Permeability of plane bilayer lipid membranes in the presence of sarcoplasmic reticulum vesicles]

Interaction between vesicles of sarcoplasmic reticulum (VSR) and bilayer lipid membranes (BLM) was investigated. VSR were added into the membrane-surrounding solution. For the formation of complex VSR-BLM the surface of BLM was charged positively by adding 10(-6) M acetyltrimethylammonium bromide and the transmembrane electrical potential was applied in the negative direction (the positive direction was chosen from inner section of camera with VSR to outer section). The formation of complex VSR-BLM proceeds via two stages. The second stage is accompanied by the formation of nonselective channels of conductivity, perhaps, aqueous pores.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.     

A new ratiometric fluorescence probe as strong sensor of surface charge of lipid vesicles and micelles

We report on the ability of a new fluorescent probe, 4-(2-pyren-1-yl-vinyl) pyridine, 1, to respond to micelles and phospholipid vesicles of different surface charge. The probe gets incorporated into micellar and membranous assemblies and shows a large red-shift in the fluorescence emission maxima especially when the surface charge of the organized media is anionic. The effect on the photo-excitation of the probe is very clear and pronounced as it can be easily visualized. The sample color upon photo-excitation changes from blue to orange/red once the probe experiences negatively charged vesicular or micellar surfaces. These results make the probe molecule useful as a reporter for sensing electrostatic environment in biological membranes and related organized assemblies.     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

Curvature-electric effect in black lipid membranes

Upon periodical bending of a BLM, by means of oscillating hydrostatic pressure with sound frequency, the generation of an a.c. electric current with the same frequency can be observed under short circuit conditions. Previously, this phenomenon was attributed by us to a displacement current due to the oscillating flexoelectric polarization. The latter is proportional to the membrane curvature and depends on the lipid dipole moment and surface charge.The theory of this effect is outlined here. Earlier results concerning dipolar and quadrupolar contributions to the total current are presented and new expressions about charge contributions are derived for the two basic regimes of free and blocked lateral lipid exchange.Further, a systematic study of the frequency dependence of the amplitude and phase of the curvature-electric signal from a bacterial phosphatidylethanolamine/n-decane BLM is reported. Constant membrane curvature at each vibration frequency was assured by a calibration of the capacitance current observed with a small transmembrane voltage.The frequency dependence of the curvature-electric current amplitude was characterized by two regions: low frequency plateau and high frequency slope, the boundary between them being about 160 Hz. Such behaviour suggested a switching of the mechanism of membrane polarization from free to blocked lateral lipid exchange. Frequency dependence of the phase shift was characterized by low frequency and high frequency plateaus and a gradual transition between them. From phase measurements on initially curved membranes the sign of the membrane flexo-coefficient was found to be negative.The influence of some modifiers of the surface charge and surface dipole, as well as of the membrane conductivity, upon the value of the effect was studied. Surface charge was separately measured by the internal field compensation method under an ionic strength gradient. The membrane flexoelectric coefficient was evaluated and compared to the theoretical predictions. A conclusion was drawn that under the present experimental conditions the main contribution to the effect comes from the curvatureinduced shift of the surface charge equilibrium.Presented at the Tenth International Liquid Crystal Conference, 15–21 July 1984, York, UK     

Effect of insulin on lipid bilayer viscoelasticity

Changes in the Young elasticity modulus in perpendicular direction to the membrane surface E perpendicular, in the coefficient of dynamic viscosity eta, in the electric capacitance C, in the surface charge U1, in the conductivity g and in the coefficient of non-linearity beta of current-voltage characteristic caused by insulin were studied in bilayer lipid membranes (BLM) prepared from a mixture of egg lecithin and cholesterol (4:1, w/w) in n-heptane. Even relatively small concentrations of insulin in electrolyte (ci approximately 4.8 x 10(-11) mol/l) caused a diminution in parameters E perpendicular and eta. Negative surface charge emerged on the membrane due to the insulin absorption, and U1 gradually increased depending on the concentration of the hormone in the electrolyte. Addition of insulin was also followed by an increase in membrane conductivity and affected the value of the coefficient of non-linearity beta of current-voltage characteristic. The effect of insulin on the BLM structure was discussed on the basis of the results obtained.     

