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Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action., (© 2021 Wiley-VCH GmbH.)

Fetal nucleated red blood cells (fNRBCs) in maternal peripheral blood containing the whole genetic information of the fetus may serve for noninvasive pregnant diagnostics (NIPD). However, the fetal cell-based NIPD is seriously limited by the poor purity of the isolated fNRBCs. Recently, the biomimetic cell membrane-camouflaged nanoparticles containing outstanding features have been widely used to detect and isolate rare cells from the peripheral blood samples. In this work, enythrocyte (RBC) and leukocyte (WBC) membranes are fused and coated onto magnet nanoparticles and then modified with anti-CD147 to isolate fNRBCs from the maternal peripheral blood with significant efficiency (∼90%) and purity (∼87%) in simulated spiked blood samples. Further, fNRBCs were isolated and identified from a series of maternal peripheral blood samples coming from pregnant women of 11-13 gestational weeks, and different chromosomal aneuploidies were diagnosed using fNRBCs isolated from maternal blood in early pregnancy. Our strategy may offer additional opportunity to overcome the limitations of current cell-based NIPD platforms.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.

Electroporation is defined as cell membrane permeabilization under the application of electric fields. The mechanism of hydrophilic pore formation is not yet well understood. When cells are exposed to electric fields, electrical stresses act on their surfaces. These electrical stresses play a crucial role in cell membrane structural changes, which lead to cell permeabilization. These electrical stresses depend on the dielectric properties of the cell, buffer solution, and the applied electric field characteristics. In the current study, the effect of electric field frequency on the electrical stresses distribution on the cell surface and cell deformation is numerically and experimentally investigated. As previous studies were mostly focused on the effect of electric fields on a group of cells, the present study focused on the behavior of a single cell exposed to an electric field. To accomplish this, the effect of cells on electrostatic potential distribution and electric field must be considered. To do this, Fast immersed interface method (IIM) was used to discretize the governing quasi-electrostatic equations. Numerical results confirmed the accuracy of fast IIM in satisfying the internal electrical boundary conditions on the cell surface. Finally, experimental results showed the effect of applied electric field on cell deformation at different frequencies.

Nanoparticles camouflaged by red blood cell (RBC) membranes have attracted considerable attention owing to reservation of structure of membrane and surface proteins, endowing prominent cell-specific function including biocompatibility, prolonged circulation lifetime, and reduced reticular endothelial system (RES) uptake ability. Considering the drawbacks of carrier-free nanomedicine including the serious drug burst release, poor stability, and lack of immune escape function, herein we developed and fabricated a novel RBC membranes biomimetic combinational therapeutic system by enveloping the small molecular drug coassemblies of 10-hydroxycamptothecin (10-HCPT) and indocyanine green (ICG) in the RBC membranes for prolonged circulation, controlled drug release, and synergistic chemo-photothermal therapy (PTT). The self-reorganized RBCs@ICG-HCPT nanoparticles (NPs) exhibited a diameter of ∼150 nm with core-shell structure, high drug payload (∼92 wt %), and reduced RES uptake function. Taking advantage of the stealth functionality of RBC membranes, RBCs@ICG-HCPT NPs remarkably enhanced the accumulation at the tumor sites by passive targeting followed by cellular endocytosis. Upon the stimuli of near-infrared laser followed by acidic stimulation, RBCs@ICG-HCPT NPs showed exceptional instability by heat-mediated membrane disruption and pH change, thereby triggering the rapid disassembly and accelerated drug release. Consequently, compared with individual treatment, RBCs@ICG-HCPT NPs under dual-stimuli accomplished highly efficient apoptosis in cancer cells and remarkable ablation of tumors by chemo-PTT. This biomimetic nanoplatform based on carrier-free, small molecular drug coassemblies integrating imaging capacity as a promising theranostic system provides potential for cancer diagnosis and combinational therapy.

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane's mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it's unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions-and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations' frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane's tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters-an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Rapid membrane damage of optically trapped red blood cells (RBCs) was observed at trapping powers ≥280 mW. An excellent agreement between the estimated laser-induced thermal gradient across trapped cell's membrane and that typically required for membrane electropermeabilization suggests a mechanism involving temperature gradient-induced electropermeabilization of membrane. Also the rapid collapse of the trapped cell due to membrane rupture was seen to cause shock waves in the surroundings permeabilizing nearby untrapped cells. When the experiments were carried out with RBCs collected from type II diabetic patients, a noticeable change in the damage rate compared to normal RBCs was seen suggesting a novel optical diagnosis method for the disease., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present a comprehensive theoretical-experimental framework for quantitative, high-throughput study of cell biomechanics. An improved electrodeformation method has been developed by combing dielectrophoresis and amplitude shift keying, a form of amplitude modulation. This method offers a potential to fully control the magnitude and rate of deformation in cell membranes. In healthy human red blood cells, nonlinear viscoelasticity of cell membranes is obtained through variable amplitude load testing. A mathematical model to predict cellular deformations is validated using the experimental results of healthy human red blood cells subjected to various types of loading. These results demonstrate new capabilities of the electrodeformation technique and the validated mathematical model to explore the effects of different loading configurations on the cellular mechanical behavior. This gives it more advantages over existing methods and can be further developed to study the effects of strain rate and loading waveform on the mechanical properties of biological cells in health and disease.

