Sphingomyelinase acts by an area-activated mechanism on the liquid-expanded phase of sphingomyelin monolayers

We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SM-enriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SM-enriched LE phase and it is not enriched at the domain boundaries. A novel mechanism is proposed for the action of SMase, which can also explain the regulatory effect of the surface topography on the enzyme activity. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer. This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. Accordingly, the number and size of Cer-enriched domains are determined by the extent of Cer supersaturation in the LE phase rather than by the SMase local activity. The kinetic barrier for nucleation, for which a compositional gap of at least 53 mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time to reaching full catalytic activity. Altogether, the data support an "area-activated mechanism," in which the enzyme is homogeneously active over the LE surface.     

Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase in lipid monolayers

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.     

Bidirectional control of sphingomyelinase activity and surface topography in lipid monolayers

Lipid lateral organization is increasingly found to modulate membrane-bound enzymes. We followed in real time the reaction course of sphingomyelin (SM) degradation by Bacillus cereus sphingomyelinase (SMase) of lipid monolayers by epifluorescence microscopy. There is evidence that formation of ceramide (Cer), a lipid second messenger, drives structural reorganization of membrane lipids. Our results provide visual evidence that SMase activity initially alters surface topography by inducing phase separation into condensed (Cer-enriched) and expanded (SM-enriched) domains. The Cer-enriched phase grows steadily as the reaction proceeds at a constant rate. The surface topography derived from the SMase-driven reaction was compared with, and found to differ from, that of premixed SM/Cer monolayers of the same lipid composition, indicating that substantial information content is stored depending on the manner in which the surface was generated. The long-range topographic changes feed back on the kinetics of Smase, and the onset of condensed-phase percolation is temporally correlated with a rapid drop of reaction rate. These observations reveal a bidirectional influence and communication between effects taking place at the local molecular level and the supramolecular organization. The results suggest a novel biocatalytic-topographic mechanism in which a surface enzymatic activity can influence the function of amphitropic proteins important for cell function.     

Sphingomyelinase-induced domain shape relaxation driven by out-of-equilibrium changes of composition

Sphingomyelinase (SMase)-induced ceramide (Cer)-enriched domains in a lipid monolayer are shown to result from an out-of-equilibrium situation. This is induced by a change of composition caused by the enzymatic production of Cer in a sphingomyelin (SM) monolayer that leads to a fast SM/Cer demixing into a liquid-condensed (LC), Cer-enriched and a liquid-expanded, SM-enriched phases. The morphological evolution and kinetic dependence of Cer-enriched domains is studied under continuous observation by epifluorescence microscopy. Domain shape annealing is observed from branched to rounded shapes after SMase activity quenching by EDTA, with a decay halftime of ∼10 min. An out-of-equilibrium fast domain growth is not the determinant factor for domain morphology. Domain shape rearrangement in nearly equilibrium conditions result from the counteraction of intradomain dipolar repulsion and line tension, according to McConnell's shape transition theory. Phase separation causes a transient compositional overshoot within the LC phase that implies an increased out-of-equilibrium enrichment of Cer into the LC domains. As a consequence, higher intradomain repulsion leads to transient branched structures that relax to rounded shapes by lowering the proportion of Cer in the domain to equilibrium values. The fast action of SMase can be taken as a compositional perturbation that brings about important consequences for the surface organization.     

Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay among SMase activity, initial membrane properties (i.e., phase behavior and lipid lateral organization) and lipid composition, and the amount of product (Cer) generated, and how it modulates membrane properties were studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that 1), the enzyme activity and extent of hydrolysis are strongly dependent on membrane physical properties but not on substrate content, and are higher in raft-like mixtures, i.e., mixtures with liquid-disordered/liquid-ordered phase separation; and 2), Cer-induced alterations are also dependent on membrane composition, specifically the cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small (∼8.5 nm) Cer/SM-gel domains. With increasing Chol, the ability of Cer to recruit SM and form gel domains strongly decreases. In the high-Chol range, a Chol-enriched/SM-depleted liquid-ordered phase predominates. Together, these data suggest that in biological membranes, Chol in particular and raft domains in general play an important role in modulating SMase activity and regulating membrane physical properties by restraining Cer-induced alterations.     

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-D-erythro-sphingosine (C16Cer), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (C16SM) and cholesterol. The amounts of DOPC, sum of C16Cer and C16SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C16Cer/(C16Cer+C16SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C16Cer (X=0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X=0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X>or=0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d-erythro-sphingosine (C₁₆Cer), N-palmitoyl-d-erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer + C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer (X = 0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X = 0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X ≥ 0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