Inhibitory effect of intracellular lipid load on macroautophagy

Degradation of intracellular components via macroautophagy is a complex multi-step process that starts with the sequestration of cytosolic cargo in a de novo formed double-membrane vesicle or autophagosome. This compartment acquires the hydrolases required for cargo digestion by fusion with lysosomes. In contrast to the detailed molecular dissection of the components that participate in the induction, regulation and execution of the early steps in macroautophagy, through the engulfment of cargo in autophagosomes, the mechanisms involved in the lysosomal clearance of autophagosomes have been poorly characterized in mammals. One of the major limitations in this respect has been the fact that autophagosome-lysosome fusion in intact cells involves several independent steps, namely binding of the molecular motors associated to the surface of the vesicles with the cytoskeletal network, directional vesicular trafficking and fusion between the two vesicular compartments. Furthermore, both lysosomes and autophagosomes are very dynamic organelles that can fuse with different vesicular structures involved in macroautophagy, but also along the endocytic and phagocytic pathways. To resolve these limitations and directly analyze the fusion step between autophagosomes and different compartments of the endocytic-lysosomal pathway, we have recently developed an in vitro fusion assay with autophagosomes, lysosomes and endosomes isolated from cells or tissues. Fluorescent labeling of these compartments allows for the tracking of fusion events by fluorescence microscopy or by fluorescence activated cell sorting (FACS). Labeling of either membrane proteins on the surface of the organelles or dye-loading of the vesicles permits the monitoring of hemi-membrane fusion and complete vesicular fusion (cargo mixing).     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Interaction between plasma lipoproteins and bilayer lipid membranes. The role of surface charge

Interactions of planar BLM with different thickness and surface charge were analysed theoretically. Drawing together of the membranes is accompanied with the appearance of intramembrane potential jumps which may cause destruction and breakdown of the membranes. The theory is extrapolated to the interaction between spherical lipoprotein particles and planar BLM. Experimentally calculated (by means of ESR) surface charges of lipoproteins of low density (LLD) (--0,3 . 10(-2) C/m2) and lipoproteins of high density (LHD) (--2 . 10(-2) C/m2) enabled calculation of the interaction energy between the particles and BLM as well as of the values of intramembrane potential jumps. The latter cause local reconstructions of the membranes in the contact region and fusion of the particles with them. The earlier obtained experimental data were proved by the finding that LHD adsorption as compared with LLD is impeded due to the existence of a high energetic barrier. These peculiarities of the particles manifested during their interactions with BLM seem to be one of the factors responsible for atherogenic function of LLD and antiatherogenic one of LHD.     

Effects of pH on the kinetics of the interaction between anthracyclines and lipid bilayers

In the present study we have analyzed the kinetics of the initial steps (first 10 seconds) of the interaction between the anthracycline daunomycin (DNM) and artificial lipid vesicles bearing opposite surface charge. The process can be monitored by measuring the fluorescence increase of the drug accompanying its association with the lipid bilayers. The results indicated that DNM consistently interacts to a larger extent with the liposomes having negative surface charge than with those having positive surface charge, suggesting the involvement of electrostatic components in the interaction. In contrast, DNM associates with the vesicles bearing positive surface charge 2 – 3 times faster (in terms of the apparent rate constants describing the process of interaction) than with those having negative surface charge, an observation probably related to the more fluid physical state of the former. Regarding the rate of access of DNM to the vesicles, rather than depending on the surface charge of the vesicles, this is critically affected by the ionization state of the drug, i. e. by the pH. Thus, the rate at which the interaction proceeds is increased nearly 15-fold when the pH of the medium increases from 7.0 to 8.3, regardless of the surface charge of the liposomes. On this basis, and taking into account the fact that the anthracyclines enter the cells by passive diffusion, possible effects of pH on the transport of these drugs through the membranes of tumor cells are discussed. Received: 3 December 1996 / Accepted: 4 July 1997     

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.     

