Solid character of membrane ceramides: a surface rheology study of their mixtures with sphingomyelin

The compression and shear viscoelasticities of egg-ceramide and its mixtures with sphingomyelin were investigated using oscillatory surface rheology performed on Langmuir monolayers. We found high values for the compression and shear moduli for ceramide, compatible with a solid-state membrane, and extremely high surface viscosities when compared to typical fluid lipids. A fluidlike rheological behavior was found for sphingomyelin. Lateral mobilities, measured from particle tracking experiments, were correlated with the monolayer viscosities through the usual hydrodynamic relationships. In conclusion, ceramide increases the solid character of sphingomyelin-based membranes and decreases their fluidity, thus drastically decreasing the lateral mobilities of embedded objects. This mechanical behavior may involve important physiological consequences in biological membranes containing ceramides.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

The influence of dioxan on the bilayer and monolayer phase transition of phospholipids

The influence of 1.4.-dioxan on the bilayer phase transition of various phospholipids was studied by differential scanning calorimetry and turbidity measurements. The addition of 1.4.-dioxan to lipid bilayers decreases the transition temperature T_m increases the transition enthalpy of the transition. The cooperativity of the transition is unaffected. The phospholipid monolayer transition from the liquid-condensed to the liquid-expanded phase was measured by recording area versus temperature curves at constant surface pressure (isobars). The monolayer transition temperature at constant surface pressure is increased when 1.4.-dioxan is added to the subphase. The change in molecular area becomes larger. A comparison of monolayer isobars on water and water/dioxan as subphase at constant surface tension rather than surface pressure leads to a decrease of the transition temperature on water/dioxan as subphase. This decrease as well as the larger change in molecular area at the monolayer transition can be correlated to the decrease in T_m and the increase in the transition enthalpy of the corresponding bilayer system. 1.4.-Dioxan seems to accumulate at the lipid head group/water interface, thus lowering the tension of the bilayer membrane. This cyclic ether can be used for altering the characteristics of bilayer membranes without disturbing the lipid chain organization.     

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.     

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 (1.80 nm thick), a liquid-condensed (1.73 nm thick, likely tilted) and a liquid-expanded (1.54 nm thick) phase over the temperature range 5–62 °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 °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.     

Cholesterol surrogates: a comparison of cholesterol and 16:0 ceramide in POPC bilayers

Experimental evidence indicates that, under some circumstances, "surrogate" molecules may play the same role as cholesterol in ordering membrane lipids. The simplest molecule in this class is Ceramide. In this article, we describe atomic-level molecular dynamics simulations designed to shed light on this phenomenon. We run simulations of hydrated phosphoryl-oleoyl phosphatidylcholine (POPC) bilayers containing cholesterol, and containing ceramide, in concentrations ranging from 5% to 33%. We also perform a simulation of a pure POPC bilayer to verify the simulation force fields against experimental structural data for POPC. Our simulation data are in good agreement with experimental data for the partial molecular volumes, areas, form factors, and order parameters. These simulations suggest that ceramide and cholesterol have a very similar effect on the POPC bilayer, although ceramide is less effective in inducing order in the bilayer compared with cholesterol at the same concentrations.     

Crystal Structures of the CERT START Domain with Inhibitors Provide Insights into the Mechanism of Ceramide Transfer

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Ω1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Ω1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.     

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.     

AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7

The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper. Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-) as a redox couple. AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface. The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion. The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit. It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements. Immunoreaction with E. coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E. coli antibodies as evidenced by AFM imaging and impedance spectroscopy. It was found that the binding of E. coli cells mainly affected the electron transfer resistance and Warburg impedance.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Ceramide channels: Influence of molecular structure on channel formation in membranes

The sphingolipid, ceramide, self-assembles in the mitochondrial outer membrane (MOM), forming large channels capable of translocating proteins. These channels are believed to be involved in protein release from mitochondria, a key decision-making step in cell death. Synthetic analogs of ceramide, bearing modifications in each of the major structural features of ceramide were used to probe the molecular basis for the stability of ceramide channels. Channel stability and mitochondrial permeabilization were disrupted by methylation of the C1-hydroxyl group whereas modifications of the C3 allylic hydroxyl group were well tolerated. A change in chirality at C2 that would influence the orientation of the C1-hydroxyl group resulted in a strong reduction of channel-forming ability. Similarly, methylation of the amide nitrogen is also detrimental to channel formation. Many changes in the degree, location and nature of the unsaturation of ceramide had little effect on mitochondrial permeabilization. Competition experiments between ceramide and analogs resulted in synergy with structures compatible with the ceramide channel model and antagonism with incompatible structures. The results are consistent with ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions, similar to the interactions responsible for protein folding.     

