Liquid-ordered Membrane Research Articles

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

The neutral Red probe location in large unilamellar vesicles: Influence of membrane composition and phase states

Biological membranes are complex and dynamic self-assembled structures composed of lipids and proteins. They depict emergent properties that are heterogeneous in the membrane plane. Therefore, their study is currently one of the greatest challenges in biophysics.The versatility of spectroscopic techniques has encouraged the development of new fluorescent probes capable of sensing different membrane properties. The Neutral Red (NR) molecule is a dye that protonates at pH < 6.8 (pKa = 6.8), leading to the charged species (NRH+). NR is widely employed in molecular biology to assess cell viability and mitochondrial function, and to sense intracellular pH. However, the photophysical behavior of NR in complex media such as membranes were not systematically characterized even though of the wide application in molecular biology.In this work, the photophysical behavior of NNR (pH 8 neutral specie) and NRH+ (pH 4) was studied by employing large unilamellar vesicles (LUVs) with different compositions, membrane phase states.The results of this study show that the spectroscopic behavior of NNR is more sensitive to the membrane composition than that of NRH+. The wavelength of the maximum emission of NNR was at shorter wavelengths in liquid-disordered (Ld) membranes compared to gel or liquid-ordered (Lo) membranes. The Red-edge excitation shifts (REES) and Anisotropy magnitudes were lower in membranes in the gel or Lo, compared to membranes in Ld. The same tendency was observed for NRH+, although the spectral changes in this case were smaller, as well as the changes in the REES effect, and anisotropy magnitudes were lower related to the NR species. We suggest that NNR and NRH+ are localized in different regions of the membrane depending on the membrane phase estate. NR and NRH+ are located in more internal regions of the interface in membranes in Ld phase state, whereas in gel or Lo phase membranes, NR is located in a more external region, and NRH+ is adsorbed on the membrane surface. Consequently, the spectral signals of both, NNR and NRH+, correspond to the species distributed in different locations of the membrane in Ld compared to gel and Lo phase. Therefore, the information provided by the probe inserted in different membranes is not directly comparable.

Characterizing the transmembrane domains of ADAM10 and BACE1 and the impact of membrane composition

The β-secretase, BACE1, and the α-secretase, ADAM10, are known to competitively cleave amyloid precursor protein (APP) in the amyloid cascades of Alzheimer’s disease. Cleavage of APP by BACE1 produces a 99-residue C-terminal peptide (APP-C99) that is subsequently cleaved by γ-secretase to form amyloid-β (Aβ) protein, whereas cleavage of APP by ADAM10 is nonamyloidogenic. It has been speculated that ADAM10/APP and BACE1/APP interactions are regulated by colocalization within and outside of liquid-ordered membrane domains; however, the mechanism of this regulation and the character of the proteins’ transmembrane domains are not well understood. In this work, we have developed and characterized minimal congener sequences for the transmembrane domains of ADAM10 and BACE1 using a multiscale modeling approach combining both temperature replica exchange and conventional molecular dynamics simulations based on the coarse-grained Martini2.2 and all-atom CHARMM36 force fields. Our results show that membrane composition impacts the character of the transmembrane domains of BACE1 and ADAM10, adding credence to the speculation that membrane domains are involved in the etiology of Alzheimer’s disease.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

Vacuole membrane contact sites regulate liquid-ordered domain formation during glucose starvation.

Liquid-ordered (Lo) membrane domains have been proposed to play important roles in a wide variety of biological processes, such as protein sorting and cell signaling. However, the mechanisms by which they are formed and maintained remain poorly understood. Lo domains are formed in the vacuolar membrane of yeast in response to glucose starvation. Here, we show that the deletion of proteins that localize to vacuole membrane contact sites (MCSs) caused a marked decrease in the number of cells with Lo domains. In addition to Lo domain formation, autophagy is induced upon glucose starvation. However, the deletion of core autophagy proteins did not inhibit Lo domain formation. Thus, we propose that vacuolar Lo domain formation during glucose restriction is regulated by MCSs but not by autophagy.

Modeling of Transmembrane Domain and Full-Length TLRs in Membrane Models.

Toll-like receptors (TLRs), classified as pattern recognition receptors, have a primordial role in the activation of the innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria, and, together withMyeloid Differentiation factor 2(MD-2) protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. Identification of TLRs has sparked great interest in the therapeutic manipulation of the innate immune system. In particular, TLR4 antagonists may be useful for the treatment of septic shock, certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain, and TLR4 agonists are under development as vaccine adjuvants in antitumoral treatments. Therefore, TLR4 has risen as a promising therapeutic target, and its modulation constitutes a highly relevant and active research area. Deep structural understanding of TLR4 signaling may help in the design and discovery of TLR4-modulating molecules with desirable therapeutic properties.Computational studies of the different independent domains composing the TLR4 were undertaken, to understand the differential domain organization of TLR4 in aqueous and membraneenvironments, including Liquid-disordered (Ld)and Liquid-ordered (Lo)membrane models, to account for the TLR4 recruitment in lipid rafts over activation. We modeled, by means of all-atom Molecular Dynamics (MD) simulations, the structural assembly of plausible full-length TLR4 models embedded into a realistic plasma membrane, accounting for the active (agonist) state of the TLR4, thus providing an analysis at both atomic/molecular and thermodynamic levels of the TLR4 assembly and biological activity. Our results unveil relevant molecular aspects involved in the mechanism of receptor activation, and adaptor recruitment in the innate immune pathways, and will promote the discovery of new TLR4 modulators and probes.

