Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.     

Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE.     

The effects of coexisting lipids on the action of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C toward liposomal substrate.

The hydrolytic activity of phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) from Bacillus thuringiensis was studied in detail toward mixed liposomes consisting of PI and one of other phospholipids and cholesterol. Among PI-liposomes, small unilamellar vesicles (SUV) were the most sensitive to PI-PLC; the enzymatic hydrolysis of PI in SUV was not less than 10-fold that in large unilamellar vesicles (LUV) or in multilamellar vesicles (MLV). Thus, in a survey of the effects of coexisting lipids on PI-PLC activity, PI-SUV was used. Phosphatidylcholine (PC) was stimulative for the enzyme activity toward PI-SUV at any molar ratio of PC to PI. Also, the effects of the addition of sphingomyelin (SM), phosphatidylethanolamine (PE) and cholesterol on the enzymatic hydrolysis of PI were studied in detail on the basis of concentration of total lipids or PI.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Lipid-based mechanisms for vesicle fission

Shape transformations and topological changes of lipid vesicles, such as fusion, budding, and fission, have important chemical physical and biological significance. In this paper, we study the fission process of lipid vesicles. Two distinct routes are considered that are both based on an asymmetry of the lipid distribution within the membrane. This asymmetry consists of a nonuniform distribution of two types of lipids. In the first mechanism, the two types of lipids are equally distributed over both leaflets of the membrane. Phase separation of the lipids within both leaflets, however, results in the formation of rafts, which form buds that can split off. In the second mechanism, the asymmetry consists of a difference in composition between the two monolayers of the membrane. This difference in composition yields a spontaneous curvature, reshaping the vesicle into a dumbbell such that it can split. Both pathways are studied with molecular dynamics simulations using a coarse-grained lipid model. For each of the pathways, the conditions required to obtain complete fission are investigated, and it is shown that for the second pathway, much smaller differences between the lipids are needed to obtain fission than for the first pathway. Furthermore, the lipid composition of the resulting split vesicles is shown to be completely different for both pathways, and essential differences between the fission pathway and the pathway of the inverse process, i.e., fusion, are shown to exist.     

Interfacial enzyme activation, non-lamellar phase formation and membrane fusion. Is there a conducting thread?

Previous studies from this laboratory have shown that the enzymic generation of diacylglycerol in bilayers by phospholipase C may lead to membrane fusion through the formation of transient non-lamellar lipidic intermediates. The present paper intends to explore the correlations existing among the three main processes involved, namely (a) the induction (or inhibition) of lamellar-to-non-lamellar phase transitions in lipid mixtures through the addition of small (< 5 mol%) proportions of other lipids, (b) the promotion, by the latter lipids, of fusion in otherwise stable phospholipid vesicles (large unilamellar liposomes) under conditions leading to inverted hexagonal/inverted cubic phase formation in bulk lipid systems, and (c) the modulation, by the same small proportions of lipids, of phospholipase C hydrolysis of phosphatidylcholine in liposome bilayers. It is concluded that phospholipase C may give rise to non-lamellar lipidic structures that in turn permit liposomal fusion to occur, but neither enzyme activity is directly modulated by non-lamellar phase formation, nor will whatever kind of enzyme-induced non-lamellar structure give rise to fusion. Moreover, only under certain kinetic conditions will the enzyme give rise to the organization of non-lamellar structures that are conducive to the fusion event.     

Process of destruction of large unilamellar vesicles by a nonionic detergent,octylglucoside, and size growth factor in vesicle formation from phospholipid–detergent mixed micelles

The process of vesicle solubilization and size growth by detergents, especially by octylglucoside, was examined in detail in order to elucidate the phenomena observed in the vesicle-to-micelle transition and to clarify the size-determining factor of vesicles prepared by removing detergent from phospholipid–detergent mixed micelles. In the vesicle solubilization process, when the detergent concentration in the vesicle membrane reached a critical value, the collapse of large unilamellar vesicles (LUV) into small unilamellar vesicles (SUV) was observed. This newly appeared SUV were named SUV*. The SUV* could be produced by adding an appropriate amount of detergent to the SUV prepared by an ultrasonication method so as to increase the concentration to a little over the critical value, such as, in the case of adding octylglucoside, a molar ratio of 1.0–1.1 to phospholipid in the membrane phase. The SUV* containing octylglucoside were fusible and grow time-dependently, but those containing sodium cholate were not fusible. On the basis of the SUV* data, the following problems were solved: the variety of the size of the vesicles prepared by detergent removal from mixed micelles composed of a phospholipid and different detergents, or by different removal methods; the complex appearance of turbidity or vesicle size observed in vesicle destruction and formation; the conflict between LUV and SUV in the partition behavior of detergent and the size change with addition of detergent.     

