Functional and pathological significance of phospholipid asymmetry in erythrocyte membranes

The normal asymmetric distribution of phospholipids in the plasma membrane is perturbed in erythrocytes from patients with chronic myelogenous leukemia. Since experimentally-produced lipid-symmetric erythrocytes are more interactive with cells of the reticuloendothelial system than are their lipid-asymmetric counterparts, the biological recognition of chronic myelogenous leukemia erythrocytes by the reticuloendothelial system was examined. With one exception, all erythrocyte samples from patients with chronic/benign chronic myelogenous leukemia were more adherent to endothelial cells and more readily phagocytosed by macrophagesin vitro than were normal erythrocytes. Thus, these naturally occurring pathological erythrocytes display the same dysfunctional intercellular interactions as the laboratory models.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.     

Membrane phospholipid asymmetry as a factor in erythrocyte-endothelial cell interactions

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.     

Transbilayer asymmetry of phospholipids in the plasma membrane regulates exocytotic release in mast cells

To investigate the role of the asymmetric distribution of phospholipids of the plasma membrane in exocytosis, we examined the effects of disruption of this asymmetrical distribution of lipids on exocytotic release from mast cells (RBL-2H3). Lipid scramblase, which is activated by divalent cations and catalyzes the transbilayer movement of phospholipids, was overexpressed in mast cells. Exogenous lipid scramblase was expressed in the plasma membrane and the cytoplasm. Activation of scramblase by divalent cations disrupted the asymmetrical distribution of phospholipids in the plasma membrane. Exocytotic release induced by calcium ionophore and phorbol ester was significantly inhibited in the cells transfected with wild-type scramblase. This inhibition was observed with time lag of about 5 min. Furthermore, when the asymmetric distribution of lipids was disrupted before induction of exocytosis, the inhibition of exocytotic release was obvious from the beginning without time lag. These results suggest that the asymmetric distribution of phospholipids in the plasma membrane plays an essential role in fusion between secretory granules and the plasma membrane. This finding also demonstrates that the transbilayer asymmetry of phospholipids regulates exocytosis and gives a new insight into the significance of lipid asymmetry in the plasma membrane.     

A model for the extracellular release of PAF: the influence of plasma membrane phospholipid asymmetry.

Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably release, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native membrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that 'acceptor' molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular 'acceptor' molecules.     

Role of membrane-associated cytoskeleton in maintenance of membrane structure

Various structural components of biological membranes are asymmetrically localized in the two surfaces of the membrane bilayer. This asymmetry is absolute for membrane (glyco) proteins, but only a partial asymmetry has been observed for membrane phospholipids. In the red cell membrane, choline-phospholipids are localized mainly in the outer monolayer whereas aminophospholipids are distributed almost exclusively in the inner monolayer. Several evidences are now available to suggest that this distribution of membrane phospholipids in red cells is directly or indirectly maintained by the membrane-associated cytoskeleton (membrane skeleton). This belief is well supported by the previous as well as recent studies carried out in the authors laboratory. Previously, it has been shown that lipid-lipid interactions play no major role in maintaining the transmembrane phospholipid asymmetry in erythrocytes, and that the asymmetry is lost upon covalent crosslinking of the major membrane skeletal protein, spectrin. The recent data presented here further shows that degradation or denaturation of spectrin indices rapid transbilayer movement of membrane phospholipids in the cells which, in turn, leads to more random phospholipid distributions across the membrane. These studies taken together strongly suggest that the skeleton-membrane associations are the major determinants of the transmembrane phospholipid asymmetry in erythrocytes, and that the dissociation of the skeleton from the membrane bilayer probably results in generation of new reorientation sites for phospholipids in the membrane. Communication No 3648 from C.D.R.I., Lucknow.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Extensive electroporation abolishes experimentally induced shape transformations of erythrocytes: a consequence of phospholipid symmetrization?

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195-204), exposure of erythrocytes to single brief electric field pulses (5-7 kV cm(-1)) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (相似文献   

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.     

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios ≥1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rhnull human erythrocytes have an abnormal membrane phospholipid organization.

Rhnull human erythrocytes lack the antigens of the Rhesus blood group system, have an abnormal shape and an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Studies with phospholipase A2 and sphingomyelinase C show that the asymmetric distribution of phosphatidylethanolamine (PtdEtn) in the membrane of these cells differs from that found in control cells. The amount of PtdEtn which can be hydrolysed by phospholipase A2 in the presence of sphingomyelinase C in intact Rhnull cells is twice as high as that in normal erythrocytes. In intact Rhnull cells all of the phosphatidylcholine (PtdCho) present in the membrane can be readily exchanged with a PtdCho-specific exchange protein, whereas in control cells 75% is readily exchanged and 25% at a much lower rate. This indicates that PtdCho experiences a relatively fast transbilayer movement in the Rhnull cells. The observation that the loss of two membrane polypeptides in the Rhnull cells leads to abnormal shape, increased osmotic fragility, abnormal PtdEtn distribution and enhanced transbilayer mobility of PtdCho strongly suggests that one or both polypeptides are essential for the maintenance of a proper membrane-membrane skeleton interaction.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

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.     

Enhancement of transbilayer mobility of a membrane lipid probe accompanies formation of membrane leaks during photodynamic treatment of erythrocytes

In order to further characterize membrane alterations in human erythrocytes subjected to photodynamic treatment the passive transbilayer mobility of a phospholipid analogue was studied in cells illuminated for various lengths of time in the presence of the photosensitizer, aluminum chlorotetrasulfophthalocyanine. These measurements were combined with the characterization of the membrane leaks for polar solutes occurring under the same conditions with respect to their apparent size, number and ion selectivity. The time-dependent photodynamic enhancement of leaks for K+ as well as choline or erythritol was paralleled by a marked increase of the transbilayer reorientation rate of the amphiphilic lipid probe, palmitoyllysophosphatidylcholine from 0.05% min-1 in native cells to 0.32% min-1 after 60 min illumination. The asymmetric orientation of native phospholipids was not affected by this treatment. The leak permeability proved to be due to the formation of pores with apparent radii of about 0.45 nm after 60 min illumination, and of 0.75 nm after 90 min. The number of pores per cell was calculated to be less than 1, the pores are slightly cation-selective (PK/PCl approximately 3:1). Since photodynamic treatment did not induce lipid peroxidation under the prevailing experimental conditions, protein modification must be the primary cause of both, leak permeability and flip enhancement. Since it is also likely that the leak permeability arises from oxidation of intrinsic membrane proteins, the results raise the interesting possibility that oxidative alteration of intrinsic membrane proteins may lead to enhanced transbilayer mobility of lipids.     

Selective association of phospholipids as a clue for the passive flip-flop diffusion through bilayer lipid membranes

We showed that the investigation of the selective association of phospholipids might contribute to the insight of the flip-flop diffusion processes. The process of selective association was studied quantitatively by testing the association probabilities for both parallel and anti-parallel orientations of the polar headgroups. The model of double chain binary mixture confirms a high capacity of phospholipids for self-association in parallel configuration of the electric dipole moments whether the cross-sectional area of the polar headgroups are in an usual range of 25–55 Ų. It is demonstrated that the aggregation of a class of phospholipids from a binary mixture is strongly dependent on the dipole-dipole interaction between the same phospholipids and is modulated by the magnitude of the electric dipole moment of the other phospholipids from that binary mixture. There are a great number of mechanisms involved in the transbilayer movement of phospholipids. We referred here only to the passive transport of lipids from one monolayer to the other. The flip-flop mechanisms raised in this paper are the breakdown of bilayer due to the increase of the packing density and the inversion of the coupled phospholipids from the opposite monolayers of the same bilayer. Thus, the pair formation promoting a drop in occupied volume decreases the packing pressure in the respective monolayer and consequently triggers a flip-flop into the other direction since the packing pressure in the other monolayer has not dropped. According to the present model for the binary mixtures of double-chain lipids, the rate of the flip-flop diffusion decreased by increasing the number of the methylene groups added to the acyl chain. This dependence may be perturbed whether the phospholipids possesses a very high cross-section area of the polar headgroups (a > 55 Ų). We think that the selective association of phospholipids is neither exclusively, nor only involved in promoting the transbilayer diffusion of phospholipids. Most probably, the selective association determines some phospholipid domains that attract certain particular proteins so that it can modulate the protein activity.     

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.     

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.     

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.     

Back and forth

Summary

That some membranes restrict certain lipid species to one side of the bilayer and others to the opposite side has been known for two decades. However, how this asymmetric transbilayer distribution is generated and controlled, how many and what type of membranes are so structured, and even the reason for its existence is just now beginning to be understood. It has been a decade since the discovery of an activity which transports in an ATP-dependent manner only the aminophospholipids from the outer to the inner leaflet of the plasma membrane. This aminophospholipid translocase has yet to be isolated, reconstituted, and identified molecularly. Elevating intracellular Ca²⁺ allows all the major classes of phospholipids to move freely across the bilayer, scrambling lipids and dissipating asymmetry. The nature of this pathway and its mode of activation by Ca²⁺ remain to be determined. Though loss of transbilayer asymmetry by blood cells clearly produces a procoagulant surface and increases interactions with the reticuloendothelial system, it remains to be elucidated whether maintenance of blood homeostasis is just one expression of a more general raison d'ětre for lipid asymmetry. It is these persisting uncertainties and gaps in our knowledge which make the field such an interesting and exciting challenge at the present time.