Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

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.     

Lipid trafficking to the outer membrane of Gram-negative bacteria

The envelope of Gram-negative bacteria is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer with an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Most of the steps of lipid synthesis occur within the cytoplasmic compartment of the cell. Lipids must then be transported across the inner membrane and delivered to the outer membrane. These topological features combined with the ability to apply the tools of biochemistry and genetics make the Gram-negative envelope a fascinating model for the study of lipid trafficking. In addition, as lipopolysaccharide is essential for growth of most strains and is a potent inducer of the mammalian innate immune response via activation of Toll-like receptors, Gram-negative lipid transport is also a promising target for the development of novel antibacterial and anti-inflammatory compounds. This review focuses on recent developments in our understanding of lipid transport across the inner membrane and to the outer membrane of Gram-negative bacteria.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Structure of the shell and tertiary membranes of eggs of softshell turtles (Trionyx spiniferus)

Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip at its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except at the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur at the intersection of four or more shell units.     

Tethered lipid bilayers on electrolessly deposited gold for bioelectronic applications

This paper presents the formation of a novel biomimetic interface consisting of an electrolessly deposited gold film overlaid with a tethered bilayer lipid membrane (tBLM). Self-assembly of colloidal gold particles was used to create an electrolessly deposited gold film on a glass slide. The properties of the film were characterized using field-effect scanning electron microscopy, energy dispersive spectroscopy, and atomic force microscopy. Bilayer lipid membranes were then tethered to the gold film by first depositing an inner molecular leaflet using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (DPGP), and cystamine in ethanol onto a freshly prepared electrolessly deposited gold surface. The outer leaflet was then formed by the fusion of liposomes made from DPGP or 1,2-dioleoyl-sn-glycero-3-phosphocholine on the inner leaflet. To provide functionality, two membrane biomolecules were also incorporated into the tBLMs: the ionophore valinomycin and a segment of neuropathy target esterase containing the esterase domain. Electrochemical impedance spectroscopy, UV/visible spectroscopy, and fluorescence recovery after pattern photobleaching were used to characterize the resulting biomimetic interfaces and confirm the biomolecule activity of the membrane. Microcontact printing was used to form arrays of electrolessly deposited gold patterns on glass slides. Subsequent deposition of lipids yielded arrays of tBLMs. This approach can be extended to form functional biomimetic interfaces on a wide range of inexpensive materials, including plastics. Potential applications include high-throughput screening of drugs and chemicals that interact with cell membranes and for probing, and possibly controlling, interactions between living cells and synthetic membranes. In addition, the gold electrode provides the possibility of electrochemical applications, including biocatalysis, bio-fuel cells, and biosensors.     

Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.     

Phospholipid Transverse Diffusion in Synaptosomes: Evidence for the Involvement of the Aminophospholipid Translocase

We have studied in Torpedo marmorata electric organ synaptosomes the equilibration kinetics of spin-labeled phospholipid analogues initially incorporated into the outer plasma membrane monolayer. As assayed by evoked releases of both ATP and acetylcholine, the nerve endings were closed vesicles containing an energy source. The aminophospholipids (phosphatidylethanolamine and phosphatidylserine) were translocated toward the inner membrane leaflet faster and to a higher extent than their choline-containing counterparts (phosphatidylcholine and sphingomyelin). This difference was abolished by incubation of synaptosomal membranes with N-ethylmaleimide, suggesting that the accumulation of aminophospholipids in the inner layer was driven by a protein. This phenomenon is comparable with what was described in plasma membranes of other eucaryotic cells (erythrocyte, lymphocyte, platelet, fibroblast), and thus we would suggest that an aminophospholipid translocase, capable of moving the aminophospholipids from the outer to the inner layer at the expense of ATP, is also present in the synaptosomal plasma membrane.     

Cu ions interact with cell membranes

The influence of Cu²⁺ ions on the physical properties of resealed human erythrocyte membranes was studied by fluorescence spectroscopy. A net ordering effect was observed at the hydrophobic–hydrophilic interface both in the bulk as well as in the lipid–protein boundary. The explanation for this result was found by X-ray diffraction performed in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Cu²⁺ did not significatively affect the structure of DMPE; however, DMPC polar head and hydrocarbon chain arrangements were perturbed at low but reordered at high Cu²⁺ concentrations. These effects were respectively explained in terms of a limited and extended interaction between Cu²⁺ ions and DMPC PO₄⁻ groups. Thus, the ordering effect in the erythrocyte membrane could be based on the interaction of this cation with phosphatidylcholine phosphate groups located in its outer leaflet. This binding, besides producing a decrease of membrane fluidity, might also induce a change in its electric field. These two effects should affect the activity of membrane proteins, particularly of ion channels. In fact, it was found that increasing concentrations of Cu²⁺ ions applied to either the mucosal or serosal surface of the isolated toad skin elicited a dose-dependent decrease of the short-circuit current (SCC) and of the potential difference (PD). These results lead to the conclusion that Cu²⁺ ions inhibited Na⁺ transport across the epithelial cell membranes.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

Characterization of biomimetic surfaces formed from cell membranes

A method for fabricating biomimetic surfaces from intact cell membranes is described. A monolayer of alkanethiol on gold is covered by a second layer derived from the components of erythrocyte membranes either by self-assembly or by Langmuir-Blodgett methods. The resulting asymmetric hybrid layer was characterized by ellipsometry, surface plasmon resonance (SPR), contact angle, capacitance, voltammetry, and electron and atomic force microscopy. The erythrocyte membrane layer was measured to be approximately 30-40 A in thickness. Using SPR, the presence of erythrocyte components on the surface was demonstrated by their selective removal by enzymatic action. The uniform deposition of membranous material on the substrate was shown by electron and atomic force microscopy. Demonstration of acetylcholinesterase (AChase) activity, a membrane-anchored enzyme, on the surface for at least 8 days, suggests that the outer leaflet of the erythrocyte membrane is present in its native form. Cyclic voltammetry demonstrates that enhanced electron transport from a solution redox species accompanies formation of the erythrocyte layer at the surface. This enhanced electron transport is blocked by 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid, a well known blocker of anion transport, suggesting that an erythrocyte anion transporter protein is incorporated into the surface layer in an active conformation.     

Transverse distribution and movement of lysophosphatidylcholine in sarcoplasmic reticulum membranes as determined by 13C NMR and lysophospholipase.

1. The transverse distribution of 1-palmitoyl-sn-glycero-3-phospho-N-[Me-13C]-choline in vitro incorporated in sarcoplasmic reticulum has been measured by means of 13C NMR and DyCl3 as an impermeable shift reagent. 2. Lysophosphatidylcholine added to the membranes equilibrates within 30 min at 20 degrees C between outer and inner membrane leaflet so that 42% is located in the inner leaflet. 3. Lysophosphatidylcholine diffuses back from the inner leaflet to the outer upon lysophospholipase action on the outer lysophosphatidylcholine pool.     

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

Understanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles.     

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.     

The Asymmetrical Structure of Golgi Apparatus Membranes Revealed by In situ Atomic Force Microscope

The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm –200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level.     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

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.     

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

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

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