Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry

We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of aminophospholipid translocase pathology.     

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

Engineering asymmetric vesicles

Vesicles are bilayers of lipid molecules enclosing a fixed volume of aqueous solution. Ubiquitous in cells, they can be produced in vitro to study the physical properties of biological membranes and for use in drug delivery and cosmetics. Biological membranes are, in fact, a fluid mosaic of lipids and other molecules; the richness of their chemical and mechanical properties in vivo is often dictated by an asymmetric distribution of these molecules. Techniques for vesicle preparation have been based on the spontaneous assembly of lipid bilayers, precluding the formation of such asymmetric structures. Partial asymmetry has been achieved only with chemical methods greatly restricting the study of the physical and chemical properties of asymmetric vesicles and their use in potential applications for drug delivery. Here we describe the systematic engineering of unilamellar vesicles assembled with two independently prepared monolayers; this process produces asymmetries as high as 95%. We demonstrate the versatility of our method by investigating the stability of the asymmetry. We also use it to engineer hybrid structures comprised of an inner leaflet of diblock copolymer and an independent lipid outer leaflet.     

Identification of an erythroid ATP-dependent aminophospholipid transporter

The asymmetric distribution of amino-containing phospholipids in plasma membranes is essential for the function and survival of mammalian cells. Phosphatidylserine (PS) is restricted to the inner leaflet of plasma membranes by an ATP-dependent transport process. Exposure of PS on the surface of cells serves as a binding site for haemostatic factors, triggers cell-cell interaction and recognition by macrophages and phospholipases. Exposure of PS on the red cell surface plays a significant role in sickle cell pathology. We report the identification of two different isoforms of the aminophospholipid translocase, Atp8a1, or flippase, in the murine red blood cell membrane.     

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.     

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal-Mueller planar bilayers in which one leaflet's composition would phase-separate in a symmetric bilayer and the other's would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions.     

An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane

The outer membranes (OMs) of Gram-negative bacteria have an asymmetric lipid distribution with lipopolysaccharides at the outer leaflet and phospholipids (PLs) at the inner leaflet. This lipid arrangement is essential for the barrier function of the OM and for the viability of most Gram-negative bacteria. Cells with OM assembly defects or cells exposed to harsh chemical treatments accumulate PLs in the outer leaflet of the OM and this disrupts lipopolysaccharide organization and increases sensitivity to small toxic molecules. We have identified an ABC transport system in Escherichia coli with predicted import function that serves to prevent PL accumulation in the outer leaflet of the OM. This highly conserved pathway, which we have termed the Mla pathway for its role in preserving OM lipid asymmetry, is composed of at least 6 proteins and contains at least 1 component in each cellular compartment. We propose that the Mla pathway constitutes a bacterial intermembrane PL trafficking system.     

Alteration of red cell membrane organization in sickle cell anaemia

Bee venom phospholipase A2 and the fluorescent probe merocyanine 540 were used to examine plasma membrane phospholipid organization in the spicules released by deoxygenation and reoxygenation of sickle red cells, as well as in reversibly and irreversibly sickled erythrocytes. Digestion of phosphatidyl ethanolamine in spicules was comparable to that of phosphatidyl choline, and these structures were stained by the fluorescent probe. Both assays suggest that membrane lipid asymmetry is disrupted in spicules. The residual cells, from which the spicules were derived, retain the normal asymmetry in phospholipid distribution between the outer and inner leaflets of the plasma membrane bilayer. Comparable experiments with cell fractions enriched in irreversibly sickled cells revealed a partial enhancement of phosphatidyl ethanolamine digestion, confirming the similar experiments of Lubin et al (1981). Staining of these cells with merocyanine 540, however, did not reveal a subfraction of stainable cells, indicating that this increase in phosphatidyl ethanolamine digestion is not due to the presence of a small fraction of cells which have completely lost their membrane asymmetry.     

Platelet procoagulant activity: physiological significance and mechanisms of exposure.