Natamycin interferes with ergosterol-dependent lipid phases in model membranes

Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.

Sensory axons induce epithelial lipid microdomain remodeling and determine the distribution of junctions in the epidermis.

Epithelial cell properties are determined by the polarized distribution of membrane lipids, the cytoskeleton, and adhesive junctions. Epithelia are often profusely innervated, but little work has addressed how neurites affect epithelial organization. We previously found that basal keratinocytes in the zebrafish epidermis enclose axons in ensheathment channels sealed by autotypic junctions. Here we characterized how axons remodel cell membranes, the cytoskeleton, and junctions in basal keratinocytes. At the apical surface of basal keratinocytes, axons organized lipid microdomains quantitatively enriched in reporters for PI(4,5)P2 and liquid-ordered (Lo) membranes. Lipid microdomains supported the formation of cadherin-enriched, F-actin protrusions, which wrapped around axons, likely initiating ensheathment. In the absence of axons, cadherin-enriched microdomains formed on basal cells but did not organize into contiguous domains. Instead, these isolated domains formed heterotypic junctions with periderm cells, a distinct epithelial cell type. Thus, axon endings dramatically remodel polarized epithelial components and regulate epidermal adhesion.

Transcriptomic Analysis Reveals Receptor Subclass-Specific Immune Regulation of CD8+ T Cells by Opioids.

Opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (OR) are expressed on immune cells. Exposure of human circulating CD8+ T cells to selective OR agonists differentially regulates thousands of genes. Gene set enrichment analysis reveals that μ-OR more strongly regulates cellular processes than δ-OR. In TCR naive T cells, triggering μ-OR exhibits stimulatory and inhibitory patterns, yet when administered prior to TCR cross-linking, a μ-OR agonist inhibits activation. μ-OR, but not δ-OR, signaling is linked to upregulation of lipid, cholesterol, and steroid hormone biosynthesis, suggesting lipid regulation is a mechanism for immune suppression. Lipid rafts are cholesterol-rich, liquid-ordered membrane domains that function as a nexus for the initiation of signal transduction from surface receptors, including TCR and μ-OR. We therefore propose that μ-OR-specific inhibition of TCR responses in human CD8+ T cells may be mediated through alterations in lipid metabolism and membrane structure.

The endosomal sorting complex required for transport complex negatively regulates Erg6 degradation under specific glucose restriction conditions.

Lipid droplets (LDs) are cytosolic fat storage organelles that play roles in lipid metabolism, trafficking and signaling. Breakdown of LDs in Saccharomyces cerevisiae is mainly achieved by lipolysis and lipophagy. In this study, we found that the endosomal sorting complex required for transport (ESCRT) in S. cerevisiae negatively regulated the turnover of a LD marker, Erg6, under both simplified glucose restriction (GR) and acute glucose restriction (AGR) conditions by monitoring the localization and degradation of Erg6. Loss of Vps27, Snf7 or Vps4, representative subunits of the ESCRT machinery, facilitated the delivery of Erg6-GFP to vacuoles and its degradation depending on the lipophagy protein Atg15 under simplified GR. Additionally, the lipolysis proteins Tgl3 and Tgl4 were also involved in the enhanced vacuolar localization and degradation of Erg6-GFP in vps4Δ cells. Furthermore, we found that Atg14, which is required for the formation of putatively liquid-ordered (Lo) membrane domains on the vacuole that act as preferential internalization sites for LDs, abundantly localized to vacuolar membranes in ESCRT mutants. Most importantly, the depletion or overexpression of Atg14 correspondingly abolished or promoted the observed Erg6 degradation in ESCRT mutant cells. We propose that Atg14 together with other proteins promotes Erg6 degradation in ESCRT mutant cells under specific glucose restriction conditions. These results shed new light on the regulation of ESCRT on LD turnover.

Real-time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells.

Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure.

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene probe behaviour in heterogeneous membranes. We characterised the pyrene fluorescence spectra in liquid-ordered (Lo) and liquid-disordered (Ld) membranes. Using statistical multivariate analysis, we found out the most appropriate wavelengths for membrane domains studies. 373 nm and 379 nm were the most discriminant wavelengths to follow the liquid-ordered and the liquid-disordered environments. Cholesterol clustering behaviour was quantified by the modulation of the cholesterol-pyrene excimers peak (474 nm). In liquid-ordered membranes at low temperature, cholesterol-pyrene was found as multimers and as monomers. At high temperature, the liquid-ordered status of the membrane decreases and cholesterol-pyrene tends to cluster. In liquid-disordered membranes, cholesterol-pyrene was present mostly as monomers and the small quantity of excimers increased with temperature. Cholesterol-pyrene was used to test the ceramide effect on membranes, and presented a behaviour in agreement with the cholesterol behaviour reported in the literature. Overall, the presented data show that cholesterol-pyrene is an efficient sensor to study liquid ordered and liquid disordered organisation in membranes.

