Lateral diffusion of ganglioside GM1 in phospholipid bilayer membranes.

The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of ganglioside GM1 in phosphatidylcholine bilayers

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.     

Receptor-mediated interaction of ricin with the lipid bilayer of ganglioside GM1-liposomes

The interaction of ricin with ganglioside GM1 or glycoprotein containing liposomes was investigated. At neutral pH, ricin bound to galactose moieties on the surface of the liposomes to form ricin-liposomes complexes, but did not associate with their lipid bilayers. When these ricin-liposomes complexes were exposed to a pH below 5, ricin bound to GM1-liposomes became associated with the lipid bilayer, whereas ricin bound to glycoprotein-liposomes (containing human erythrocyte Band 3) was only rarely associated. Association of ricin with the lipid bilayer of GM1-liposomes did not occur in the presence of lactose, which inhibits the binding of ricin to ganglioside GM1. Using a hydrophobic probe, 8-amino-1-naphthalene sulfonic acid (ANS), it was revealed that an acidity below pH 5 resulted in exposure of hydrophobic regions on the ricin molecule. These results strongly suggest that association of ricin with the lipid bilayer of GM1-liposomes at acidic pH is mediated by the binding of ricin to ganglioside GM1 at neutral pH and occurs through interaction between the exposed hydrophobic region on the ricin molecule and the lipid bilayer of GM1-liposomes at low pH.     

Specific interaction between follitropin and GM1 ganglioside incorporated into lipid membranes.

A specific interaction was demonstrated between a monosialo-ganglioside (galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide) incorporated in glycerol monoleate planar bilayer membranes and follitropin. Conductance of glycerol monoleate planar bilayer membranes containing gangliosides were measured before and after addition of follitropin in the aqueous phase. A 5 fold increase of membrane conductance was observed in presence of a specific monosialo-ganglioside. No significant effect was obtained with di- and trisialogangliosides. Membrane conductance changes were suppressed by the presence of an equimolar mixture of the saccharide residues (galactose, N-acetylgalactosamine and N-acetylneuraminic acid) present in the hydrophilic moeity of the ganglioside. The results favour the hypothesis that gangliosides may contribute to the formation of functional glycoproteins receptors.     

Thermotropic characterization of phosphatidylcholine vesicles containing ganglioside GM1 with homogeneous ceramide chain length

The thermotropic behavior of dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine large unilamellar vesicles containing ganglioside GM1 of homogeneous long chain base composition has been studied by high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. At neutral pH and in the absence of Ca2+, the thermotropic behavior of these systems is independent of the ganglioside chain length composition. The presence of Ca2+ at concentrations higher than 5 mM induces ganglioside phase separation in a manner dependent upon the length difference between the ganglioside long chain base and the phosphatidylcholine acyl chains. The analysis of the chain length dependence of the thermotropic behavior suggests that the driving force for ganglioside phase separation is not a Ca2+-induced cross-bridging of the ganglioside head group but a passive ganglioside exclusion from Ca2+-perturbed phosphatidylcholine-rich regions within the bilayer. Experiments with native ganglioside GM1, primarily a mixture of C18:1 and C20:1 long chain bases, indicate that the individual components of the mixture maintain their characteristic behavior within the lipid bilayer matrix. These results, together with the presence of a phase transition in native GM1 micellar dispersions, absent in purified C18:1 or C20:1 ganglioside micelles, strengthen the idea of a possible role of chain length composition in the modulation of ganglioside function.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin—along with ganglioside—within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin - along with ganglioside - within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.     

The adsorption of divalent cations to phosphatidylcholine bilayer membranes

Electrophoretic mobility and ³¹P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations to phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the ³¹P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the ³¹P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Sr²⁺, Ba²⁺. If we assume that a lipid molecule occupies an area of 60 Ų and that there is a 1: 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.     

Bilayer membranes containing the ganglioside GM1: models for electrostatic potentials adjacent to biological membranes

Although the Gouy-Chapman-Stern theory of the aqueous diffuse double layer describes well the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes, it does not describe adequately the zeta potential of biological membranes: the zeta potential of an erythrocyte is about half the value predicted from the theory by using the known density of negatively charged sialic acid residues. To investigate the factors responsible for this low electrophoretic mobility, we formed membranes from mixtures of the zwitterionic lipid phosphatidylcholine, PC, and the glycolipid galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl) -galactosylglucosylceramide, GM1. This glycolipid differs from phospholipids in two respects. First, the negative charge on GM1 is located about 1 nm from the surface, which tends to increase the electrophoretic mobility of vesicles. Second, the head group of GM1 contains five sugar groups that exert a hydrodynamic drag, which tends to decrease the mobility of the vesicles. In a decimolar monovalent salt solution, where the Debye length is about 1 nm, the electrophoretic mobility of the PC-GM1 vesicles is about half the mobility of PC-phosphatidylserine or PC-phosphatidylglycerol vesicles of equivalent composition. In addition, conductance measurements with planar bilayer membranes as well as 31P nuclear magnetic resonance and fluorescence measurements with sonicated vesicles indicate that the potential at the surface of PC-GM1 membranes is about half the value measured for PC-phosphatidylserine membranes in a 0.1 M monovalent salt solution.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.     

GM1 ganglioside enhances Ret signaling in striatum

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell‐derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co‐receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration‐ and time‐dependent manner. GM1‐induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration‐dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP‐mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits.