Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.     

Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens

Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overexpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally transduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vitro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulating during apoptosis induced by ligation of the death receptor CD95, treatment with the anti-cancer drug etoposide, or exposure to gamma-radiation was not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane by bacterial SMase was not converted by the enzyme. Thus, GCS, located at the Golgi, is topologically segregated from Cer produced in the plasma membrane. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different Cer topology and distinct physicochemical behavior of the synthetic Cer species, respectively. Exogenous cell-permeable Cer species, despite their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conclusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and, therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of Cer accumulation is limited.     

Phospholipid scramblase: An update

The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.     

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids

Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.     

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.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure

Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP‐dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.     

Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.     

Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent,but apoptosis-unrelated,exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.     

Protection against amyloid beta-peptide (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes by tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester: implications for Alzheimer's disease

Amyloid-beta (1-42) [Abeta (1-42)] deposition in the brain is a hallmark of Alzheimer's disease (AD) and has been shown to induce apoptosis and disrupt cellular ion homeostasis. Abeta (1-42) induces membrane lipid peroxidation, and 4-hydroxynonenal (HNE) and 2-propenal (acrolein) are the two reactive products of lipid peroxidation, which structurally modify proteins by covalent interaction and inhibit enzyme function. Phosphatidylserine (PS), an aminophospholipid, is sequestered in the inner leaflet of the plasma membrane in nonstimulated cells. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine in the outer leaflet of the membrane. The ATP-requiring enzyme, flippase, maintains phospholipid asymmetry of PS. Here, we have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by Abeta (1-42). Flippase activity depends on a critical cysteine residue, a putative site of covalent modification by the Abeta (1-42)-induced lipid peroxidation products, HNE or acrolein. The present study is aimed to investigate the protective effects of tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester (FAEE) on Abeta (1-42) induced modulation in phospholipid asymmetry in the synaptosomal membranes. Pretreatment of synaptosomes with D609 and FAEE significantly protected Abeta (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes. Our results suggest that D609 and FAEE exert protective effects against Abeta (1-42) induced apoptosis. The increase in intracellular Ca(2+) might not be the sole cause for the loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in the modulation of phospholipid asymmetry. The results of this study are discussed with relevance to neuronal loss in the AD brain.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

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

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

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.     

Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells

Only capacitated sperm cells are able to fertilize egg cells, and this process is triggered by high levels of bicarbonate. Bicarbonate renders the plasma membrane more fluid, which is caused by protein kinase A (PKA)-mediated alterations in the phospholipid (PL) bilayer. We studied exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in human sperm cells. Surface exposure of PS and PE on sperm cell activation in vitro was found to be bicarbonate dependent and restricted to the apical area of the head plasma membrane. The PL scrambling in bicarbonate-triggered human sperm was not related to apoptosis, because the incubated cells did not show any signs of caspases or degeneration of mitochondria or DNA. The PL scramblase (PLSCR) gene family has been implicated in this nonspecific, bidirectional PL movement. A 25-kDa isoform of PLSCR was identified that was homogeneously distributed in human sperm cells. We propose that compartment-dependent activation of PKA is required for the surface exposure of aminophospholipids at the apical plasma membrane of sperm cells. Bicarbonate-induced PL scrambling appears to be an important event in the capacitation process, because the entire intact scrambling sperm subpopulation showed extensive tyrosine phosphorylation, which was absent in the nonscrambling subpopulation. The proportion of live cells with PL scrambling corresponded with that showing capacitation-specific chlortetracyclin staining.     

Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.     

Stimulation of suicidal erythrocyte death by lipoxygenase inhibitor Bay-Y5884.