Cyclization has been recognized as a valuable technique for increasing the efficacy of small molecule and peptide therapeutics. Here we report the application of a hydrocarbon staple to a rationally-designed cationic antimicrobial peptide (CAP) that acquires increased membrane targeting and interaction vs. its linear counterpart. The previously-described CAP, 6K-F17 (KKKKKK-AAFAAWAAFAA-NH 2 ) was used as the backbone for incorporation of an i to i + 4 helical hydrocarbon staple through olefin ring closing metathesis. Stapled versions of 6K-F17 showed an increase in non-selective membrane interaction, where the staple itself enhances the degree of membrane interaction and rate of cell death while maintaining high potency against bacterial membranes. However, the higher averaged hydrophobicity imparted by the staple also significantly increases toxicity to mammalian cells. This deleterious effect is countered through stepwise reduction of the stapled 6K-F17's backbone hydrophobicity through polar amino acid substitutions. Circular dichroism assessment of secondary structure in various bacterial membrane mimetics reveals that a helical structure may improve - but is not an absolute requirement for - antimicrobial activity of 6K-F17. Further, phosphorus-31 static solid state NMR spectra revealed that both non-toxic stapled and linear peptides bind bacterial membranes in a similar manner that does not involve a detergent-like mechanism of lipid removal. The overall results suggest that the technique of hydrocarbon stapling can be readily applied to membrane-interactive CAPs to modulate how they interact and target biological membranes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Memantine is an NMDA receptor antagonist clinically used for the treatment of moderate to severe Alzheimer's disease. Currently, it is the only NMDA receptor antagonist drug marketed against this disease. Despite the large number of publications regarding its clinical and therapeutic use, studies related to its mechanism of action are still inconclusive. Knowledge of drug interactions with cell membranes may lead to the development of novel drugs for neurodegenerative diseases. The present mini-review aims to give an overview of the latest findings regarding the interaction of memantine with cell membranes, specifically with that of the human erythrocyte., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Spectrin-based proteinaceous membrane skeletal network has been found to be implicated in membrane disorders like hereditary spherocytosis (HS). HS greatly affects eryptosis via loss of membrane asymmetry which is seen to be the case in haemoglobin disorders like thalassemia and sickle cell disease as well. The biological implications of the status of membrane asymmetry are strongly correlated to spectrin interactions with aminophospholipids, e.g. PE and PS. Fluorescence and X-ray reflectivity (XRR) measurements of spectrin interactions with small unilamellar vesicles (SUVs) and cushioned bilayers of phospholipids, respectively, were studied. Both the XRR and fluorescence measurements led to the characterization of spectrin orientation on the surface of lipid bilayer of phosphatidylcholine (PC) and PC/aminophospholipid mixed membrane systems showing formation of a uniform layer of spectrin on top of the mixed phospholipid bilayer. Fluorescence studies show that spectrin interacts with PC and phosphatidylethanolamine (PE)/phosphatidylserine (PS) membranes with binding dissociation constants (K d ) in the nanomolar range indicating the role of spectrin in the maintenance of the overall membrane asymmetry of erythrocytes.

Transfusion of packed red blood cells (PRBC) to patients in critical states is often accompanied by post-transfusion complications. This may be related with disturbance of properties of PRBC and their membranes during long-term storage in the hemopreservative solution. The purpose of our work is the study of transformation of morphology, membranes stiffness and nanostructure for assessment of PRBC quality, in vitro. By atomic force microscopy we studied the transformation of cell morphology, the appearance of topological nanodefects of membranes and by atomic force spectroscopy studied the change of membrane stiffness during 40 days of storage of PRBC. It was shown that there is a transition period (20-26 days), in which we observed an increase in the Young's modulus of the membranes 1.6-2 times and transition of cells into irreversible forms. This process was preceded by the appearance of topological nanodefects of membranes. These parameters can be used for quality assessment of PRBC and for improvement of transfusion rules.

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid-osmotic model on the grounds of time-resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid-osmotic model can predict time-dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model-predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid-osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum-infected erythrocytes. The contribution of vesiculation to the malaria-protective function of hemoglobin S is discussed., (© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

The shape of the human red blood cell is known to be a biconcave disk. It is evident from a variety of theoretical work that known physical properties of the membrane, such as its bending energy and elasticity, can explain the red-blood-cell biconcave shape as well as other shapes that red blood cells assume. But these analyses do not provide information on the underlying molecular causes. This paper describes experiments that attempt to identify some of the underlying determinates of cell shape. To this end, red-blood-cell ghosts were made by hypotonic hemolysis and then reconstituted such that they were smooth spheres in hypo-osmotic solutions and smooth biconcave discs in iso-osmotic solutions. The spherical ghosts were centrifuged onto a coated coverslip upon which they adhered. When the attached spheres were changed to biconcave discs by flushing with an iso-osmotic solution, the ghosts were observed to be mainly oriented in a flat alignment on the coverslip. This was interpreted to mean that, during centrifugation, the spherical ghosts were oriented by a dense band in its equatorial plane, parallel to the centrifugal field. This appears to be evidence that the difference in the densities between the rim and the dimple regions of red blood cells and their ghosts may be responsible for their biconcave shape., Competing Interests: The author declares no conflict of interest.

Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 μM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016., (© 2016 Wiley Periodicals, Inc.)

Buckwheat is a valuable source of many biologically active compounds and nutrients. It has properties that reduce blood cholesterol levels, and so reduces the risk of atherosclerosis, seals the capillaries, and lowers blood pressure. The aim of the study was to determine quantitative and qualitative characteristics of polyphenols contained in extracts from buckwheat husks and stalks, the biological activity of the extracts, and biophysical effects of their interaction with the erythrocyte membrane, treated as a model of the cell. An analysis of the extract's composition has shown that buckwheat husk and stalk extracts are a rich source of polyphenolic compounds, the stalk extracts showing more compounds than the husk extract. The study allowed to determine the location which incorporated polyphenols occupy in the erythrocyte membrane and changes in the membrane properties caused by them. It was found that the extracts do not induce hemolysis of red blood cells, causing an increase in osmotic resistance of erythrocytes. They affect mainly the hydrophilic region by changing the degree of order of the polar heads of lipids, but do little to change the fluidity of the membrane and its hydration. The results showed also that polyphenolic substances included in the extracts well protect the membranes of red blood cells against oxidation and exhibit anti-inflammatory effect.

Staphylococcus aureus (S. aureus) is notorious for its ability to acquire antibiotic-resistance, and antibiotic-resistant S. aureus has become a wide-spread cause of high mortality rate. Novel antimicrobials capable of eradicating S. aureus cells including antibiotic-resistant ones are thus highly desired. Membrane-active bactericides and species-specific antimicrobials are two promising sources of novel anti-infective agents for fighting against bacterial antibiotic-resistance. We herein show that Ca(2+) and Mg(2+), two alkaline-earth-metal ions physiologically essential for diverse living organisms, both disrupt model S. aureus membranes and kill stationary-phase S. aureus cells, indicative of membrane-activity. In contrast to S. aureus, Escherichia coli and Bacillus subtilis exhibit unaffected survival after similar treatment with these two cations, indicative of species-specific activity against S. aureus. Moreover, neither Ca(2+) nor Mg(2+) lyses mouse red blood cells, indicative of hemo-compatibility. This works suggests that Ca(2+) and Mg(2+) may have implications in targeted eradication of S. aureus pathogen including the antibiotic-resistant ones.

A ceramide commonly found in mammalian cells, C16-ceramide (N-palmitoyl-d-erythro-sphingosine), is capable of forming large, protein-permeable channels in the mitochondrial outer membrane (MOM). However, C16-ceramide is unable to permeabilize the plasma membrane of erythrocytes. This specificity is unexpected considering that ceramide forms channels in simple phosphoglycerolipid membranes. Synthetic analogs of C16-ceramide with targeted changes at each of the functional regions of the molecule including methylation, altered hydrocarbon chain length, and changes in the stereochemistry, were tested to probe the role of ceramide's molecular features on its ability to form channels in these two different membrane types. The ability to permeabilize the MOM was relatively insensitive to modifications of the various functional groups of ceramide whereas the same modifications resulted in plasma membrane permeabilization (a gain of function rather than a loss of function). Some analogs (ceramine, NBD-labeled ceramide, C18,1 ceramide) gained another function, the ability to inhibit cytochrome oxidase. The gain of deleterious functions indicates that constraints on the structure of ceramide that is formed by the cell's synthetic machinery includes the avoidance of deleterious interactions. We propose that the specific structure of ceramide limits the size of its interactome (both proteins and lipids) thus reducing the likelihood of unwanted side effects., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Vibrio cholerae cytolysin (VCC) permeabilizes target cell membranes by forming transmembrane oligomeric β-barrel pores. VCC has been shown to associate with the target membranes via amphipathicity-driven spontaneous partitioning into the membrane environment. More specific interaction(s) of VCC with the membrane components have also been documented. In particular, specific binding of VCC with the membrane lipid components is believed to play a crucial role in determining the efficacy of the pore-formation process. However, the structural basis and the functional implications of the VCC interaction with the membrane lipids remain unclear. Here we show that the distinct loop sequences within the membrane-proximal region of VCC play critical roles to determine the functional interactions of the toxin with the membrane lipids. Alterations of the loop sequences via structure-guided mutagenesis allow amphipathicity-driven partitioning of VCC to the membrane lipid bilayer. Alterations of the loop sequences, however, block specific interactions of VCC with the membrane lipids and abort the oligomerization, membrane insertion, pore-formation and cytotoxic activity of the toxin. Present study identifies the structural signatures in VCC implicated for its functional interactions with the membrane lipid components, a process that presumably acts to drive the subsequent steps of the oligomeric β-barrel pore-formation and cytotoxic responses., (© 2015 John Wiley & Sons Ltd.)