Aggregation properties of mycolic acid molecules in monolayer films: a comparative study of compounds from various acid-fast bacterial species

Three kinds of mycolic acids (MAs) (alpha-, keto and methoxy-MAs) extracted from several species of mycobacteria were used to prepare monolayer films on water, and the surface pressure-area (pi-A) isotherms of the monolayers have been compared, so that the monolayer characteristics of the MAs as in cell walls would be revealed, since the monolayer molecular aggregation is related to drug permeability via the molecular packing. It was expected that the limiting molecular areas of the isotherms would be changed only a little, which reflects the minor difference in chemical structure and conformation of the mycobacteria. Nevertheless, the results are largely different from the expectation, and two greatly different patterns of the limiting molecular area have been observed. In a new model for elucidation of the results, two parts in an MA molecule are separately considered, and both contributions to the molecular unfolding by the monolayer compression have been suggested. This model is found to be useful to totally understand the isotherm behaviors of MAs. The relationship between monolayer properties and chemical structures for MAs has been summarized for the first time.     

A comparative study of the lipid phase in native and circulating multiple-modified low-density lipoproteins of human blood by the use of the spin probe method

Different approaches based on the spin probe method were used to compare the physical state of the surface lipid monolayer in subfractions of low-density lipoproteins: in native low-density lipoproteins constituting the bulk of human blood low-density lipoproteins and in circulating multiple-modified low-density lipoproteins whose portion is minor in healthy persons but significantly increases in atherosclerotic patients. The data obtained in in vitro experiments suggest that circulating multiple-modified low-density lipoproteins possess atherogenic properties. The order parameter S, rotational correlation time tau, and hydrophobicity parameter h were calculated from electron spin resonance spectra of a series of spin probes whose paramagnetic groups are located at different depths of the lipid monolayer. These parameters characterize the molecular packing, fluidity, and polarity in the microenvironment of paramagnetic groups. The kinetics of the reduction of paramagnetic groups by ascorbate and oxidation by hypochlorite were obtained for the spin probe whose paramagnetic group is located deeply in the lipid monolayer at the level of the terminal segments of phospholipid acyl chains. No difference between native low-density lipoproteins and circulating multiple-modified low-density lipoproteins was revealed in respect of the physical properties of the lipid domain of surface proteolipid layer, as sampled by spin probes.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Effect of the surface charge density of nanoparticles on their translocation across pulmonary surfactant monolayer: a molecular dynamics simulation

Interaction between nanoparticles (NPs) and pulmonary surfactant monolayer plays a very significant role in nanoparticle-based pulmonary drug delivery system. Previous researches have indicated that different properties of nanoparticles can affect their translocation across pulmonary surfactant monolayer. Here we performed coarse-grained molecular dynamics simulation aimed at nanoparticles’ surface charge density effect on their penetration behaviours. Several hydrophilic nanoparticles with different surface charge densities were modelled in the simulations. The results show that NPs’ surface charge density affects their translocation capability: the higher the surface charge densities of NPs are, the worse their translocation capability is. It will cause the structural changes of pulmonary surfactant monolayer, and inhibit the normal phase transition of the monolayer during the compression process. Besides, charged NPs can be adsorbed on the surface of the monolayer after translocation as a stable state, and the adsorption capability of NPs increases generally with the increase of surface charge densities. Our simulation results suggest that the study of nanoparticle-based pulmonary drug delivery system should consider the nanoparticles’ surface charge density effect in order to avoid biological toxicity and improve efficacy.     

Increases in ceramide levels in normal human mesangial cells subjected to different cellular stresses result from changes in distinct enzyme activities and can influence cellular responses to other stimuli

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.     

The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.     

Enzyme-catalyzed oxidation of cholesterol in pure monolayers at the air/water interface.