The prostaglandin PGE(2), a metabolite of the cyclooxygenase pathway, activates Ca(2+)-permeable cation channels in erythrocyte cell membranes leading to entry of Ca(2+) with subsequent eryptosis, i.e. cell shrinkage, breakdown of phosphatidylserine (PS) asymmetry and membrane blebbing, all features typical for apoptosis in nucleated cells. PS exposing cells are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether the specific lipoxygenase inhibitor Bay-Y5884 influences eryptosis. As determined by competitive ELISA, Bay-Y5884 (20 microM) enhanced the release of PGE(2) from human erythrocytes. According to whole-cell patch-clamp, Bay-Y5884 (20 microM) activated nonselective cation channels. The effect of Bay-Y5884 on cation channels was abolished by the cyclooxygenase inhibitor diclophenac (10 microM). Bay-Y5884 (30-40 microM) significantly increased erythrocyte free Ca(2+) concentration and PS exposure as analyzed in flow cytometry by Fluo3 fluorescence and annexin-V binding, respectively. PS exposure triggered by 20 microM (but not by 40 microM) Bay-Y5884 was blunted by cyclooxygenase inhibitors acetylsalicylic acid (50 microM) and diclophenac (10 microM). In conclusion, the lipoxygenase inhibitor Bay-Y5884 enhances erythrocyte PGE(2) formation with subsequent activation of non-selective cation channels, Ca(2+) entry and phospholipid scrambling.     

The role of phosphatidylserine in recognition and removal of erythrocytes.

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.     

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.     