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μM) than against E. coli (64 μM) and an intermediate cytotoxicity against hRBC (30 μM). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica-labelling immunoelectron microscopy in combination with lipid-binding proteins and a peptide to examine both transbilayer distribution and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells, such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBCs, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine and/or phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane., (© 2015. Published by The Company of Biologists Ltd.)

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.

In-depth understanding the toxicity of nanomaterials in red blood cells (RBCs) is of great interest, because of the importance of RBCs in transporting oxygen in blood circulation. Although the toxic effects of nanoparticles in RBCs have been revealed, the conclusions from the literature are conflicting, and in particular, the toxic mechanism is still at the infant stage. Herein, we investigated the size-dependent toxicity of well-known CdTe semiconductor quantum dots (QDs) and revealed the exact toxic mechanism at the molecular level by confocal microscopy and Fourier transform infrared (FT-IR) spectroscopy techniques. We found that smaller mercaptosuccinic acid-capped CdTe QDs (MSA-QDs) with the green-emitting color could cause hemagglutination whereas the middle-size yellow-emitting MSA-QDs induced the formation of stomatocytes and echinocytes and the bigger size red-emitting MSA-QDs induced heavy hemolysis and the formation of lots of ghost cells. The FT-IR data proved that all the MSA-QDs were likely to bond to the RBCs membranes and caused the structural changes of lipid and protein in RBCs. But only the red-emitting MSA-QDs caused the breakage of the phosphodiester bond, which might cause the heavy hemolysis. To some extent, this is the first example that reveals the hemolysis mechanism at the molecular level.

Background: The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i) and ceramide formation. The present study tested whether PRIMA-1 stimulates eryptosis., Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence., Results: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca(2+)]i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation., Conclusions: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation., (© 2015 S. Karger AG, Basel.)

Background/aims: Curcuminoids are the main bioactive constituents of the rhizome of turmeric. Erythrocytes lesions in diabetes are probably related to hyperglycemia and protein glycation. It has been reported that curcumin prevent lipid peroxidation. However, reports on the effects of demethoxycurcumin and bis-demethoxycurcumin on human erythrocytes at high glucose levels are scarce. Our aim is to investigate the effect of curcuminoids on oxidative stress and membrane of erythrocytes exposed to hyperglycemic condition., Methods: In this study, the different blood samples were treated with two doses of glucose (10 or 30 mM) to mimic hyperglycemia in the presence or absence of three kinds of curcuminoids (5 or 10 μM) in a medium at 37 °C for 24 h (Each experiment consists of 20 blood samples from 10 male and 10 female volunteers). The malondialdehyde was checked by HPLC, antioxidase (GSH and GSSG) were measured by LC/MS, SOD was checked by WST-1 kit, morphology and phospholipid symmetry were detected by flow cytometry, confocal scanning microscope and scanning electron microscope., Results: The results illustrated that all three curcuminoids reduce oxidative stress damage on the membrane and maintain a better profile for erythrocytes. Furthermore, three curcuminoids had benefit effects on antioxidase., Conclusion: The three kinds of curcuminoids supplementation may prevent lipid peroxidation at different intensity and membrane dysfunction of human erythrocytes in hyperglycemia., (© 2015 S. Karger AG, Basel.)

Biological membranes are complex structures whose mechanics are usually described at a mesoscopic level, such as the Helfrich bending theory. In this article, we present the phase-field methods, a useful tool for studying complex membrane problems which can be applied to very different phenomena. We start with an overview of the general theory of elasticity, paying special attention to its derivation from a molecular scale. We then study the particular case of membrane elasticity, explicitly obtaining the Helfrich bending energy. Within the framework of this theory, we derive a phase-field model for biological membranes and explore its physical basis and interpretation in terms of membrane elasticity. We finally explain three examples of applications of these methods to membrane related problems. First, the case of vesicle pearling and tubulation, when lipidic vesicles are exposed to the presence of hydrophobic polymers that anchor to the membrane, inducing a shape instability. Finally, we study the behavior of red blood cells while flowing in narrow microchannels, focusing on the importance of membrane elasticity to the cell flow capabilities., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Saponins, amphiphiles of natural origin with numerous biological activities, are widely used in the cosmetic and pharmaceutical industry. Some saponins exhibit relatively selective cytotoxic effects on cancer cells but the tendency of saponins to induce hemolysis limits their anticancer potential. This review focused on the effects of saponin activity on membranes and consequent implications for red blood and cancer cells. This activity seems to be strongly related to the amphiphilic character of saponins that gives them the ability to self-aggregate and interact with membrane components such as cholesterol and phospholipids. Membrane interactions of saponins with artificial membrane models, red blood and cancer cells are reviewed with respect to their molecular structures. The review considered the mechanisms of these membrane interactions and their consequences including the modulation of membrane dynamics, interaction with membrane rafts, and membrane lysis. We summarized current knowledge concerning the mechanisms involved in the interactions of saponins with membrane lipids and examined the structure activity relationship of saponins regarding hemolysis and cancer cell death. A critical analysis of these findings speculates on their potential to further develop new anticancer compounds.

