of the inner and outer leaflets of a cellular membrane to investigate lipid-protein and lipid-lipid interactions. Films composed of phospholipids, cholesterol and an integral membrane protein were utilized. The results show the outer membrane leaflet mimic (DPPC/cholesterol/glycophorin A LB film) consisting of a single homogeneous phase whereas the inner membrane leaflet mimic (DPPE/cholesterol/glycophorin A LB film) displays heterogeneity in the form of two separate phases. A DPPE/cholesterol phase and a glycophorin A phase. This points to differences in membrane domain formation based upon the different chemical composition of the leaflets of a cell membrane. The reliability of the measurements was enhanced by establishing the influence of the matrix effect upon the measurement and by utlilizing PCA to enhance the contrast of the images.

Ceramide in bacterial infections and cystic fibrosis

Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhoeae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.