Dietary ganglioside decreases cholesterol content, caveolin expression and inflammatory mediators in rat intestinal microdomains

Membrane microdomains rich in cholesterol and sphingolipids, including gangliosides (GGs), are known to be important regions for cell signaling and binding sites for various pathogens. Cholesterol depletion inhibits the cellular entry of pathogens and also reduces inflammatory signals by disrupting microdomain structure. Our previous study showed that dietary gangliosides increased total ganglioside incorporation while decreasing cholesterol in the intestinal mucosa. We hypothesized that diet-induced reduction in cholesterol content in the intestinal mucosa disrupts microdomain structure resulting in reduced pro-inflammatory signals. Male weanling Sprague-Dawley rats were fed semipurified diets for 2 weeks. Experimental diets were formulated to include either ganglioside-enriched lipid (GG diet, 0.02% gangliosides [w/w of diet] ) or polyunsaturated fatty acid (PUFA diet, 1% arachidonic acid and 0.5% docosahexaenoic acid, w/w of total fat), in a control diet containing 20% fat. Levels of cholesterol, GG, caveolin, platelet activating factor (PAF), and diglyceride (DG) were measured in the microdomain isolated from the intestinal brush border. The GG diet increased total gangliosides by 50% with a relative increase in GD3 and a relative decrease in GM3. Cholesterol content was also reduced by 23% in the intestinal microdomain. These changes resulted in a significant decrease in the ratio of cholesterol to ganglioside. The GG diet and the PUFA diet were both associated with reduction in caveolin, PAF, and DG content in microdomains, whereas no change occurred in the ganglioside profile of animals fed the PUFA diet. Dietary gangliosides decrease the cholesterol/ganglioside ratio, caveolin, PAF and DG content in microdomains thus exerting a potential anti-inflammatory effect during gut development.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L_o) and liquid-disordered (L_d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L_o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In ¹³C-¹³C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.     

Application of highly purified anti-galactocerebroside antibody to the analysis of lipid-lipid or lipid-protein interactions. Significance of lipid matrix

The significance of the lipid matrix in the reaction of liposomal antigen, antibody and complement (Ag-Ab-C) was analyzed using purified anti-glactocerebroside antibody and synthetic lipids and the following results were obtained. 1. For the optimal Ag-Ab-C reaction it was necesary that galactocerebroside (galxCMH) and lecithin molecules were well dispersed by virtue of cholesterol (Chol) and that the molar ratio of cholesterol to the sum of galactocerebroside and lecithin was more than one. 2. The Ag-Ab-C reactivity changed depending upon the chain length of lecithin, and the maximal reaction was observed in the case of dilauroylphosphatidylcholine. When the fatty acyl chain of lecithin was either shorter or longer than that of dilaurosylphosphatidylcholine, the reactivity was reduced. 3. The Ag-Ab-C reactivity was increased by elongation of the fatty acyl chain of galactocerebroside and an abrupt change was found to be around the carbon number 8 to 10 of the fatty acyl chain. 4. The Ag-Ab-C reactivity was elevated by the increase in unsaturation of fatty acyl moiety. 5. There is a tendency that an increase in the charge of the lipid matrix leads to the reduction of the Ag-Ab-C reactivity. 6. The results suggest that the physicochemical properties of lipids and especially the lipid-lipid interaction in the hydrophobic region of the lipid matrix play an important role in the Ag-Ab-C reaction.     

Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.     

Roles for L o microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (L_o) microdomains, sterol-rich raftlike regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during L_o formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here we report that ER stress induced by lipid imbalance and other stressors induces L_o microdomain formation. This process is ESCRT independent and dependent on Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or L_o microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce L_o microdomains, µLP in response to these stressors is ESCRT dependent and L_o microdomain independent. Our findings reveal that L_o microdomain formation is a yeast stress response, and stress-induced L_o microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and L_o microdomains play functionally distinct roles in LD uptake during stress-induced µLP.     