Background: Our previous studies showed that hypothermic storage (HS) induces red blood cell (RBC) microparticle (RMP) generation and changes in phosphatidylserine (PS) and CD47 expression on RBCs and RMPs. The aim of this study was to evaluate the effect of cold rejuvenation treatment at multiple time points during storage on these prehemolytic indicators of RBC membrane storage lesion., Study Design and Methods: Leukoreduced RBC units in saline-adenine-glucose-mannitol were used to generate three groups: untreated controls, sham-treated units, and units treated with a cold (1-6°C) rejuvenation solution on Day 28, 35, or 42 of HS. Units were assessed for hemolysis, adenosine triphosphate (ATP) concentration, lipid composition, and RMP generation, as well as PS and CD47 expression throughout 49 days of HS., Results: Rejuvenation treatment led to a significant increase in ATP concentration in all units, irrespective of treatment day. There were no significant differences between sham- and rejuvenation-treated RBC samples in the levels of PS externalization, CD47 expression, or the rate of RMP formation. RBCs rejuvenated on Day 28 were enriched in glycerophosphocholine (+23.5%), depleted in sphingomyelin (-14%), and slightly depleted in cholesterol (-3.5%)., Conclusion: Cold rejuvenation in hypothermically stored RBCs affects the lipid composition of RBCs and respective RMPs in a time-dependent fashion., (© 2013 American Association of Blood Banks.)

A new monomer, 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl methacrylate (AEO4 MA), and its direct atom transfer radical polymerization (ATRP) into poly(AEO4 MA), then "clicked" with prop-2-ynyle choline phosphate (CP) to produce a poly(choline phosphate) are described. This polymer exhibits a lower critical solution temperature (LCST) at ≈ 32 °C, and provides a universal thermally reversible biomembrane adhesive, which can rapidly both bind to any mammalian cell membrane and internalize into the cytoplasm of nucleated cells below the LCST. Moving above the LCST reverses cell surface binding. The use of ATRP implies that such polymers can be applied to modify the surfaces of a wide range of biomaterials. The capacity to bind and immobilize cells at room temperature and release them above the LCST should be particularly useful for in vitro cell manipulation and tissue engineering applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Patients diagnosed with fulminant hepatic failure face high mortality rates. A potential therapeutic approach for these patients is the use of extracorporeal porcine liver perfusion, to serve as a form of "liver dialysis." Previously, our laboratory has shown that, during a 72-hour extracorporeal perfusion with human blood, porcine Kupffer cells bind to and phagocytose human erythrocytes causing the hematocrit to fall to 2.5% of the original value. Subsequently, erythrocyte binding has been shown to involve N-acetylneuraminic acid (Neu5Ac) on the surface of human erythrocytes and sialoadhesin on the surface of the porcine Kupffer cells., Methods: Given that no primate other than the human is known to express the majority of its sialic acid as Neu5Ac, we evaluated whether nonhuman primates would provide adequate evaluation of the loss of erythrocytes that might be expected in a clinical trial of extracorporeal porcine liver perfusion., Results: We found that while porcine macrophages readily bound human erythrocytes, binding of nonhuman primate erythrocytes was significantly reduced (P<0.001)., Conclusions: This study suggests that nonhuman primates may fail to serve as an adequate model for studying extracorporeal porcine liver perfusion because of the fact that porcine macrophages do not bind nonhuman primate erythrocytes.

Proteins from the oyster mushroom, 15 kDa ostreolysin A (OlyA), and 59 kDa pleurotolysin B (PlyB) with a membrane attack complex/perforin (MACPF) domain, damage cell membranes as a binary cytolytic pore-forming complex. Measurements of single-channel conductance and transmembrane macroscopic current reveal that OlyA/PlyB form non-selective ion-conducting pores with broad, skewed conductance distributions in N18 neuroblastoma and CHO-K1 cell membranes. Polyethylene-glycol 8000 (hydrodynamic radius of 3.78 nm) provides almost complete osmotic protection against haemolysis, which strongly suggests a colloid-osmotic type of erythrocyte lysis. Our data indicate that OlyA/PlyB form transmembrane pores of varied sizes, as other pore-forming proteins with a MACPF domain., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium., Methods: RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay., Results: Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction., Conclusion: These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids., General Significance: Our findings should therefore be of great value in understanding abnormal RBC-EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions., (© 2013.)

Plasmodium falciparum, the causative agent of malaria, completely remodels the infected human erythrocyte to acquire nutrients and to evade the immune system. For this process, the parasite exports more than 10% of all its proteins into the host cell cytosol, including the major virulence factor PfEMP1 (P. falciparum erythrocyte surface protein 1). This unusual protein trafficking system involves long-known parasite-derived membranous structures in the host cell cytosol, called Maurer's clefts. However, the genesis, role, and function of Maurer's clefts remain elusive. Similarly unclear is how proteins are sorted and how they are transported to and from these structures. Recent years have seen a large increase of knowledge but, as yet, no functional model has been established. In this perspective we review the most important findings and conclude with potential possibilities to shed light into the enigma of Maurer's clefts. Understanding the mechanism and function of these structures, as well as their involvement in protein export in P. falciparum, might lead to innovative control strategies and might give us a handle with which to help to eliminate this deadly parasite.