Stable DOPG/Glycyrrhizin Vesicles with a Wide Range of Mixing Ratios: Structure and Stability as Seen by Scattering Experiments and Cryo-TEM

Phosphatidylglycerols represent a large share of the lipids in the plasmamembrane of procaryotes. Therefore, this study investigates the role of charged lipids in the plasma membrane with respect to the interaction of the antiviral saponin glycyrrhizin with such membranes. Glycyrrhizin is a natural triterpenic-based surfactant found in licorice. Vesicles made of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1’-glycerol) (DOPG)/glycyrrhizin are characterized by small-angle scattering with neutrons and X-rays (SANS and SAXS). Small-angle scattering data are first evaluated by the model-independent modified Kratky–Porod method and afterwards fitted by a model describing the shape of small unilamellar vesicles (SUV) with an internal head-tail contrast. Complete miscibility of DOPG and glycyrrhizin was revealed even at a ratio of lipid:saponin of 1:1. Additional information about the chain-chain correlation distance of the lipid/saponin mixtures in the SUV structures is obtained from wide-angle X-ray scattering (WAXS).     

Physical properties of phosphatidylcholine vesicles containing small amount of sodium cholate and consideration on the initial stage of vesicle solubilization

The effects of sub-solubilizing concentrations of sodium cholate (Na-chol) on several physicochemical properties of phosphatidylcholine (PC) small unilamellar vesicles (SUV) were considered in connection with the initial stage of membrane solubilization. ESR spectra of 12-doxylstearic acid (12-DS) in phosphatidylcholine from egg yolk (EPC) or dimyristoylphosphatidylcholine (DMPC) SUV at low concentrations (insufficient to destroy the vesicles) of Na-chol were composed of two (a strongly immobilized and an additional weakly immobilized) immiscible components. The origin of the additional bands was phase separation which occurred in the hydrophobic parts of PC SUV in the presence of Na-chol. Differential scanning calorimetry measurements demonstrated that the mixed DMPC/Na-chol SUV possessed two (a sharp low-temperature and a broad high-temperature) endothermic peaks, which is consistent with the coexistence of two immiscible phases in the vesicular membranes. zeta Potentials of the EPC/Na-chol SUV revealed that high anionic densities appeared on the surfaces of the SUV at a Na-chol concentration slightly below the upper boundary of the vesicle region. Thus, the initial stage of the solubilization of PC SUV by Na-chol was caused by the aggregation of hydrophobic parts of PC membranes, followed by the occurrence of high anionic densities on the surfaces of the vesicles. The fact that removal of Na-chol from PC/Na-chol mixed systems preferentially resulted in the formation of small vesicles might originate from these anionic charges.     

Incorporation of Na(+),K(+)-ATP-ase into the thiolipid biomimetic assemblies via the fusion of proteoliposomes

The black lipid membranes (BLMs) are artificial membrane systems that have been widely used in the study of different biological processes. In this paper the planar bilayer lipid membranes have been used to study the behavior of thiolipid molecules-dipalmitoyl-phosphatidyl-ethanolamine-mercaptopropionamide (DPPE-MPA) and cholesteryl 3-mercaptopropionate (Chs-MPA)-as compared to classical BLM made of natural lipids. We present our experiments on black thiolipid bilayer (BTM) formation from a thiolipid solution and basic results of pump currents generated by sodium-potassium pump-Na(+),K(+)-ATP-ase-introduced to such bilayer systems via proteoliposome adsorption with subsequent fusion. Our results imply that no substantial difference exists between BLMs formed from classical lipids and those made from thiolipids used in this study. The same thiolipid molecules were subsequently used for the formation of covalently bound, tethered bilayer lipid membranes (t-BLMs) on polycrystalline gold electrodes. Similarly, as in the case of BLMs, we took advantage of proteoliposome adsorption/fusion to obtain a t-BLM system with reconstituted enzyme. The vesicle fusion on hydrophobic or hydrophilic substrates is one of the main ways to obtain a bilayer system with incorporated biological species. In this paper we present also our preliminary results of electrochemical experiments using rapid solution exchange technique on such t-BLMs systems and their comparison with painted solid supported membranes (SSMs) and BLMs. We have also followed the process of vesicles fusion onto thiolipid monolayer by means of in situ atomic force microscopy in tapping mode (TM-AFM). On the basis of these experiments, we conclude that DPPE-MPA and Chs-MPA molecules used in our experiments preserve lipid properties, allowing for at least partial reconstitution of Na(+),K(+)-ATP-ase into such t-BLMs. On the other hand, the relatively compact organization on polycrystalline gold and the hydrophobic nature of the first monolayer of tethered thiolipids slows down the proteoliposome fusion onto such monolayers and consequently hinders the protein insertion. However, this effect can be overcome by mechanical stimulus that facilitates proteoliposome delamination onto the self-assembled monolayer.     

