Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.     

How Cholesterol Could Be Drawn to the Cytoplasmic Leaf of the Plasma Membrane by Phosphatidylethanolamine

In the mammalian plasma membrane, cholesterol can translocate rapidly between the exoplasmic and cytoplasmic leaves, so that its distribution between them should be given by the equality of its chemical potential in the leaves. Due to its favorable interaction with sphingomyelin, which is almost entirely in the outer leaf, one expects the great majority of cholesterol to be there also. Experimental results do not support this, implying that there is some mechanism attracting cholesterol to the inner leaf. We hypothesize that it is drawn there to reduce the bending free energy of the membrane caused by the presence of PE (phosphatidylethanolamine). It does this in two ways: first by simply diluting the amount of PE in the inner leaf, and second by ordering the tails of the PE to reduce its spontaneous curvature. Incorporating this mechanism into a model free energy for the bilayer, we find that between 50 and 60% of the total cholesterol should be in the inner leaf of human erythrocytes.     

New reagents for phosphatidylserine recognition and detection of apoptosis

The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36 kDa, Ca2+-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis.     

Cholesterol-Dependent Bending Energy Is Important in Cholesterol Distribution of the Plasma Membrane

We consider the plasma membrane that contains a cholesterol molar fraction of 0.4 and ask how that cholesterol is distributed between the two leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical potentials in the two leaves. When we consider only the contributions of entropy and interactions to the cholesterol chemical potential in our model system, we find, not surprisingly, that the cholesterol is mostly in the outer leaf because of the strong attraction between cholesterol and sphingomyelin (SM), which is predominantly in that leaf. We find 72% there. We then include the contribution from the bending energy in each leaf that must be overcome to join the leaves in a flat bilayer. The product of bending modulus and spontaneous curvature is obtained from simulation. We find that the addition of cholesterol to the outer leaf reduces the spontaneous curvature, which is initially positive, until it passes through zero when the molar fraction of cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward the inner leaf by the sphingomyelin phosphatidylcholine mixture. This is resisted by the bending energy contribution to the inner leaf. We find, again by simulation, that the addition of cholesterol monotonically increases the magnitude of the spontaneous curvature of the inner leaf, which is negative. This increases its bending energy. We conclude that, as a result of these competing effects, the percentage of cholesterol in the outer leaf is reduced to ∼63 ± 6%.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.     

Effect of Lead on Lipid Peroxidation, Phospholipids Composition, and Methylation in Erythrocyte of Human

Lead (Pb) is one of the most abundant heavy metals on earth considered as number one environmental persistent toxin and health hazard affecting millions of people in all age groups. After entering bloodstream, 99 % of Pb is accumulated in erythrocytes and causes poisoning. Toxic Pb effects on erythrocytes membrane’s composition of phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), and sphingomyelin (SM), and phospholipids transmethylation were determined. Lipid peroxidation in Pb-exposed erythrocytes was evaluated as malondialdehyde (MDA) formation in presence of Fe and vitamin E to understand severity of Pb toxicity and its mitigation. Pb (0.5–5.0 μM) degraded PS (12 to 31 %, P?<?0.05–0.001) and elevated SM (19–51 %, P?<?0.05–0.001). Composition of PC and PE were diminished (22 %) and elevated (29 %), respectively, with higher Pb exposure (5.0 μM, P?<?0.001). Pb toxicity suppressed (P?<?0.001) transmethylation of phospholipids in membranes (34, 41, and 50 %, respectively, with 0.5, 2.5, and 5.0 μM). Pb-induced dose-related MDA production (P?<?0.05–0.001) in erythrocytes was obtained, which was accentuated in presence of Fe (P?<?0.05–0.001). The vitamin E mitigated (P?<?0.05–0.01) the severity of Pb-induced lipid peroxidation. The ratio PS/SM showed maximum change of ?27 (P?<?0.01), ?30 (P?<?0.01), and ?54 % (P?<?0.001), respectively at 0.5, 2.5, and 5.0 μM Pb exposures. Ratios PC/SM and PS/PE were at the second, whereas PE/PS at the third order. The study suggests that the mechanisms underlying distortion of compositional phospholipids, inhibition of transmethylation, and exasperated phospholipid peroxidative damage are the active phenomena of Pb toxicity in erythrocytes.

