Mechanisms of attack and defence at the cell surface: The use of phospholipid bilayers as models for cell membrane

Electrical conductivity across phospholipid bilayers induced by various cytotoxic proteins has been used to analyse the damaging action of such proteins on cells; the protective effect of divalent cations and protons against such attack has also been investigated. The predominant effect of divalent cations and protons is to promote the closed state of membrane pores, i.e. to gate protein-induced lesions.     

The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.     

Changes in membrane permeability during semliki forest virus induced cell fusion

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (<6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influex of protons into the cells (Kempfet al. Biosci. Rep. 7, 761–769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (Mr<1000) was observed when infected cells were exposed to a pH<6.2. The pH dependence of the peremability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.     

Ion permeability of maize root membrane vesicles: Studies with light scattering

A possible modulation of permeabilities of membrane vesicles to anions and cations was explored by light scattering techniques, evaluated by measuring the capacity of the vesicles to shrink and swell in response to changes of the osmolarity of the incubation medium. Membrane fractions were obtained by phase partition. Purity was evaluated by detection and quantification of membrane enzyme markers: vanadate-sensitive ATPase for the plasma membrane, nitrate-sensitive ATPase for the tonoplast and azide-sensitive ATPase for mitochondria. Membrane vesicles (250 g protein) were exposed to hypertonic solutions of salts (0.6 osmolar). Kinetics of the changes in apparent absorbance at 546 nm were observed by the addition of potassium, nitrate and chloride salts. The diffusion of ions into vesicles was induced by an osmotic gradient across the membrane and brought about volume changes of vesicles. Upon addition of vesicles to the different solutions the following ion permselectivity sequences were observed: PNO ₃ ^– >PCl ^–>PSO ₄ ^2– and PK ⁺PNa ⁺>PNH ₄ ⁺.Abbreviations ATP adenosine 5-triphosphate - EDTA ethylene diaminetetraacetic acid - Tris-Mes (Tris[hydroxymethyl]aminomethane, Mes-(2-[N-Morpholino]ethanesulfonic acid) - PEG polyethylene glycol     

Interactions of oritavancin, a new lipoglycopeptide derived from vancomycin, with phospholipid bilayers: Effect on membrane permeability and nanoscale lipid membrane organization

Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (< 5 min) and complete (> 95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0-600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 μM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. I. Transport of non-electrolytes via the lipid domain

In order to further elucidate the influence of membrane lipids on transport via the lipid domain of the erythrocyte membrane, simple non-electrolyte diffusion was investigated by tracer flux measurements in whole cells after cleavage of up to 65% of phosphatidylcholine or sphingomyelin by phospholipase A₂ from Naja naja, or by sphingomyelinase.A new type of labelled model non-electrolyte was used in this study, readily available by reacting a non-labelled thiol with a labelled alkylating SH-reagent.In spite of the marked enzymatic alterations of the membrane, which lead to the occurrence of large quantities of lysophosphatidylcholine and long chain fatty acids, or of ceramide, the permeability of the lipid domain remained unaffected.This finding is very surprising, since the physical properties of the lipid phase (microviscosity, structure of the membrane interface) are likely to be perturbed in the enzyme-treated membranes.Sphingomyelinase-treated cells undergo stomatocytic shape changes followed by deep invaginations of the membrane and finally endocytosis, while phospholipase A₂-treated cells essentially maintain their normal shape.     

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (L_p), cryoprotectant permeability (P_CPA), and reflection coefficient (σ) for immature (germinal vesicle stage, GV) and in vitro–matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 ± 2°C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The L_p values for GV and MII oocytes in DMSO (L_p^DMSO) were 0.70 ± 0.06 and 1.14 ± 0.07 μm/min/atm (mean ± SEM) and in EG (L_p^EG) were 0.50 ± 0.06 and 0.83 ± 0.07 μm/min/atm, respectively. Estimates of P_DMSO for GV and MII oocytes were 0.36 ± 0.03 and 0.48 ± 0.03 μm/sec, and P_EG values for GV and MII oocytes were 0.22 ± 0.03, 0.37 ± 0.03 μm/sec, respectively. The σ values for GV and MII oocytes in DMSO (σ_DMSO) were 0.86 ± 0.03 and 0.90 ± 0.04 and in EG (σ_EG) were 0.94 ± 0.03 and 0.76 ± 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte P_DMSO is higher than the P_EG. These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages. Mol. Reprod. Dev. 49:408–415, 1998. © 1998 Wiley-Liss, Inc.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.     