Mean molecular area vs. lateral surface pressure isotherms were determined for monolayers containing cholesterol, 4-cholesten-3-one (cholestenone), or binary mixtures of the two. At all lateral surface pressures examined, cholestenone had a larger mean molecular area requirement than cholesterol. Results with the binary mixtures of cholesterol and cholestenone suggested that the sterols did not mix ideally (non additive mean molecular area) with each other in the monolayer; the observed mean molecular area for mixtures was less than would be expected based on ideal mixing. The mixed sterol monolayers also displayed a reduction in the lateral collapse pressure which appeared to be a linear function of the mole fraction of cholestenone in the monolayer, suggesting that cholesterol and cholestenone were completely miscible in the mixed monolayer. The pure cholesterol monolayer was next used to examine the cholesterol oxidase-catalyzed (Brevibacterium sp.) oxidation of cholesterol to cholestenone at different lateral surface pressures at 22 degrees C. The difference in mean molecular area requirements of cholesterol and cholestenone was directly used to convert monolayer area changes (at constant lateral surface pressure) into average reaction rates. It was observed that the average catalytic activity of cholesterol oxidase increased linearly with increased lateral surface pressure in the range of 1 to 20 mN/m. In addition, the enzyme was capable to oxidize cholesterol in monolayers with a lateral surface pressure close to the collapse pressure of cholesterol monolayers (collapse pressure 45 mN/m; oxidation was observed at 40 mN/m). The adsorption of cholesterol oxidase to an inert sterol monolayer film at low surface pressures (around 9 mN/m) was marginal, although clearly detectable at very low (0.5-4 mN/m) lateral surface pressures, suggesting that the enzyme did not penetrate deeply into the monolayer in order to reach the 3 beta-hydroxy group of cholesterol. This interpretation is further supported by the finding that a maximally compressed cholesterol monolayer (40 mN/m) was readily susceptible to enzyme-catalyzed oxidation. It is concluded that cholesterol oxidase is capable of oxidizing cholesterol in laterally expanded monolayers as well as in tightly packed monolayers, where the lateral surface pressure is close to the collapse pressure. The kinetic results suggested that the rate-limiting step in the overall process was the substrate availability per surface area (or surface concentration) at the water/lipid interface.     

Biophysical Interactions of Membrane Anionic Phospholipids with pH, Calcium and Auxins

A series of phospholipid monolayers—phosphatidylinositol,phosphatidylserine and phosphatidyl 4, 5-bisphosphate, possessingan increasingly negative headgroup potential, were spread andcompressed over aqueous sub-phases and, under varying conditions,assayed for changes in surface parameters. These included surfacetension and degree of molecular compression expressed by surfacepressure/molecular area isotherms. Experimentation employingWilhelmy-Du Nouy tensio-metry and the Langmuir-trough compressionprocedure indicated that with increasing electrostatic Ca²⁺cross-linking, this being a direct function of Ca²⁺ concentrationin the aqueous sub-phase, there was a concomitant increase insurface tension, i.e. monolayer condensation. This effect wasgreater with the more negatively charged phospholipids and wasmore pronounced at pH 7–0 than at pH 4–5. Applicationof the auxins indoleacetic acid, indolebutyric acid and naphthaleneaceticacid (which presumably act as proton donors which may dislodgecross-linking Ca²⁺ ) causes a significant decrease in surfacetension when applied at physiological Ca²⁺ concentrations. Asevidenced by surface pressure/molecular area isotherms, Ca²⁺increases monolayer rigidification and decreases phosphatidyl4, 5-bisphosphate molecular area. The auxins tested possessan opposite and de-ngidifying effect as indicated by increaseof the phospholipid’s molecular area and by the observedconsiderable lowering of monolayer collapse pressures. The surfactiveeffectivity of the auxins increased with decreasing sub-phaseCa²⁺ concentration in the 10^–4 to 10^–6mol dm^–3range. These data collectively suggest that surface-associatedbiophysical changes should be taken into account during interpretationof transmembrane hormonal signal transduction. Key words: Membrane phospholipids, monolayers, surface properties     

Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts

Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fcgamma receptor II (FcgammaRII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of FcgammaRII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of FcgammaRII cross-linking. The time course of ceramide production preceded the recruitment of FcgammaRII to rafts and the receptor phosphorylation. Exogenous C(16)-ceramide facilitated clustering of FcgammaRII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of cross-linked FcgammaRII. Under these conditions, tyrosine phosphorylation of FcgammaRII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked FcgammaRII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.