Chondrocytes utilize a cholesterol-dependent lipid translocator to externalize phosphatidylserine

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Spontaneous insertion of lipopolysaccharide into lipid membranes from aqueous solution

Lipopolysaccharide (LPS), one of the main components of outer membranes of Gram-negative bacteria, consists of a hydrophobic lipid (lipid A) with six hydrocarbon chains and a large hydrophilic polysaccharide chain. LPS plays endotoxic roles and can stimulate macrophages and B cells. To elucidate the mechanism of the interaction of LPS with various cell membranes, it is important to investigate the interaction of wild type LPS in a buffer with lipid membranes. In this report we investigated the interaction of low concentrations of LPS in a buffer with giant unilamellar vesicles (GUVs) of dioleoylphosphatidylcholine (DOPC) membrane in the liquid-crystalline (L_α) phase and sphingomyelin (SM)/cholesterol(chol) (molar ration; 6/4) membrane in the liquid-ordered (lo) phase. We found that low concentrations (less than critical micelle concentration) of LPS in aqueous solution induced the shape changes such as the transformation from a prolate to a two-spheres-connected by a very narrow neck in the DOPC-GUVs and also in the SM/chol (6/4)-GUVs above their threshold concentrations. The analysis of the shape changes of the GUVs indicates that the monomers of LPS can insert spontaneously into the external monolayer of the lipid membranes of these GUVs from the aqueous solution. Moreover, higher concentrations of LPS induced the vesicle fission of SM/chol(6/4)-GUVs above its higher threshold concentration. The vesicle fission of GUVs is similar to those induced by single long chain amphiphiles such as lysophosphatidylcholine. On the basis of these results, we discuss the interaction of wild type LPS with lipid membranes and cell membranes. These results suggest that LPS molecules can insert spontaneously into the external monolayer of the plasma membranes composed of the L_α phase-membrane and the microdomain in the lo phase.     

Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering

We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S_x-ray. When 40% cholesterol is added to either DOPC or DPPC, S_x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.     

Neuronal glycoprotein M6a induces filopodia formation via association with cholesterol-rich lipid rafts

A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca(2+) transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-β-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation.     

N-3 fatty acids and membrane microdomains: From model membranes to lymphocyte function

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease.     

Lipid phase behavior and stabilization of domains in membranes of platelets

Lipid domains are acquiring increasing importance in our understanding of the regulation of several key functions in living cells. We present here a discussion of the physical mechanisms driving the phase separation of membrane lipid components that make up these domains, including phase behavior of the lipids and the role of cholesterol. In addition, we discuss phenomena that regulate domain geometry and dimensions. We present evidence that these mechanisms apply to the regulation of domains in intact cells. For example, the observation that physiologically functional microdomains present at 37°C aggregate into macrodomains in human blood platelets when they are chilled below membrane lipid phase transition temperatures is predictable from the known behavior of the constituent lipids in vitro. Finally, we show that the principles developed from studies on these lipids in model systems can be used to develop techniques to stabilize the physiological, resting microdomain structure of platelets during freeze-drying. These latter findings have immediate applications in clinical medicine for the development of methods for storing platelets for therapeutic use.     

Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by 1H NMR

Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several ¹H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2–9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the ¹H NMR spectra of TNM-A with sodium dodecyl sulfate-d₂₅ (SDS-d₂₅) micelles and small dimyristoylphosphatidylcholine (DMPC)-d₅₄/dihexanoylphosphatidylcholine (DHPC)-d₂₂ bicelles in the presence of a paramagnetic quencher Mn²⁺. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader ¹H signals of TNM-A were observed in 10 mol % cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn²⁺ to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid–water interface (close to the C2′ portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.     

Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains

Bovine rhodopsin was reconstituted into mixtures of didocosahexaenoylphosphatidylcholine (di22:6-PC), dipalmitoylphosphatidylcholine (di16:0-PC), sn-1-palmitoyl-sn-2-docosahexaenoylphosphatidylcholine (16:0, 22:6-PC) and cholesterol. Rhodopsin denaturation was examined by using high-sensitivity differential scanning calorimetry. The unfolding temperature was increased at lower levels of lipid unsaturation, but the highest temperature was detected for native disk membranes: di22:6-PC < 16:0,22:6-PC < di16:0,18:1-PC < native disks. The incorporation of 30 mol% of cholesterol resulted in 2-4 degrees C increase of denaturation temperature in all reconstituted systems examined. From the analysis of van't Hoff's and calorimetric enthalpies, it was concluded that the presence of cholesterol in di22:6-PC-containing bilayers induces a level of cooperativity in rhodopsin unfolding. Fluorescence resonance energy transfer (FRET), using lipids labeled at the headgroup with pyrene (Py) as donors and rhodopsin retinal group as acceptor of fluorescence, was used to study rhodopsin association with lipids. Higher FRET efficiencies detected for di22:6-PE-Py, compared to di16:0-PE-Py, in mixed di22:6-PC-di16:0-PC-cholesterol bilayers, indicate preferential segregation of rhodopsin with polyunsaturated lipids. The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.     

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. ¹H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3 ppm peak intensity and a discontinuity at ~18 °C, for which the simplest explanation is a phase transition from L_d to L_o phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3 ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance.     

Neural membrane microdomains as computational systems: Toward molecular modeling in the study of neural disease

Several studies indicate that the lipid biological membrane contains discrete regions known as rafts or microdomains. These structures range in size from approximately 50 to 70nm to nearly a mum and play important roles in cell signaling. In the neuron, computational models suggest that transiently polarized microdomain ethenes may regulate ion-channel dynamics and control impulse propagation. Thus the microdomain is nominated as the fundamental unit of nervous system signaling. Based on this model, the article proposes a first-approximation design for a supported-membrane device which would mimic microdomain properties. The basic architecture would consist of an electrically addressable biotemplated nanowire crossing an artificial membrane corralled in a vertical carbon nanofiber barrier. Advantages and disadvantages of model components are discussed at length. It is proposed that artificial devices of this type would be medically useful in simulating membrane states correlated with neural disease. This possibility is examined with reference to the A-current potassium channel, implicated in epilepsy.     

The self-organization of lipids and proteins of myelin at the membrane interface. Molecular factors underlying the microheterogeneity of domain segregation

The advances over the last 10 years on the understanding of myelin heterogeneity are reviewed. The main focus is on the applicability of Langmuir monolayers, Langmuir-Blodgett films and some associated techniques to unravelling the behaviour of interfaces formed with all the components of a natural membrane. Lipid-protein lateral segregation appears as a major driving force to determine surface patterns that can change under compression from circular domains to two-dimensional fractal structures. The major proteins of the myelin membrane induce lateral segregation in an otherwise homogeneous surface formed by the mixture of total myelin lipids. The lipid and protein components appear to distribute in the surface domains according to their charge, compressibility and relative molecular weight: myelin proteins, ganglioside GM1 and fluorescent lipid probes partition into liquid-expanded phase domains; other components such as phosphatidylserine and galactocerebroside partition into another liquid phase enriched in cholesterol. Simplified protein-lipid mixtures allow assessment of the participation of the major proteins in the two dimensional pattern development. One of the major myelin proteins, the Folch-Lees proteolipid, self-segregates into, and determines formation of, fractal-like patterns. The presence of the second major protein, myelin basic protein, leads to round liquid-expanded domains in the absence of Folch-Lees proteolipid and softens the boundaries of the fractal structures in its presence. The location of myelin basic protein in the interface is surface pressure sensitive, being slightly squeezed out at high surface pressure, allowing the fractal domains enriched in Folch-Lees proteolipid to evolve.