Mechanisms of apoptotic phosphatidylserine exposure

It has been a long-standing enigma which scramblase causes phosphatidylserine residues to be exposed on the surface of apoptotic cells, thereby facilitating the phagocytic recognition, engulfment and destruction of apoptotic corpses. In a recent paper in Science, Nagata and coworkers reveal that the scramblases Xkr8 and its C. elegans ortholog, CED-8, are activated by caspase cleavage in apoptotic cells.All cells are separated from the extracellular environment by the plasma membrane, a phospholipid bilayer that prevents diffusion of proteins, ions and other essential molecules into the extracellular space and constitutes the structure in which membrane proteins are embedded. In animal cells, the lipid composition of the outer and inner leaflets of the plasma membrane is not symmetrical. Phosphatidylcholine (PC) and sphingomyelin (SM) are mainly present in the outer leaflet of the plasma membrane, whereas phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are restricted to the inner leaflet. This lipid asymmetry is maintained by the combined action of ATP-dependent enzymes called flippases and floppases, which specifically translocate phospholipids and other molecules from the outer to the inner membrane leaflet and from the inner to the outer membrane leaflet, respectively¹. Lipid composition asymmetry not only defines the curvature and electrochemical properties of the plasma membrane, but is also essential for the correct function of determined lipids, as for instance, PI, which only functions as a second messenger if present in the inner leaflet². Nonetheless, several physiologically relevant processes as diverse as platelet activation, neurotransmitter release, sperm capacitation or apoptosis, require dissipation of plasma membrane lipid asymmetry, a process known as scrambling. The enzymes responsible for this activity are called scramblases, and function to randomize the distribution of phospholipids between both membrane leaflets in an ATP-independent manner^2,3,4.Although plasma membrane asymmetry and the existence of flippases, floppases and scramblases have been known for decades, the identity of the specific enzymes involved in these activities has only begun to be revealed during the last few years. Very recently, the group of Shigekazu Nagata identified TMEM16F as the long sought-after calcium-dependent phospholipid scramblase³. However, to date, the identity of the scramblase(s) involved in apoptosis-related (and calcium-independent) PS exposure had remained elusive. Cell surface PS exposure is a classic feature of apoptotic cells and acts as an “eat me” signal allowing phagocytosis of post-apoptotic bodies. In a recent paper in Science, Nagata''s group identified Xk-Related Protein 8 (Xkr8) as the enzyme responsible for this activity and demonstrated an evolutionarily conserved role of this protein in apoptosis-induced lipid scrambling⁵.To identify enzymes involved in membrane lipid scrambling, Nagata''s group took advantage of their previously generated mouse Ba/F3 pro-B cell line³, which presented a high basal level of PS exposure. They then generated a cDNA library from Ba/F3 cells and overexpressed it in the parental cell line. Through sequential enrichment of cells with increased PS exposure, they were able to isolate a cDNA encoding the Xkr8 protein, which enhanced PS scrambling when overexpressed. Xkr8 overexpression (but not that of TMEM16F) was able to increase apoptosis-associated PS exposure. The authors then noticed that both impaired apoptosis-induced PS exposure and deficient post-apoptotic body clearance were correlated with low Xkr8 expression in leukemia and lymphoma cell lines, which was linked to hypermethylation of its promoter. Interestingly, these two alterations were reverted either by overexpressing Xkr8 or by restitution of endogenous Xkr8 expression after treatment with the demethylating agent 5-aza-2′-deoxycytidine (DAC), suggesting that methylation of the Xrk8 promoter may be a mechanism by which tumor cells evade their phagocytosis after apoptotic death, which may result in increased local inflammation, thus favoring tumor progression. Far from being restricted only to PS exposure, Xrk8 overexpression was able to promote scrambling of multiple lipid species during apoptosis, which was demonstrated by incorporation of fluorescent PC and SM analogues. This scrambling activity was restricted to apoptotic events, as Xkr8 overexpression had no effect on Ca²⁺-induced PS exposure. This specificity may be explained by the presence of an evolutionarily conserved caspase recognition site near Xkr8 C-terminal region, whose mutation prevented both Xkr8 cleavage by caspase-3 or -7 and PS exposure during the course of apoptosis (Figure 1). These results from human cell lines were confirmed in Xkr8^−/− mouse embryonic fibroblasts and fetal thymocytes, which were unable to expose PS upon induction of apoptosis, underscoring the broad physiological relevance of Xkr8 in the apoptotic process. Finally, the authors moved to the nematode Caenorhabditis elegans to analyze whether the role of Xpr8 as lipid scramblase is evolutionarily conserved. C. elegans harbors only one ortholog of Xk proteins, CED-8, known to participate in the phagocytic removal of apoptotic corpses⁶. To determine the role of CED-8 in PS exposure, the authors took advantage of the “floater” assay, which is based on the appearance of floating cells (“floaters”) that have detached from developing C. elegans embryos defective for apoptotic cell phagocytosis⁷. Nagata''s group discovered that ced-8 deficiency leads to the accumulation of floaters. Moreover, ced-8 deficiency synergistically enhanced the number of floaters found in other engulfment mutants, which suggests that CED-8 function is not redundant to that developed by previously known engulfment mutants. This enhancing effect of ced-8 deletion was dependent on CED-3, the C. elegans ortholog of caspase-3, confirming the aforementioned results in mammalian cells. The authors then characterized that floaters resulting from ced-8 deletion show a largely deficient PS exposure after developmental apoptosis, confirming the evolutionarily conserved role of Xk-related proteins in apoptosis-induced lipid scrambling. However, they observed that ced-8 deletion does not lead to a total impairment in apoptotic PS presentation, suggesting that additional proteins must be involved in this process. Indeed, apoptosis-inducing factor can induce PS exposure in mammalian cells in a caspase-independent fashion⁸, and the C. elegans AIF ortholog, WAF-1, physically interacts with and activates another scramblase, SCRM-1⁴.Open in a separate windowFigure 1Xrp8 acts as apoptosis-induced lipid scramblase. Under normal conditions, the combined action of multiple mechanisms, including the activity of flippases and floppases, maintains lipid asymmetry between the outer and inner leaflets of the plasma membrane. Once apoptotic program is activated, caspases-3 and -7 are able to cleave and activate Xrp8 protein, which acts as a lipid scramblase and leads to the loss of lipid asymmetry, resulting in PS exposure to the extracellular space. This acts as the “eat-me” signal that will allow phagocytosis of post-apoptotic cell corpses. PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PS, phosphatidylserine.In summary, through a series of elegant manipulations, Nagata''s group has found the long-sought caspase-activated lipid scramblase that mediates the exposure of “eat-me” signals in post-apoptotic cell corpses. Further studies involving Xkr8 protein, including the mechanisms participating in its epigenetic repression may open new roads for the study of autoimmune diseases, such as lupus erythematosus, which is associated with failure in the post-apoptotic corpse clearance system.