In this study, the antifungal activity and mode of action(s) of hibicuslide C derived from Abutilon theophrasti were investigated. Antifungal susceptibility testing showed that hibicuslide C possessed potent activities toward various fungal strains and less hemolytic activity than amphotericin B. To understand the antifungal mechanism(s) of hibicuslide C in Candida albicans, flow cytometric analysis with propidium iodide was done. The results showed that hibicuslide C perturbed the plasma membrane of the C. albicans. The analysis of the transmembrane electrical potential with 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] indicated that hibicuslide C induced membrane depolarization. Furthermore, model membrane studies were performed with calcein encapsulating large unilamellar vesicles (LUVs) and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of hibicuslide C on the fungal plasma membrane were through the formation of pores with radii between 2.3 nm and 3.3 nm. Finally, in three dimensional flow cytometric contour plots, a reduced cell sizes by the pore-forming action of hibicuslide C were observed. Therefore, the present study suggests that hibicuslide C exerts its antifungal effect by membrane-active mechanism., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In this study, we isolated (-)-olivil-9'-O-β-d-glucopyranoside (OLI9G), a phytochemical from the stem bark of Sambucus williamsii, and investigated the antifungal mechanism of OLI9G against Candida albicans. First of all, the antifungal susceptibility testing and hemolysis assay showed that OLI9G exerted a potent activity without hemolysis compared to the activity of amphotericin B. To investigate the mechanism of action of OLI9G, we first examined membrane depolarization using cyanine dye, 3,3'-dipropylthiacarbocyanine iodide (diSC35). The results showed that OLI9G significantly changed the fungal membrane potential. To further understand this activity on the membrane, we did the propidium iodide (PI) influx assay. From the results, OLI9G caused membrane permeabilization in the fungal membrane, and the three dimensional (3D) flow cytometric contour plot from the PI influx assay further showed that the cells had shrunk due to the membrane damage. Finally, the membrane-active mechanism of OLI9G was confirmed by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs). The calcein leakage showed the membrane-disruptive effects caused by direct action of OLI9G. In conclusion, the current study suggests that OLI9G exerts its antifungal activity through a membrane-disruptive action., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Atomic force microscopy (AFM) allows a researcher to obtain images of red blood cells (RBC) and their membranes. Various effects on blood lead to surface alterations of cell membranes. Such alterations are estimated by a corrugation of membrane surface. This problem is complicated for statistical analysis because the membrane is the ensemble of structures with different sizes. In the present work we used the space Fourier transform to decompose the complex AFM image of the surface into three simpler ones. The parameters of spectral windows were selected according to the natural structures of RBC membranes. This method allowed us to obtain high resolution images for the corresponding spectral windows, to establish specificity of alterations from each effect, to estimate quantitatively the membrane nanostructures at different space scales and to compare their sizes statistically after actions of different agents. The blood intoxication was modeled by adding hemin, furosemide, chlorpromazine and zinc ions into blood, in vitro., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Spectrin and ankyrin are membrane skeletal proteins that contribute to mechanical support of plasma membranes and micron-scale organization of diverse membrane-spanning proteins. This chapter provides a plausible scenario for the evolution of ankyrin- and spectrin-based membrane domains with a focus on vertebrates. The analysis integrates recent phylogenetic information with functional analyses of spectrin and ankyrin in erythrocytes, axon initial segments and nodes of Ranvier in neurons, T-tubules and intercalated disks of cardiomyocytes, lateral membrane domains of epithelial cells, and costameres of striated muscle. A core spectrin-ankyrin mechanism for coordinating membrane-spanning proteins and mechanically stabilizing membrane bilayers was expanded in vertebrates by gene duplication events, insertion of giant alternately spliced exons of axonal ankyrins, and a versatile peptide-binding fold of ANK repeats that facilitated acquisition of new protein partners. Cell adhesion molecules (CAM), including dystroglycan, L1 CAM family members, and cadherins, are the earliest examples of membrane-spanning proteins with ankyrin-binding motifs and were all present in urochordates. In contrast, ion channels have continued to evolve ankyrin-binding sites in vertebrates. These considerations suggest a model where proto-domains formed through interaction of ankyrin and spectrin with CAMs. These proto-domains then became populated with ion channels that developed ankyrin-binding activity with selective pressure provided by optimization of physiological function. The best example is the axon initial segment where ankyrin-binding activity evolved sequentially and independently first in L1 CAMs, then in voltage-gated sodium channels, and finally in KCNQ2/3 channels, with the selective advantage of fast and precisely regulated signaling., (© 2013 Elsevier Inc. All rights reserved.)

