Bruno Maggio

Thermodynamic-geometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine

The morphology of aqueous dispersions of five neutral glycosphingolipids (GalCer, GlcCer, LacCer, asialo-GM2, asialo-GM1), sulfatide, and five gangliosides (GM3, GM2, GM1, GD1a and GT1b) and their mixtures with dipalmitoylphosphatidylcholine was studied by negative staining electron microscopy. The morphological features are interpreted on the basis of thermodynamic and geometric constraints previously studied in these systems (Maggio, B (1985) Biochim. Biophys. Acta 815, 245-258). The correlation between the theoretical predictions and the experimental findings are in reasonable agreement. Small changes in the molecular parameters of the individual glycosphingolipids or in their proportion in mixtures with dipalmitoylphosphatidylcholine bring about remarkable variations on the type of structure formed, its radius of curvature and thermodynamic stability.

Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).

Molecular interactions and thermotropic behavior of glycosphingolipids in model membrane systems.

The oligosaccharide chain of glycosphingolipids (GSLs) has a marked influence on their thermotropic behavior, intermolecular packing and surface electrical potential. The transition temperature and enthalpy of GSLs decrease proportionally to the complexity of the polar head group and show a linear dependence with the intermolecular spacings. Interactions occurring among GSLs and phospholipids induce changes of the molecular area and surface potential that depend on the type of GSLs. Increasing proportions of phospholipids perturb the thermodynamic properties of the GSLs up to a point where phase separated phospholipid domains separate out but no phase separation of pure GSLs occurs. Heterogeneous equilibria among different structures occur for some systems. Large changes of the molecular free energy, eccentricity, asymmetry ratio and phase state of the GSLs-containing structure can be triggered by small changes of the molecular parameters, lipid composition and lateral surface pressure. The thermotropic behavior of GSLs is considerably perturbed by myelin basic protein. Phase separation occurs depending on the amount of protein and type of GSLs. The protein induces a decrease of the lipid molecular area, the more so the more complex the oligosaccharide chain in the GSLs. These membrane systems can not be described only on the basis of the individual properties of the molecules involved in a simple causal manner. Still scarcely explored long range thermodynamic, geometric and field effects that belong simultaneously to the intervening molecules, to the morphological properties of the structure involved and to the aqueous environment, are important determinants of their behavior.

The action of sphingomyelinase in lipid monolayers as revealed by microscopic image analysis

In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase)-driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the mum level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.

Interaction of myelin basic protein, melittin and bovine serum albumin with gangliosides, sulphatide and neutral glycosphingolipids in mixed monolayers.

Some parameters that may regulate the miscibility and stability of mixed lipid-protein monolayers at the air-145 mM NaCl interface were studied employing six glycosphingolipids (acidic or neutral), three different types of proteins (soluble, extrinsic or highly amphipathic) and some phospholipids. The results obtained show that the percentage of the total area occupied by the protein at the interface is an important parameter leading to lateral phase separations; the amount and area contribution of the protein accepted in the film before the components become immiscible increase with the complexity of the polar head group of the glycosphingolipids. The interactions occur with progressive reductions of the intermolecular packing as the polar head group of the glycosphingolipid becomes more complex and this is accompanied by more negative values of the excess free energy of mixing. The lipid component seems to be the major responsible for the reduction in mean molecular area.

Micropolarity of interfaces containing anionic and neutral glycosphingolipids as sensed by Merocyanine 540

Abstract Spectral properties of Merocyanine 540 (MC 540) were used in an attempt to investigate the interfacial micropolarity of liposomes constituted by egg phosphatidylcholine (PC) with phosphatidic acid (PA) or different neutral glycosphingolipids and gangliosides, MC 540 reveals a more polar microenvironment for interfaces containing anionic glycosphingolipids or PA compared to interfaces constituted by PC alone or PC with neutral glycosphingolipids. The interfacial microenvironment detected by the dye in liposomes containing anionic lipids can be reversibly modified by low pH or the addition of Ca 2+ , myelin basic protein and melittin. Studies of penetration of MC 540 into various lipid monolayers suggest that, in some cases, the distinct microenvironments detected may be accounted for by a combination of effects. These refer to the interfacial polarity detected by the dye and differences in the ability of the probe to interact with and penetrate interfaces of different composition.

Interaction of melittin with glycosphingolipids and phospholipids in mixed monolayers at different temperatures. Effect of the lipid physical state

Abstract The influence of the liquid-expanded or liquid-condensed state of the lipid interface induced by changes of temperature on the lipid-protein interactions and their two-dimensional miscibility was studied for mixtures of melittin with different phospholipids (DPPC, DMPC, DOPC egg PC) and gangliosides (GM1, GD1a) in mixed monolayers at the air/145 mM NaCl interface. The critical amount of melittin at which a phase separation takes place in the mixed film increases as the glycosphingolipid or phospholipid is more liquid-expanded. The lipid-protein interaction increases the stability of both melittin and the lipid. The interaction of melittin with gangliosides is thermodynamically more favorable as these are more liquid-expanded. The interaction of melittin with phospholipids, on the other hand, is more favorable when the lipids are in the liquid-condensed state even if these films show lateral immiscibility at a lower proportion of protein compared to lipids in the liquid-expanded state. Hydration-dehydration effects in the polar head group region are likely to participate in these lipid-protein interactions.

Phase state and surface topography of palmitoyl-ceramide monolayers

In cell biology (and in many biophysical) studies there is a natural tendency to consider ceramide as a highly condensed, solid-type lipid conferring rigidity and close packing to biomembranes. In the present work we advanced the understanding of the phase behavior of palmitoyl-ceramide restricted to a planar interface using Langmuir monolayers under strictly controlled and known surface packing conditions. Surface pressure-molecular area isotherms were complemented with molecular area-temperature isobars and with observations of the surface topography by Brewster Angle Microscopy. The results described herein indicate that palmitoyl-ceramide can exhibit expanded, as well as condensed phase states. Formation of three phases was found, depending on the surface pressure and temperature: a solid (1.80nm thick), a liquid-condensed (1.73nm thick, likely tilted) and a liquid-expanded (1.54nm thick) phase over the temperature range 5-62 degrees C. A large hysteretic behavior is observed for the S phase monolayer that may indicate high resistance to domain boundary deformation. A second (or higher) order S-->LC phase transition is observed at about room temperature while a first order LC-->LE transition occurs in a range of temperature encompassing the physiological one (observed above 30 degrees C at low surface pressure). This phase behavior broadens the view of ceramide as a type of lipid not-always-rigid but able to exhibit polymorphic properties.

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale-both in situ and in real-time-the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.