This review describes an important function of blood platelets in the hemostatic process: the formation of a procoagulant surface. Two essential steps of the coagulation cascade, the formation of factor Xa and the formation of thrombin, require a catalytic surface on which the enzyme complexes can be assembled. This catalytic surface is provided by the phospholipids of the platelet plasma membrane. However, in the quiescent platelet, the negatively charged phospholipids which are essential to the catalytic properties of the surface, are located in the cytoplasmic leaflet of the membrane. Dependent on the activator, the normal asymmetric distribution of phospholipids is lost, resulting in the formation of a procoagulant surface. Although platelets are primarily predestined to exhibit this function, certain pathological conditions can lead to exposure of a procoagulant surface in other cells as well. Current views to explain the mechanisms of exposure of a procoagulant surface include the role of the cytoskeleton, the formation of microvesicles from the plasma membrane as well as the contribution of a membrane protein, which actively transports specific phospholipids from the outer-to inner leaflet of the membrane bilayer.     

Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.     

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.     

Sickled erythrocytes accelerate clotting in vitro: an effect of abnormal membrane lipid asymmetry

A membrane lipid abnormality induced by sickling and found as a permanent alteration in the irreversibly sickled cell (ISC) is the rearrangement of phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) from the inner to the exterior side of the lipid bilayer. Since PS can provide a catalytic surface for the binding of blood coagulation factors and thus can exhibit procoagulant activity, we investigated the influence of oxy and deoxy reversibly sickled cells (RSC) ass well as ISC on clotting in vitro. Red blood cells (RBC), as the source of phospholipid, were added to platelet-poor citrated plasma containing Russell's viper venom (RVV) and clotting time was measured after recalcification. The clotting time after addition of normal RBC and oxy- RSC was similar to the saline blank (100 sec). In contrast, both oxy- ISC and deoxy completely sickled RSC shortened clotting time by 30%. Using liposomes prepared with identical phospholipid composition to the outer lipid leaflet of either normal RBC, RSC or ISC clotting times similar to those with intact cells were achieved. Since the liposomes did not contain protein, accentuation of clotting appears to be related to abnormal phospholipid organization, in particular to the abnormal exposure to aminophospholipids on the outer surface of the membrane. This abnormality may contribute to the pathogenesis of the vaso- occlusive episode in sickle cell anemia.     

Hereditary spherocytosis and elliptocytosis erythrocytes show a normal transbilayer phospholipid distribution.

Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment with N-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells.     

Scott syndrome: an inherited defect of the procoagulant activity of platelets

Anionic phospholipids, chiefly phosphatidylserine, are essential for the assembly of the characteristic enzyme complexes of the blood coagulation cascade at the surface of stimulated platelets and derived microparticles. In the resting cell, these phospholipids are sequestered in the inner leaflet of the plasma membrane. Scott syndrome is an extremely rare bleeding disorder that confirms the essential role of these anionic procoagulant phospholipids. In Scott patients, phosphatidylserine externalization and microparticle shedding are dramatically impaired. This functional deficiency is clearly evidenced by the measurement of residual prothrombin in serum. The recent detection of a familial Scott syndrome testifies to the genetic origin of the defect. Symptomatic Scott patients present provoked hemorrhages and are probably homozygous for the disorder whereas asymptomatic children are probably heterozygous. The Scott phenotype can be detected in platelets, red cells and lymphocytes by functional prothrombinase assay and flow cytometry. Intermediate degrees of phosphatidylserine exposure and vesiculation are observed in cells from the asymptomatic heterozygous offspring when compared to those from their homozygous defective parent and healthy subjects. The functional and molecular characterization of mutated element(s) in Scott syndrome should be of valuable help for the understanding of phospholipid transmembrane migration, also termed flip-flop, its possible links with membrane vesiculation, and the eventual implications in thrombotic or apoptotic processes.     

Membrane phospholipid asymmetry as a determinant of erythrocyte recognition by macrophages.

To assess the role of transbilayer phospholipid distribution in the recognition and phagocytosis of erythrocytes by macrophages, human erythrocytes with either a symmetric or asymmetric distribution of membrane phospholipids were prepared by hypotonic hemolysis and then incubated with cultures of human monocyte-derived macrophages. Erythrocytes with an abnormal, symmetric distribution were phagocytosed 4 times more readily than their counterparts with an asymmetric distribution or than normal, asymmetric intact erythrocytes. This enhanced phagocytosis correlated with two biophysical properties of the membrane: the spacing of phospholipids, as assessed by binding of the dye merocyanine 540, and the relative hydrophobicity, as measured by aqueous two-phase polymer partitioning. These results suggest a mechanism by which loss of membrane asymmetry is translated into recognition by macrophages and provide guidelines in loading erythrocytes that may be useful in manipulating the mode of delivery when erythrocytes are used as drug carriers in vivo.     

Plasma lipids and composition of red cells in four cases of Werner's syndrome

Four cases of accelerated aging, known as Werner's syndrome (WS), are presented. Plasma lipid and lipoprotein findings and lipid compositions of red cell membranes are compared to those of old and young healthy people. Typical clinical findings, as reported in the literature, confirmed the diagnosis. Furthermore, an elevated osmotic fragility of red blood cells (RBC) was observed. RBC membranes of WS patients showed phospholipid concentrations similar to those found in people aged over 70 years, which have been reported to be lower than those of mature donors aged below 30 years. Fatty acid patterns of RBC membranes and plasma phospholipids were indicative of some disturbance in phospholipid subclass distributions in WS-patients.     

Introduction of antigenic phospholipids into the plasma membrane of mammalian cells: organization and antibody-induced lipid redistribution.

Phosphatidylethanolamine bearing the 2,4,6-trinitrophenyl hapten was introduced into the surface membrane of mammalian fibroblasts by incubating the cells with small unilamellar vesicles containing this hapten-conjugated lipid. Consistent with integration of the antigen into the plasma membrane lipid bilayer, the exogenously supplied lipid was observed by immunofluorescence to diffuse rapidly (D greater than or equal to 0.6 X 10(-8) cm2/sec) over the surface of polykaryons formed between vesicle- and non-vesicle-treated cells. Association of the exogenous lipids with cells via adsorption of vesicles to the plasma membrane was rigorously excluded by a combination of ultrastructural and immunofluorescence studies. The distribution of the integrated antigenic lipid in the plasma membranes of vesicle-treated cells was followed by immunofluorescence microscopy. The exogenously supplied hapten-conjugated phospholipid was observed to be uniformly distributed and remained so for up to 1 hr at 37 degrees C. However, upon the addition of bivalent, but not monovalent, antihapten antibodies, the phospholipid underwent a rapid temperature-dependent redistribution, forming small patches that eventually coalesced into one or more large aggregates. This unexpected finding is discussed in terms of the mode of insertion of the lipid into the cell surface and the possible mechanisms by which bivalent ligands might alter the mobility and distribution of cell surface phospholipids.     

Phosphatidylserine exposure in B lymphocytes: a role for lipid packing

Plasma membrane lipids are usually distributed asymmetrically, with phosphatidylserine (PS) confined to the inner leaflet. PS exposure at the outer leaflet occurs early in apoptosis, but it is also constitutive on some nonapoptotic cell populations where it plays a role in cell signaling. How PS is transported ("flopped") to the cell surface is unknown. Contrary to previous reports that normal murine B lymphocytes lack lipid asymmetry, we show that PS is normally restricted to the inner leaflet of these cells. PS exposure on normal B cells did, however, occur spontaneously ex vivo. Consistent with the hypothesis that loss of PS asymmetry is regulated by CD45, PS is constitutively exposed on viable, CD45-deficient B cells. We show that calcium-stimulated PS exposure in B cells is strain variable, ABCA1 independent, and both preceded by and dependent on a decrease in lipid packing. This decrease in lipid packing is concomitant with cell shrinkage and consequent membrane distortion, both of which are potently inhibited by blockers of volume-regulatory K+ and Cl- ion channels. Thus, changes in plasma membrane organization precede PS translocation. The data suggest a model in which PS redistribution may occur by a translocase-independent mechanism at energetically favorable sites of membrane perturbation where lipid packing is decreased.