Nuclear magnetic resonance studies of cation transport across vesicle bilayer membranes.

We analyze an increasingly popular NMR method analogous to the black lipid membrane (BLM) isotopic tracer experiment for the study of mediated cation transport but involving the preparation of vesicles with an environment asymmetric in that paramagnetic metal ions are present only outside the vesicles. This asymmetry is manifest in the NMR spectrum as two distinct resonances for magnetic nuclei in outside and inside lipid headgroups. As mediated transport begins and for the paramagnetic metal ions enter the vesicles, the inner headgroup resonance line shifts and changes shape with a time course containing much information on the actual ion transport mechanism. Processes by which the ions enter the vesicles one or a few at a time (such as via a diffusive carrier) are easily distinguishable from those by which the ions enter in large bursts (such as by pore activation). The limiting case where intervesicular mediator exchange is slow relative to cation transport (the situation for integral membrane proteins) is treated analytically. Computer simulated curves indicate conditions necessary for certain changes in the line shape which are analogous to the "current jumps" observed in BLM conductance studies. The theory derived allows estimates of the average number of ions entering the first few bursts, how often the bursts occur, and how they depend on the concentration of the mediating species in the vesicular membrane. Preliminary experimental spectra illustrating some of the various possible line shape behaviors are presented.     

Electrophoretic mobility of bimolecular lipid membranes

The floating membrane vesicle is fixed by the counter solution flow in different points along the radius of a cylinder electrophoretic chamber, which permits to measure the vesicle electrophoretic mobility (EM). Close state condition of the chamber is provided for by the capillary system successively connected with the chamber. Relationship between EM of bimolecular lipid membranes (BLM) and pH and ionic concentration of aqueous solution qualitatively coincides with similar relationship for liposomes. The EM value of BLM essentially decreases in solution containing polyene antibiotics nystatine and levorin when derivative of cholesterol having 3betaOH-groups is present in the membrane.     

Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.     

Intermolecular interactions of influenza M1 proteins on the model lipid membrane surface: A study using the inner field compensation method

M1 protein binding to the lipid bilayer membrane (BLM) was recorded by the inner field compensation technique as a change of the boundary potential. After the protein was added to the bulk solution, the M1 adsorption produced a slow increase in boundary potential to a stationary value that was reached within the time period dependent on the quantity of the added protein. The stationary value of the potential grew with the decrease of pH or KCl concentration in the medium and was higher in the presence of negatively charged lipids in the BLM. It was shown that the potential growth with the decrease of pH is due to an increase of M1 molecule charge and not due to the increase of the M1 surface concentration or to the change of lipid charge. As the potential did not change after the removal of the protein from the bulk solution, we consider the protein adsorption on the BLM irreversible. The obtained results suggest that the protein adsorption is influenced by both electrostatic and hydrophobic interactions of M1 molecules with each other and with lipid membrane. We offer a mechanism of dissociation of the viral shell formed by M1 matrix protein. The protein shell is destabilized due to electrostatic repulsion of protein molecules caused by the increase of their positive charge.     

Interaction of the nuclear proteins protamine and histone with charged bilayer membranes]

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.     

Estimation of membrane surface potential and charge density from the phase equilibrium of a paramagnetic amphiphile.

The distribution of a paramagnetic amphiphile, N,N-dimethyl-N-nonyl-N-tempoylammonium ion, between the membranes of charged phospholipid vesicles and the surrounding aqueous medium was studied by electron paramagnetic resonance spectroscopy. By systematically varying the surface charge of the vesicles and the aqueous electrolyte concentration, the distribution was shown to indicate vesicle surface potential. At each fixed phospholipid composition, the surface potential exhibited a dependence on aqueous NaCl concentration very similar to that predicted by the Gouy equation. The ability to sense and quantitate surface potentials makes this facile and sensitive technique of value in the study of cell and organelle surfaces.