Model Plasma Membrane Exhibits a Microemulsion in Both Leaves Providing a Foundation for “Rafts”

We consider a model lipid plasma membrane, one that describes the outer leaf as consisting of sphingomyelin, phosphatidylcholine, and cholesterol and the inner leaf of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and cholesterol. Their relative compositions are taken from experiment; the cholesterol freely interchanges between leaves. Fluctuations in local composition are coupled to fluctuations in the local membrane curvature, as in the Leibler-Andelman mechanism. Structure factors of components in both leaves display a peak at nonzero wavevector. This indicates that the disordered fluid membrane is characterized by structure of the corresponding wavelength. The scale is given by membrane properties: its bending modulus and its surface tension, which arises from the membrane’s connections to the cytoskeleton. From measurements on the plasma membrane, this scale is on the order of 100 nm. We find that the membrane can be divided into two different kinds of domains that differ not only in their composition but also in their curvature. The first domain in the outer, exoplasmic leaf is rich in cholesterol and sphingomyelin, whereas the inner, cytoplasmic leaf is rich in phosphatidylserine and phosphatidylcholine. The second kind of domain is rich in phosphatidylcholine in the outer leaf and in cholesterol and phosphatidylethanolamine in the inner leaf. The theory provides a tenable basis for the origin of structure in the plasma membrane and an illuminating picture of the organization of lipids therein.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes

The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.     

Phase separation in bilayer lipid membranes: effects on the inner leaf due to coupling to the outer leaf

The combined effects of the tendency of cholesterol to order lipids in the liquid phase and the coupling between lipids in the two leaves of a bilayer are investigated theoretically utilizing a Landau free energy. We show that as a consequence of these combined effects, lateral phase separation in the outer leaf between cholesterol-rich and -poor liquids causes a similar, but weaker, phase separation in the inner leaf. Just as the areal density of lipids in the outer leaf increases in the cholesterol-rich regions, so the areal density of lipids also increases in the inner leaf. Thus, the areal density in the inner leaf varies spatially, reflecting spatial variations of the areal density in the outer leaf. This provides a mechanism for proteins attached to the inner leaf via a hydrocarbon tether to respond to variations in the composition of the outer leaf. We also note that the effect of coupling between the leaves should be observable in artificial bilayers.     

Acyl Chain Length and Saturation Modulate Interleaflet Coupling in Asymmetric Bilayers: Effects on Dynamics and Structural Order

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.     

Lipid composition of the main fractions of rabbit semen

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 microg/10(9) but was only 21-23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp(p)) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE+PC ratios than in the germinal cells. Some physiological implications are discussed.     

Relationship between the transverse distribution of phospholipids in plasma membrane and shape change of human platelets

ESR spectroscopy was used to investigate the distribution of spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the presence of human platelets. Three rates were determined: hydrolysis of the ester bond at position 2, reduction of labels by cytoplasm, and internalization of labels situated in the outer leaflet of the plasma membrane. We found that the half-time for transverse diffusion of added phospholipids was shorter for aminophospholipids (40 min and less than 10 min for PE and PS, respectively) than for the choline derivatives (greater than 120 min for PC, not measurable for SM). Addition of any of the phospholipids led to a considerable change in the initial platelet shape (assessed by electron microscopy) from a discoid form to a smaller body with very long pseudopods. When aminophospholipids were used, the platelets quickly returned to the initial shape [half-time of 20 min and less than 5 min for (0.2)PE and (0.2)PS, respectively]. Conversely, there was no relaxation after (0.2)PC or (0.2)SM was added. We conclude that there is a relationship between the excess of phospholipids in the outer leaflet of the plasma membrane and cytoskeletal organization presumably via actin polymerization, which is responsible for platelet shape.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.