氮素水平对作物膜透性等生理特性的影响

以向日葵和玉米为供试作物,进行沙培试验,设置3个氮素水平,研究了氮素水平对双子叶植物的向日葵和单子叶植物玉米耗水量,吸N量,细胞膜透性,根系活力,NR活性,游离氨基酸含量的影响。结果表明氮素供应不足,吸N量减小,细胞膜透性增大,根系活力增强：NR活性与植物叶片中游离氨基酸含量减小,2种作物的耗水量均降低,但水分利用率提高。2种作物相比较,氮素胁迫对双子叶植物向日葵的影响大于对单子叶植物玉米的影响。     

Effect of high pH on the plasma membrane potential and conductance in Elodea densa

In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions. Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about -160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance. The application of pH 11, which caused a momentary hyper- or depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around -160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Microtubule organization differs between acid and alkaline bands in internodal cells ofChara but bands can develop in the absence of microtubules

We have studied the relationship between pH banding and the organization of cortical microtubules in the alga Chara corallina Klein ex Willd. Microtubules were visualized by immunofluorescence and also by imunogold-silver enhancement to allow immediate comparison of microtubule arrangement with visible structural cell features. In cells that are nearing growth completion, microtubule number and alignment change between acidic and alkaline bands over a distance of a few micrometres. Thus, it appears that the still unknown mechanisms for microtubule organization respond to the localized differences in membrane properties. Band formation was not prevented when microtubules were depolymerized with the herbicide oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in these cells.Abbreviations DMSO dimethylsulfoxide - MT microtubule We thank Frank Gubler for helpful advice on immunogold-silver enhancement procedures, Brian Gunning for tuition in confocal microscopy, Ann Cork for assistance with photography and Dean Price for helpful discussions. G.O.W. gratefully acknowledges the receipt of a National Research Fellowship and a Queen Elizabeth II Fellowship from the Australian Research Council.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. II. Protein-mediated transfer of monosaccharides and anions

In order to investigate the influence of membrane lipids on transport via the protein domain of the erythrocyte membrane, a number of facilitated diffusion processes was studied by tracer flux techniques in whole cells after cleavage of up to 65% of the phosphatidylcholine or the sphingomyelin by phospholipase A₂ from Naja naja or bee venom, or by sphingomyelinase, respectively.The mediated fluxes of l-arabinose, which is transported by the glucose carrier, and of l-lactate, which uses a specific monocarboxylate carrier, were markedly inhibited by cleavage of either phosphatidylcholine or sphingomyelin. These phospholipid dependencies are in line with earlier data on cholesterol dependencies (Deuticke, B. (1977) Rev. Physiol. Biochem. Pharmacol. 78, 1–97). They can only in part be explained by changes of membrane fluidity. More specific interactions of the degradation products with the carrier proteins seem also to play a role.Sulfate and oxalate transfer, which proceed via the inorganic anion-exchange system, are essentially unaffected by cleavage of phosphatidylcholine and less sensitive to sphingomyelin cleavage than the two other processes. This also agrees with earlier data on cholesterol independency of sulfate transfer. The inorganic anion-exchange protein thus seems to be less dependent on the surrounding lipids in its conformation and its mode of action than the two other carriers.     

Effect of environmental factors on the limpid composition membrane structure and permeability ofMicrosporum gypseum

Supplementation with unsaturated fatty acids, substitution of glucose by glycerol as carbon source and lowered growth temperature (20°C) increased the total phospholipid content ofMicrosporum gypseum spheroplasts. Levels of sterols increased with glycerol substitution and decreased in other growth conditions. Substantial changes were seen in the ratios of unsaturated to saturated fatty acids and phosphatidylcholine to phosphatidylethanolafne under all the experimental conditions. Changed lipid composition resulted in altered uptake of amino acids (L-lysine, L-aspartic acid and L-glycine) and increased number of binding sites for a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.     