Aggregation and fusion of phospholipid vesicles

Membrane fusion and aggregation of phospholipid vesicles are reviewed and discussed. The fusion process is viewed as consisting of several stages: aggregation and close apposition of the particles, destabilization, and finally, merging of the bilayers. A procedure is presented which yields both the rate constant of the dimerization (C₁₁) and the rate constant for fusion of the dimers (f₁₁), which is a direct measure of the probability that two apposed vesicles will fuse. Experimental methods used in the study of membrane fusion are reviewed, primarily with respect to their capacity to monitor the kinetics of vesicle fusion. A few kinetic studies on the mixing of aqueous contents, leakage and increase in size of fusing vesicles are presented in detail.The range of C₁₁ values for Ca²⁺-induced aggregation and fusion of small unilamellar vesicles (SUV, ～ 125 Å radius) composed of phosphatidylserine (PS) is 10⁶ to 5 × 10⁷ M^-1 in the presence of Ca²⁺ concentrations from 1.15 to 2 mM, respectively. For larger PS vesicles (LUV, ～ 500 Å radius) C₁₁ = 6.5 × 10⁷ M^-1s^-1 in the presence of 5 mM Ca²⁺. These values are in good agreement with theoretical calculations based on van der Waals and electrostatic interactions, in which binding of cations is explicitly taken into account. The rate constants of fusion, f₁₁, are 5 s^-1 for PS SUV and 0.08 s^-1 for LUV in the presence of 2 mM and 5 mM Ca²⁺, respectively. The significance of these fusion rate constants to the duration of the fusion event is discussed.Factors affecting fusion such as cations, temperature, membrane composition vesicle concentration and size are reviewed and analyzed. Di- or tri-valent cations induce fusion of acidic phospholipid vesicles (except for phosphatidylinositol) in either pure or mixed form. Among the neutral phospholipids, phosphatidylcholine (PC) inhibits but phosphatidylethanolamine (PE) sustains or enhances the fusion capacity of acidic phospholipid vesicles. Monovalent cations induce reversible aggregation of negatively charged vesicles, but they inhibit the fusion induced by divalent cations such as Ca²⁺ or Mg²⁺. Fusion of neutral phospholipid vesicles, and it occurs the cation-induced fusion of acidic phospholipid vesicles, and it occurs only at temperatures below the gel to liquid crystalline phase transition temperature Tc. This is in contrast to the acidic phospholipid vesicle fusion which is greatly enhanced when the temperature is above the Tc of the phospholipid.     

Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks

We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.     

Characterization of pore formation by streptolysin O on supported lipid membranes by impedance spectroscopy and surface plasmon resonance spectroscopy

We report the study of the interactions of bacterial toxin streptolysin O (SLO) and cholesterol-containing membranes using electrochemical impedance and surface plasmon resonance (SPR) spectroscopy at low hemolytic units on a novel supported membrane interface. The detailed understanding of the process will aid significantly the construction of nanoscale transport channels for biosensing applications. Cholesterol-containing egg PC vesicles, pristine and incubated with SLO toxin, were fused onto a hexyl thioctate (HT)-modified gold substrate. The charge-transfer resistance of the resulting lipid membrane, which is related to the formation of the transmembrane pores, is measured with the aid of an electroactive probe. Impedance spectra were collected over a range of 0.1-100 kHz, and the obtained complex resistance was fit to an equivalent circuit. The charge-transfer resistance decreases for increasing SLO concentration, following a first-order exponential decay. The two-part membrane interface was further characterized with SPR spectroscopy. For the hexyl thioctate support layer, an equivalent monolayer thickness of 1.3 nm was determined. This value suggests a loosely packed structure of the monolayer on gold, presenting an ideal platform for permeability studies. A comparative study on the fusion behavior of vesicles with and without SLO induced pores revealed no substantial difference for the two systems, indicating that the pore formation has no adverse effect on the integrity of the vesicles. The resulting lipid membrane thickness from pre-perforated lipids was found to be 3.2 nm, suggesting that one leaflet is knocked off during the fusion process and a hybrid membrane is formed. A slightly higher thickness value of 3.4 nm was obtained for membranes from non-perforated vesicles. Deposition of lipids and subsequent incubation with SLO, as monitored by SPR, shows that the HT surface chemistry allows partial insertion of the toxin into the membrane, indicating unique properties as compared to the previously explored long-chain alkylthiols.     