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.

How to minimize dye-induced perturbations while studying biomembrane structure and dynamics: PEG linkers as a rational alternative

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.

Anticancer Agent Edelfosine Exhibits a High Affinity for Cholesterol and Disorganizes Liquid-Ordered Membrane Structures.

Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.

The Lipid Raft Proteome of African Trypanosomes Contains Many Flagellar Proteins.

Lipid rafts are liquid-ordered membrane microdomains that form by preferential association of 3-β-hydroxysterols, sphingolipids and raft-associated proteins often having acyl modifications. We isolated lipid rafts of the protozoan parasite Trypanosoma brucei and determined the protein composition of lipid rafts in the cell. This analysis revealed a striking enrichment of flagellar proteins and several putative signaling proteins in the lipid raft proteome. Calpains and intraflagellar transport proteins, in particular, were found to be abundant in the lipid raft proteome. These findings provide additional evidence supporting the notion that the eukaryotic cilium/flagellum is a lipid raft-enriched specialized structure with high concentrations of sterols, sphingolipids and palmitoylated proteins involved in environmental sensing and cell signaling.

Colocalization of receptors for Shiga toxins with lipid rafts in primary human renal glomerular endothelial cells and influence of D-PDMP on synthesis and distribution of glycosphingolipid receptors.

Damage of human renal glomerular endothelial cells (HRGECs) of the kidney represents the linchpin in the pathogenesis of the hemolytic uremic syndrome caused by Shiga toxins of enterohemorrhagic Escherichia coli (EHEC). We performed a comprehensive structural analysis of the Stx-receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer) and their distribution in lipid raft analog detergent-resistant membranes (DRMs) and nonDRMs prepared from primary HRGECs. Predominant receptor lipoforms were Gb3Cer and Gb4Cer with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0). Stx-receptor GSLs co-distribute with sphingomyelin (SM) and cholesterol as well as flotillin-2 in DRMs, representing the liquid-ordered membrane phase and indicating lipid raft association. Lyso-phosphatidylcholine (lyso-PC) was identified as a nonDRM marker phospholipid of the liquid-disordered membrane phase. Exposure of primary HRGECs to the ceramide analogon d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) reduced total Gb3Cer and Gb4Cer content, roughly calculated from two biological replicates, down to half and quarter of its primordial content, respectively, but strengthened their prevalence and cholesterol preponderance in DRMs. At the same time, the distribution of PC, SM and lyso-PC to subcellular membrane fractions remained unaffected by D-PDMP treatment. Defining the GSL composition and precise microdomain structures of primary HRGECs may help to develop novel therapeutic options to combat life-threatening EHEC infections.

Effects of seaweed sterols fucosterol and desmosterol on lipid membranes

Higher sterols are universally present in large amounts (20–30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

Elucidating PI(4,5)P2 Aqueous Micelle Behavior and PI(4,5)P2 Cluster Formation in an Inner Leaflet Model Membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important component of the inner leaflet of the plasma membrane (PM) in eukaryotic cells. In model membranes, others have found that PI(4,5)P2 can form segregated clusters at high non-physiological concentrations. How PI(4,5)P2 behaves in membranes with inner leaflet compositions (i.e. PE/PS/PI/Chol) at physiological concentrations of 1-2 mol% is not known. We used several PI(4,5)P2 species, including brain-PI(4,5)P2 and the acyl-chain fluorescently labeled TopFluor-PI(4,5)P2 in fluorometric and microscopic assays. To better understand the behavior of this unusual phospholipid, we studied the partitioning of PI(4,5)P2 between an aqueous environment and membrane bilayers. In the absence of other lipids, PI(4,5)P2 forms micelles spontaneously at 1-2μM at pH = 7.2, detected by light scattering. In the presence of approximately 300-500 μM total lipids, PI(4,5)P2 incorporates efficiently into bilayers within minutes and does not partition out of bilayers after hours. Partitioning of brain-PI(4,5)P2 from solution into bilayer favors liquid-disordered compared to liquid-ordered membranes. Next, we found that TopFluor-PI(4,5)P2 is highly quenched in inner leaflet model membranes below 0.2%, suggesting strong clustering. In stark contrast, TopFluor labeled PE, PC and PS exhibited no quenching at these concentrations. We are currently studying whether other lipids, such as PI, or acyl chain composition or cholesterol concentration, influence PI(4,5)P2 clustering behavior. We are also studying the effects of PI(4,5)P2 clustering on cellular peripheral proteins and how these PI(4,5)P2-specific binding partners influence PI(4,5)P2 behaviors. For example, does protein binding promote or disrupt PI(4,5)P2 clustering? These findings may be important for understanding key functions of PI(4,5)P2 in cellular biology and virus research.