Parallel and antiparallel dimers of magainin 2: their interaction with phospholipid membrane and antibacterial activity.

Magainin 2 (M2) forms pores by associating with several other M2 molecules in lipid membranes and shows antibacterial activity. To examine the effect of M2 dimerization on biological activity and membrane interaction, parallel and antiparallel M2 dimers were prepared from two monomeric precursors. Antibacterial and haemolytic activities were enhanced by dimerization. CD measurements showed that both dimers and monomers have an alpha-helical structure in the presence of lipid vesicles. Tryptophan fluorescence shift and KI quenching studies showed that all the peptides were more deeply embedded in acidic liposomes than in neutral liposomes. Experiments on dye-leakage activity and membrane translocation of peptides suggest that dimers and monomers form pores through lipid membranes, although the pore formation may be accompanied by membrane disturbance. Although dimerization of M2 increased the interaction activity with lipid membranes, no appreciable difference between the activities of parallel and antiparallel M2 dimers was observed.     

Chloride permeability in human red cells: Influence of membrane protein rearrangement resulting from ATP depletion and calcium accumulation

Summary As 15% of band 3 protein, the assumed chloride channel, is associated with spectrin, the major peripheral protein of a lattice located at the red cell membrane-cytosol interface, the present study was undertaken to evaluate whether a rearrangement of the lattice modifies the functional property of band 3 protein. Such a rearrangement was modulated by depletion of cell ATP and/or by accumulation of Ca²⁺ ions within the cell.ATP depletion induces an inhibition of the electroneutral one-for-one chloride exchanges. Neither the modification of red cell morphology due to ATP depletion (discocyte-echinocyte transformation) nor a direct effect of the decrease in internal ATP level can account for this inhibition. On the other hand, it seems reasonable to consider that inhibition is related to the changes in membrane protein organization (formation of heteropolymers) induced by the decrease in ATP level. But it does not appear that the degree of inhibition is modified when this altered assembly of membrane protein is stabilized by disulfide linkages.Accumulation of Ca²⁺ ions in the cell at a relatively low concentration (10m range) inhibits chloride exchange without apparent modification of the assembly of membrane proteins. This effect of calcium on chloride exchanges is speculatively denoted as a direct effect of calcium.Calcium loading of fresh red cells at higher concentrations (500 to 1000 m) obtained by use of the ionophore A23187 induces a very strong inhibition of chloride exchanges. In this case, inhibition can be reasonably accounted for by two simultaneous effects of calcium: a direct effect which explains half of the inhibition and an indirect effect due to the formation of membrane protein complexes stabilized by covalent crosslinkages (activation by Ca²⁺ ions of a transglutaminase).It is interesting to note that intracellular calcium, whatever the level, inhibits electroneutral exchanges of chloride but increases net chloride movements.     

Effects of poly(ethylene glycol) on liposomes and erythrocytes: Permeability changes and membrane fusion

Poly(ethylene glycol) 6000 induced a concentration-dependent, time-dependent decrease in the latency of the reaction between Arsenazo III sequestered in liposomes and extraliposomal Ca²⁺. This was mediated by a gross change in liposomal permeability, i.e. by a release of Arsenazo III from liposomes rather than simply by an entry of Ca²⁺. The loss of latency was strongly temperature-dependent, and it was markedly diminished on increasing the cholesterol content of the liposomes. It was apparently not due to an osmotic stress of the polymer. The high activation energy found (63 kJ · mol^?1) is thought to indicate that the loss of latency resulted from local discontinuities in the lipid bilayers, caused by dehydration, rather than from partial or total lysis. Related microscopy experiments indicated that the polymer also caused the liposomes to fuse, and it is suggested that membrane fusion may have occurred at the sites of dehydration-induced discontinuities in adjacent bilayers, in addition the polymer was found to enhance the permeability of hen erythrocytes to Ca²⁺ in a manner that was comparable to its effect on liposomal latency, and it is proposed that cell fusion induced by poly(ethylene glycol) may occur at the sites of similarly induced discontinuities in the phospholipid bilayers of two closely adjacent cells.