Product-retardation and -activation of catalytic hydrolysis by phospholipase D in small unilamellar vesicles of egg yolk phosphatidylcholine

 We evaluated the hydrolysis of egg yolk phosphatidylcholine (PC) by phospholipase D from Streptomyces chromofuscus (PLD) in small unilamellar vesicles (SUV) in presence of 50 μM Ca²⁺. After initial choline production (hydrolysis of 1.5% of the PC at the outer leaflets of the vesicle bilayers), the hydrolysis was reduced to 5% of the initial velocity. The kinetic behavior in SUV of premixed PC and a low percentage of the hydrolysis product, phosphatidic acid (PA), was similar to that of PC SUV. The reduced velocity disappeared when the membrane structure was disintegrated by means of a nonionic surfactant. In the retardation phase, the partially hydrolyzed vesicles (postsubstrates) had much higher affinity for PLD than fresh PC SUV. These results indicated that small clusters of the product, PA, at the vesicle surface were responsible for the reduced velocity of hydrolysis. The initial velocity increased in a biphasic manner with the substrate concentration. At a PC concentration range up to 4 mM, the experimental data fit Michaelis–Menten kinetics. At concentrations above 6 mM, the velocity again markedly increased. Negatively charged mixed vesicles of PC and PA did not have such kinetics. Furthermore, adding PC SUV to the postsubstrates, where the fraction of free PLD was less than 0.05, induced steep choline production. These results showed that PLD bound to vesicles had higher activity than free PLD. We speculated that PLD bound to vesicles collided with and was directly transferred to PC SUV when the fraction of free PLD in aqueous medium was very small. Received: 5 November 1996 Accepted: 26 February 1997     

Comparison of phosphatidylcholine vesicle properties related to geometrical isomerism

Glycerophosphatidylcholine containing trans-unsaturated fatty acid residues was prepared by reaction of the corresponding naturally occurring cis lipid with photochemically generated thiyl radicals. This modified lipid was chosen as the simplest model for gaining some insights of the complex scenario of membrane formation, in connection with the role of lipid geometry and the predominance of cis lipids in eukaryotic cells. The critical aggregation concentration for the spontaneous formation of vesicles was determined for cis and trans isomers with cis-parinaric acid used as a fluorescent probe and it was found to be similar for both lipids. Vesicle dimensions were investigated by light scattering and electron microscopy, and the type of fatty acid residues influenced the vesicle diameter, with a decrease along the series cis > trans > saturated. Fluorescence measurement of dye release from trans and cis vesicles showed also a different permeability. A picture emerged of the geometrical isomer preference in cells as a process driven by natural selection during the life evolution of different organisms, both in terms of compartment dimensions and membrane functionality.     

Photo-sensitive lipid membrane perturbation by a single chain lipid having terminal spiropyran group

The synthesis a novel single chain lipid (SP-16A), containing a photo-sensitive spiropyran group at its hydrophobic terminus is described. The photoisomerization behavior of SP-16A has been studied by UV-Vis spectroscopy. The lipid has been incorporated into small unilamellar vesicles (SUVs) in quantities up to 10 mol% by mixing with dipalmitoylphosphatidylcholine (DPPC) and consequent sonication. The photo-induced membrane perturbation of the SP-16A/DPPC mixed SUV liposome was observed by monitoring of leakage of carboxyfluorescein(CF), a water soluble fluorescent dye, from the interior aqueous phase of the SUV. SUVs containing 5–10 mol% of SP-16A showed drastic leakage of CF by UV irradiation at 465 nm, however, neither liposome rupture nor release of the lipid from the SUV was observed after the UV irradiation. On/off switching of the leakage of interior material from the vesicle could also be demonstrated. In order to discuss the mechanism and kinetics of the photo-sensitive membrane perturbation behavior for the SP-16A/DPPC mixed SUV, the isomerization behavior of the SP-16A molecule and the leakage of CF from the SUV over time were investigated using a pulsed excimer laser. These experiments suggested that the main factor involved in membrane perturbation by SP-16A is a conformational change of the lipid in the membrane, which occurs after isomerization of the molecule. This new lipid molecule is thus a novel and useful photo-sensitive switching system, and may have future applications in drug delivery systems.     

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID VESICLE SYSTEMS: COMPARISON OF INSIDE-OUTSIDE ASYMMETRY BETWEEN LARGE AND SMALL UNILAMELLAR VESICLES*

Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV.     

Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles

Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.