Side effects of gossypol, a polyphenolic component of Gossypium, with male contraceptive, anticancer, antimicrobial and antiviral activities include anemia due to accelerated demise of erythrocytes. Erythrocytes may be cleared from circulating blood following apoptosis-like suicidal death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether gossypol stimulates eryptosis of human erythrocytes. Utilizing flow cytometry, [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. A 48 h exposure to gossypol (0.75 μM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Gossypol exposure was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) significantly blunted the effect of gossypol (1 μM) on annexin-V-binding. The present observations reveal a novel effect of gossypol on human erythrocytes, which contributes to or even accounts for the triggering of anemia by this substance., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Typical peptides composed of Phe, Ile, and Arg residues have not been reported, and the effect of the helix-forming unit (HFU) composed of the tripeptide core on biological activity remains unclear. In this study, multimers of the 3-residue HFU were designed to investigate the structure-function relationships. The in vitro biological activities of the peptides were determined. We used synthetic lipid vesicles and intact bacteria to assess the interactions of the peptides with cell membranes. The well-studied peptide melittin was chosen as a control peptide. The results showed that the antimicrobial and hemolytic activities of the peptides increased with the number of HFUs. HFU3 had optimal cell selectivity as determined by the therapeutic index. HFU3 and HFU4 exhibited strong resistance to salts, pH, and heat. CD spectra revealed that the peptides except HFU2 displayed α-helix-rich secondary structures in the presence of SDS or trifluoroethanol (TFE). The peptides interacted weakly with zwitterionic phospholipids (mimicking mammalian membranes) but strongly with negatively charged phospholipids (mimicking bacterial membranes), which corresponds well with the data for the biological activities. There was a correlation between the cell selectivity of the peptides and their high binding affinity with negatively charged phospholipids. Cell membrane permeability experiments suggest that the peptides targeted the cell membrane, and HFU3 showed higher permeabilization of the inner membrane but lower permeabilization of the outer membrane than melittin. These findings provide the new insights to design antimicrobial peptides with antimicrobial potency by trimers.

This paper presents a novel discrete model for cell membranes and electrodes contact impedances alternative to the widely used finite elements. The finite element approach can be considered as a tool for constructing finite dimensional systems of equations that approximate the specific electroquasistatic biological problem on the discrete level. Although the finite element technique is explained typically in terms of variational or weighted-residual approaches, another, less familiar way is available to reformulate geometrically the same physical problem. This approach, referred to as discrete geometric approach, allows a direct link between geometry and the degrees of freedom describing the specific biological problem. It is straightforward to implement in any finite element open software and it assures a correct modeling of voltages and currents playing a fundamental role in a biological problem. The validation has been performed, as a first step, against analytical solutions; then, we considered impedance measurements regarding erythrocytes in whole blood flowing in microchannels at high shear rates.

Amino acid-based surfactants constitute an important class of natural surface-active biomolecules with an unpredictable number of industrial applications. To gain a better mechanistic understanding of surfactant-induced membrane destabilization, we assessed the phospholipid bilayer-perturbing properties of new cationic lysine-based surfactants. We used erythrocytes as biomembrane models to study the hemolytic activity of surfactants and their effects on cells' osmotic resistance and morphology, as well as on membrane fluidity and membrane protein profile with varying pH. The antihemolytic capacity of amphiphiles correlated negatively with the length of the alkyl chain. Anisotropy measurements showed that the pH-sensitive surfactants, with the positive charge on the α-amino group of lysine, significantly increased membrane fluidity at acidic conditions. SDS-PAGE analysis revealed that surfactants induced significant degradation of membrane proteins in hypo-osmotic medium and at pH 5.4. By scanning electron microscopy examinations, we corroborated the interaction of surfactants with lipid bilayer. We found that varying the surfactant chemical structure is a way to modulate the positioning of the molecule inside bilayer and, thus, the overall effect on the membrane. Our work showed that pH-sensitive lysine-based surfactants significantly disturb the lipid bilayer of biomembranes especially at acidic conditions, which suggests that these compounds are promising as a new class of multifunctional bioactive excipients for active intracellular drug delivery.

Purpose: The interactions of poly(ethylene oxide)-co-poly(propylene oxide) tri-block copolymers (PEO-PPO-PEO block copolymers, Pluronics®, Synperonics®, Poloxamers) of differing chemical composition with cell membranes were systematically investigated in order to clarify the mechanisms behind their previously reported various cellular responses., Methods: Relationships between the structural components of a defined series of PEO-PPO-PEO block copolymers and i) their interactions with biological membranes; ii) their cytotoxic potential were probed using a combination of haemolysis studies and cytotoxicity assays in the Caco-2 and HMEC-1 cell lines., Results: The length of the PPO block as well as the PEO/PPO ratio were determinants of their membrane binding constant and cytotoxicity endpoints measured in the MTS and LDH assays. Similar 2D parabolic relationships were found between polymer composition and their affinity for membranes or their cytotoxicity potential. Cytotoxicity was related to the ability of the copolymers to form ion transversable pores within the cell membrane., Conclusions: The data suggest a link between the affinity of certain Pluronics for biological membranes and their cellular adverse effects. This first cell-based investigation of the interactions of Pluronics with biological membranes is an important step towards unravelling the complex mechanisms which govern the biological effects of widely used amphiphilic materials.