Transbilayer movement of ceramide in the plasma membrane of live cells

Ceramide (Cer) is the precursor for sphingolipids and functions as a second messenger in a variety of cellular processes including apoptosis. However, no direct target of Cer leading to apoptosis has been identified. Understanding the movement and trafficking of Cer is important for fully understanding Cer signaling. In this study, we identified, for the first time, the transbilayer movement of Cer in the plasma membrane (PM) of living cells. We developed a new method to monitor transbilayer Cer movement using ceramide kinase activity. To produce Cer on the extracellular leaflet of the PM, bacterial sphingomyelinase (SMase) was added to rat basophilic leukemia cells. Interestingly, the dramatic elevation of ceramide 1-phosphate (C1P), the product of CerK, was observed following the increase of Cer induced by SMase treatment. Since we determined that both the protein and catalytic activity of CerK exists in the intracellular compartment, the all conversion of Cer to C1P by CerK should be occur intracellularly. This result indicates the rapid transbilayer movement of Cer from the outer leaflet to the inner leaflet of the PM of living cells. Furthermore, protease digestion of membrane proteins, inhibition of ABC transporters (by glibencramide) and of cation channels (by carbonyl cyanide m-chlorophenylhydrozone), and modification of cholesterol content did not affect the transbilayer movement of Cer. Thus, this movement might occur spontaneously. Our findings indicate not only Cer movement in the PM, but also identify an intrinsic property of Cer enabling Cer signaling.     

Fluidization of a dipalmitoyl phosphatidylcholine monolayer by fluorocarbon gases: potential use in lung surfactant therapy

Fluorocarbon gases (gFCs) were found to inhibit the liquid-expanded (LE)/liquid-condensed (LC) phase transition of dipalmitoyl phosphatidylcholine (DPPC) Langmuir monolayers. The formation of domains of an LC phase, which typically occurs in the LE/LC coexistence region upon compression of DPPC, is prevented when the atmosphere above the DPPC monolayer is saturated with a gFC. When contacted with gFC, the DPPC monolayer remains in the LE phase for surface pressures lower than 38 mN m(-1), as assessed by compression isotherms and fluorescence microscopy (FM). Moreover, gFCs can induce the dissolution of preexisting LC phase domains and facilitate the respreading of the DPPC molecules on the water surface, as shown by FM and grazing incidence x-ray diffraction. gFCs have thus a highly effective fluidizing effect on the DPPC monolayer. This gFC-induced fluidizing effect was compared with the fluidizing effect brought about by a mixture of unsaturated lipids and proteins, namely the two commercially available lung surfactant substitutes, Curosurf and Survanta, which are derived from porcine and bovine lung extracts, respectively. The candidate FCs were chosen among those already investigated for biomedical applications, and in particular for intravascular oxygen transport, i.e., perfluorooctyl bromide, perfluorooctylethane, bis(perfluorobutyl)ethene, perfluorodecalin, and perfluorooctane. The fluidizing effect is most effective with the linear FCs. This study suggests that FCs, whose biocompatibility is well documented, may be useful in lung surfactant substitute compositions.     

Detergent-resistant, ceramide-enriched domains in sphingomyelin/ceramide bilayers

When cell membranes are treated with Triton X-100 or other detergents at 4 degrees C, a nonsolubilized fraction can often be recovered, the "detergent-resistant membranes", that is not found when detergent treatment takes place at 37 degrees C. Detergent-resistant membranes may be related in some cases to membrane "rafts". However, several basic aspects of the formation of detergent-resistant membranes are poorly understood. To answer some of the relevant questions, a simple bilayer composition that would mimic detergent-resistant membranes was required. The screening of multiple lipid compositions has shown that the binary mixture egg sphingomyelin/egg ceramide (SM/Cer) exhibits the required detergent resistance. In detergent-free membranes composed of different mixtures of SM and Cer (5-30 mol % of Cer) differential scanning calorimetry, fluorescence spectroscopy, and fluorescence microscopy experiments reveal the presence of discrete, Cer-enriched gel domains in a broad temperature range. In particular, at temperatures below SM phase transition ( approximately 40 degrees C) two gel (respectively Cer-rich and SM-rich) phases are directly observed using fluorescence microscopy. Although pure SM membranes are fully solubilized by Triton X-100 at room temperature, 5 mol % Cer is also enough to induce detergent resistance, even with a large detergent excess and lengthy equilibration times. Short-chain Cers do not give rise to detergent resistance. SM/Cer mixtures containing up to 30 mol % Cer become fully soluble at approximately 50 degrees C, i.e., well above the gel-fluid transition temperature of SM. The combined results of temperature-dependent solubilization and differential scanning calorimetry reveal that SM-rich domains are preferentially solubilized over the Cer-rich ones as soon as the former melt (i.e., at approximately 40 degrees C). As a consequence, at temperatures allowing only partial solubilization, the nonsolubilized residue is enriched in Cer with respect to the original bilayer composition. Fluorescence microscopy of giant unilamellar vesicles at room temperature clearly shows that SM-rich domains are preferentially solubilized over the Cer-rich ones and that the latter become more rigid and extensive as a consequence of the detergent effects. These observations may be relevant to the phenomena of sphingomyelinase-dependent signaling, generation of "raft platforms", and detergent-resistant cell membranes.     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers

Fluorescence, polarized fluorescence, and Brewster angle microscopy reveal that human lung surfactant protein SP-B and its amino terminus (SP-B[1-25]) alter the phase behavior of palmitic acid monolayers by inhibiting the formation of condensed phases and creating a new fluid protein-rich phase. This fluid phase forms a network that separates condensed phase domains at coexistence and persists to high surface pressures. The network changes the monolayer collapse mechanism from heterogeneous nucleation/growth and fracturing processes to a more homogeneous process through isolating individual condensed phase domains. This results in higher surface pressures at collapse, and monolayers easier to respread on expansion, factors essential to the in vivo function of lung surfactant. The network is stabilized by a low-line tension between the coexisting phases, as confirmed by the observation of extended linear domains, or "stripe" phases, and a Gouy-Chapman analysis of protein-containing monolayers. Comparison of isotherm data and observed morphologies of monolayers containing SP-B(1-25) with those containing the full SP-B sequence show that the shortened peptide retains most of the native activity of the full-length protein, which may lead to cheaper and more effective synthetic replacement formulations.     

Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol.

The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid condensed (LC) and liquid expanded (LE) phases. Some TR-SP-A appeared in the LE phase but not in the LC phase. At intermediate surface pressures (10-20 mN/m), the protein caused the occurrence of more, smaller condensed domains, and it appeared to be excluded from the monolayers at surface pressure in the range of 30-40 mN/m. TR-SP-A interaction with DPPC/dipalmitoylphosphatidylglycerol monolayers was different. The protein did not appear in either LE or LC but only in large aggregates at the LC-LE boundary regions, a distribution visually similar to that of fluorescently labeled concanavalin A adsorbed onto monolayers of DPPC. The observations are consistent with a selectivity of interaction of SP-A with DPPC and for its accumulation in boundaries between LC and LE phase.     

Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin-enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.     

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.     

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.     

Coexistence of Immiscible Mixtures of Palmitoylsphingomyelin and Palmitoylceramide in Monolayers and Bilayers

A combination of lipid monolayer- and bilayer-based model systems has been applied to explore in detail the interactions between and organization of palmitoylsphingomyelin (pSM) and the related lipid palmitoylceramide (pCer). Langmuir balance measurements of the binary mixture reveal favorable interactions between the lipid molecules. A thermodynamically stable point is observed in the range ∼30-40 mol % pCer. The pSM monolayer undergoes hyperpolarization and condensation with small concentrations of pCer, narrowing the liquid-expanded (LE) to liquid-condensed (LC) pSM main phase transition by inducing intermolecular interactions and chain ordering. Beyond this point, the phase diagram no longer reveals the presence of the pSM-enriched phase. Differential scanning calorimetry (DSC) of multilamellar vesicles reveals a widening of the pSM main gel-fluid phase transition (41°C) upon pCer incorporation, with formation of a further endotherm at higher temperatures that can be deconvoluted into two components. DSC data reflect the presence of pCer-enriched domains coexisting, in different proportions, with a pSM-enriched phase. The pSM-enriched phase is no longer detected in DSC thermograms containing >30 mol % pCer. Direct domain visualization has been carried out by fluorescence techniques on both lipid model systems. Epifluorescence microscopy of mixed monolayers at low pCer content shows concentration-dependent, morphologically different pCer-enriched LC domain formation over a pSM-enriched LE phase, in which pCer content close to 5 and 30 mol % can be determined for the LE and LC phases, respectively. In addition, fluorescence confocal microscopy of giant vesicles further confirms the formation of segregated pCer-enriched lipid domains. Vesicles cannot form at >40 mol % pCer content. Altogether, the presence of at least two immiscible phase-segregated pSM-pCer mixtures of different compositions is proposed at high pSM content. A condensed phase (with domains segregated from the liquid-expanded phase) showing enhanced thermodynamic stability occurs near a compositional ratio of 2:1 (pSM/pCer). These observations become significant on the basis of the ceramide-induced microdomain aggregation and platform formation upon sphingomyelinase enzymatic activity on cellular membranes.     

Membrane domain formation, interdigitation, and morphological alterations induced by the very long chain asymmetric C24:1 ceramide

Ceramide (Cer) is involved in the regulation of several biological processes, such as apoptosis and cell signaling. The alterations induced by Cer in the biophysical properties of membranes are thought to be one of the major routes of Cer action. To gain further knowledge about the alterations induced by Cer, membrane reorganization by the very long chain asymmetric nervonoylceramide (NCer) was studied. The application of an established fluorescence multiprobe approach, together with x-ray diffraction, differential scanning calorimetry, and confocal fluorescence microscopy, allowed the characterization of NCer and the determination of the phase diagram of palmitoyloleoylphosphatidylcholine (POPC)/NCer binary mixtures. Nervonoylceramide undergoes a transition from a mixed interdigitated gel phase to a partially interdigitated gel phase at ∼20°C, and a broad main transition to the fluid phase at ∼52°C. The solubility of NCer in the fluid POPC is low, driving gel-fluid phase separation, and the binary-phase diagram is characterized by multiple and large coexistence regions between the interdigitated gel phases and the fluid phase. At 37°C, the relevant phases are the fluid and the partially interdigitated gel. Moreover, the formation of NCer interdigitated gel phases leads to strong morphological alterations in the lipid vesicles, driving the formation of cochleate-type tubular structures.     

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.