Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

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.     

Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS     

Divalent cation-induced surface tension increase in acidic phospholipid membranes: Ion binding and membrane fusion

Chelation binding of divalent cations to phospholipid membranes may cause deformation in the headgroup regions of these lipid molecules. This deformation may be responsible for the observed large increase in surface tension of acidic phospholipid membranes induced by divalent cations. On the other hand, simple binding of monovalent cations without being followed by such a deformation of membrane molecules, does not result in a large surface tension increase in the membrane. A theoretical explanation for the above situation is given and the divalent cation-induced acidic phospholipid membrane fusion as well as other lipid membrane fusions are discussed in terms of the increased surface energy of membranes.     

Use of a fluorescence assay to monitor the kinetics of fusion between erythrocyte ghosts,as induced by sendai virus

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendal virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R₁₈) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R₁₈ insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.     

The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease

A common feature of all eukaryotic membranes is the non-random distribution of different lipid species in the lipid bilayer (lipid asymmetry). Lipid asymmetry provides the two sides of the plasma membrane with different biophysical properties and influences numerous cellular functions. Alteration of lipid asymmetry plays a prominent role during cell fusion, activation of the coagulation cascade, and recognition and removal of apoptotic cell corpses by macrophages (programmed cell clearance). Here we discuss the origin and maintenance of phospholipid asymmetry, based on recent studies in mammalian systems as well as in Caenhorhabditis elegans and other model organisms, along with emerging evidence for a conserved role of mitochondria in the loss of lipid asymmetry during apoptosis. The functional significance of lipid asymmetry and its disruption during health and disease is also discussed.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

Effects of N-phosphorylated amino acids on membrane phospholipid of human erythrocytes

Abtract Raman spectra were used to study the effects of the phosphorylated amino acids on the erythrocyte membrane. It was found that some phosphorylated amino acids might cause the polar part of the membrane phospholipid to become less ordered, the packing of the chains to become looser, and the end of the chain more ordered. Some of the phosphoamino acids cause the phospholipids' all-trans/gauche ratio to increase and some cause them to decrease. This could give some clues to the function of phosphorylated proteins in the biological process concerning the change in membrane mobility.     

Membrane fusion: Lipid vesicles as a model system

In many cellular functions the process of membrane fusion is of vital importance. It occurs in a highly specific and strictly controlled fashion. Proteins are likely to play a key role in the induction and modulation of membrane fusion reactions. Aimed at providing insight into the molecular mechanisms of membrane fusion, numerous studies have been carried out on model membrane systems. For example, the divalent-cation induced aggregation and fusion of vesicles consisting of negatively charged phospholipids, such as phosphatidylserine (PS) or cardiolipin (CL), have been characterized in detail. It is important to note that these systems largely lack specificity and control. Therefore conclusions derived from their investigation can not be extrapolated directly to a seemingly comparable counterpart in biology. Yet, the study of model membrane systems does reveal the general requirements of lipid bilayer fusion. The most prominent barrier to molecular contact between two apposing bilayers appears to be due to the hydration of the polar groups of the lipid molecules. Thus, dehydration of the bilayer surface and fluctuations in lipid packing, allowing direct hydrophobic interactions, are critical to the induction of membrane fusion. These membrane alterations are likely to occur only locally, at the site of intermembrane contact. Current views on the way membrane proteins may induce fusion under physiological conditions also emphasize the notion of local surface dehydration and perturbation of lipid packing, possibly through penetration of apolar amino acid segments into the hydrophobic membrane interior.     

Effect of acidosis on bilirubin-induced toxicity to human erythrocytes

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0–8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0–7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.     

Time sequence and morphological evaluations of cells fused by polyethylene glycol 6000

Summary Mouse L cells (clone 1D) were fused with polyethylene glycol (PEG). The fusion sequence was determined by using sequential light microscopy of the same group of cells, scanning electron microscopy (SEM), transmission electron microscopy, and freeze-etching. The cells were found to fuse only 1 min after PEG had been washed off at small localized areas. Larger fusion images were found after 3 min. Intramembrane particles were observed to have a tendency to aggregate after PEG treatment, but a direct correlation of this activity with the fusion process could not be made. No pathological changes were noted at longer times after PEG removal, except for the extensive widening of the rough-surface endoplasmic reticulum (RER) in some cells. It is proposed that fusion does not occur if apposing cells have many microvilli at the area of apparent contact. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This work was supported by U.S. Public Health Service research grants CA 10815 from the National Cancer Institute and GM 21615 from the Institute of General Medical Sciences.     

Influence of the spectrin network on fusion of influenza virus with red blood cells

We examined the influence of the physical state of the membrane skeleton on low pH fusion of influenza virus A/PR 8/34 with intact human red blood cells. Spectrin, the major component of the skeleton, is known to become denaturated at 50°C. After heat treatment of erythrocytes at 50°C we observed an enhanced kinetics of fusion monitored spectrofluorometrically by the octadecylrhodamine fluorescence dequenching assay, while the extent of fusion was not affected. The accelerated fusion of influenza virus after preincubation of red blood cells at 50°C is not mediated by alterations of the lipid phase of the target. From ESR measurements using spin-labelled phospholipids we conclude that heat-induced alterations of the spectrin network did not affect either the phospholipid asymmetry or the fluidity of the exoplasmic and the cytoplasmic leaflets of the erythrocyte membrane. Moreover, as deduced from our previous investigations, the swelling behaviour of red blood cells could not be responsible for the observed effect. Possible mechanisms for the spectrin effect include a change in the ability of the target membrane to bend locally, and a change in the rate of formation and development of the fusion pore.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Abstract Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.