In this paper, we describe a thiol-mediated and energy-dependent membrane transport of selenium by erythroid anion exchanger 1 (AE1, also known as band 3 protein). The AE1 is the most abundant integral protein of red cell membranes and plays a critical role in the carbon dioxide transport system in which carbon dioxide is carried as bicarbonate in the plasma. This protein mediates the membrane transport of selenium, an essential antioxidant micronutrient, from red cells to the plasma in a manner that is distinct from the already known anion exchange mechanism. In this pathway, selenium bound to the cysteine 93 of the hemoglobin β chain (Hb-Cysβ93) is transported by the relay mechanism to the Cys317 of the amino-terminal cytoplasmic domain of the AE1 on the basis of the intrinsic interaction between the two proteins and is subsequently exported to the plasma via the Cys843 of the membrane-spanning domain. The selenium export did not occur in plain isotonic buffer solutions and required thiols, such as albumin, in the outer medium. Such a membrane transport mechanism would also participate in the export pathways of the nitric oxide vasodilator activity and other thiol-reactive substances bound to the Hb-Cysβ93 from red cells to the plasma and/or peripherals.

We develop a biomimetic cell-on-membrane architecture in close-volume format which allows the interfacial biocompatibility and the reagent delivery capability for on-chip bioassays. The key concept lies in the microfluidic engraving of lipid membranes together with biological cells on a supported substrate with topographic patterns. The simultaneous engraving process of a different class of fluids is promoted by the front propagation of an air-water interface inside a flow-cell. This highly parallel, microfluidic cell-on-membrane approach opens a door to the natural biocompatibility in mimicking cellular stimuli-response behavior essential for diverse on-chip bioassays that can be precisely controlled in the spatial and temporal manner.

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Unlabelled: Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis in P. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule-membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describe P. aeruginosa OMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation., Importance: Despite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation in P. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This "bilayer-couple" model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.

We analyzed the photoinactivation of the membrane functions of bacteria and erythrocytes induced by xanthene dyes. The dyes tested were rose bengal, phloxine B, erythrosine B and eosin B. These dyes induced the leakage of K(+) from Staphylococcus aureus cells within minutes of photoirradiation, in the order of rose bengal > phloxine B > erythrosine B > eosin B. The ability of dyes to inhibit respiration was weak, except for rose bengal, and the dyes dissipated the membrane potential in similar time traces with changes in K(+) permeability. The xanthene dyes also induced the leakage of K(+) from bovine erythrocytes upon photoirradiation in the same order as that observed with bacteria. Furthermore, we found that the ability to cause the leakage of K(+) from erythrocytes was associated with dye-induced morphological changes, forming a crenated form from the normal discoid. These results are discussed in connection with the ability of xanthene dyes to generate singlet oxygen and bind to bacterial cells, and further compared with the actions of cationic porphyrins, which induced photoinactivation of bacteria through respiratory inhibition., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

It is well-known that nanomaterials are capable of entering living cells, often by utilizing the cells' endocytic mechanisms. Passive penetration of the lipid bilayer may, however, occur as an alternative process. Here we have focused on the passive transport of small nanoparticles across the plasma membranes of red blood cells, which are incapable of endocytosis. By using fluorescence microscopy, we have observed that zwitterionic quantum dots penetrate through the cell membranes so that they can be found inside the cells. The penetration-induced structural changes of the lipid bilayer were explored by surface-enhanced infrared absorption spectroscopy and electrochemistry studies of model membranes prepared on solid supports with lipid compositions identical to those of red blood cell membranes. A detailed analysis of the infrared spectra revealed a markedly enhanced flexibility of the lipid bilayers in the presence of nanoparticles. The electrochemistry data showed that the overall membrane structure remained intact; however, no persistent holes were formed in the bilayers.

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.

RBCs (red blood cells) circulating through narrow blood capillaries withstand major deformation. The mechanical and chemical stresses commonly exerted on RBCs continue to attract interest for the study of membrane structure and function. Snake venoms are lethal biochemical 'cocktails' that often contain haemotoxins, metalloproteinases, myotoxins, neurotoxins, phosphodiesterases, phospholipases and proteases. We have monitored the effects of 4 snake venoms (Pseudechis guttatus, Oxyuranus scutellatus, Notechis scutatus and Naja kaouthia) on human RBCs using NMR spectroscopy, DIC (differential interference contrast) and confocal light microscopy. RBCs underwent reproducible stomatocytosis, with unusual geographical-like indentations, spherocytosis, followed by rapid lysis. Confocal micrographs using a fluorescent dye linked to phalloidin showed that the change in morphology was associated with the aggregation of actin in the cytoskeleton. (31)P NMR saturation transfer experiments recorded transport of the univalent anion HPA (hypophosphite) on a subsecond time scale, thereby reporting on the function of capnophorin or Band 3 linked to the cytoskeleton; anion-exchange activity was substantially reduced by venom treatment. We propose a molecular-cytological hypothesis for the shape and functional changes that is different from, or supplementary to, the more 'traditional' bilayer-couple hypothesis more often used to account for similar morphological changes invoked by other reagents., (© The Author(s) Journal compilation © 2012 Portland Press Limited)