Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues

Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar-inverse hexagonal (L(alpha)-H(II)) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.     

Destabilization and Fusion of Zwitterionic Large Unilamellar Lipid Vesicles Induced by a β-Type Structure of the Hiv-1 Fusion Peptide

Abstract

The peptide HIVarg, corresponding to a sequence of 23 amino acid residues at the N-terminus of HIV-1 gp41, has the capacity to induce fusion of large unilamellar vesicles (LUV) consisting of negatively charged or zwitter-ionic phospholipids. In the present study, we further characterize this destabilization and fusion process using LUV consisting of phosphatidylcholine, phosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Evidence for fusion includes a demonstration of membrane lipid mixing as well as mixing of aqueous vesicle contents. Kinetic analysis of the overall process of vesicle aggregation and fusion revealed that the rate constant of the fusion step per se increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide acts as a true fusogen. The peptide caused the release of small molecules (Ants/DPX), whereas large solutes (Fitc-dextran, MW_av 19,600) were partly retained. The estimated critical number of peptides per vesicle necessary to release vesicle contents, M = 2-4, indicates that leakage does not involve the formation of classical pores. Infrared spectroscopy of the peptide in the presence of liposomes demonstrated that the equilibrium conformation of the membrane-bound peptide is an antiparallel β-structure. This finding supports the notion that the HTV fusion peptide in a β-conformation has the capacity to perturb vesicle bilayers, inducing initial permeabilization and subsequent membrane fusion.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Bilayer Mixing,Fusion, and Lysis Following the Interaction of Populations of Cationic and Anionic Phospholipid Bilayer Vesicles

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.     

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca²⁺ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca²⁺ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca²⁺ accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t_mix < 1 s) while an ensemble assay revealed two slow mixing processes with t_mix ∼ 1 min and t_mix ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Evaluation of Meloxicam-Loaded Cationic Transfersomes as Transdermal Drug Delivery Carriers

The aim of this study is to develop meloxicam (MX)-loaded cationic transfersomes as skin delivery carriers and to investigate the influence of formulation factors such as cholesterol and cationic surfactants on the physicochemical properties of transfersomes (i.e., particle size, size distribution, droplet surface charge and morphology), entrapment efficiency, stability of formulations and in vitro skin permeation of MX. The transfersomes displayed a spherical structure. Their size, charge, and entrapment efficiency depended on the composition of cholesterol and cationic surfactants in the formulation. Transfersomes provided greater MX skin permeation than conventional liposomes and MX suspensions. The penetration-enhancing mechanism of skin permeation by the vesicles prepared in this study may be due to the vesicle adsorption to and/or fusion with the stratum corneum. Our results suggest that cationic transfersomes may be promising dermal delivery carriers of MX.     

Calculation of free energy barriers to the fusion of small vesicles

The fusion of small vesicles, either with a planar bilayer or with one another, is studied using a microscopic model in which the bilayers are composed of hexagonal- and lamellar-forming amphiphiles. The free energy of the system is obtained within the self-consistent field approximation. We find that the free energy barrier to form the initial stalk is hardly affected by the radius of the vesicle, but that the barrier to expand the hemifusion diaphragm and form a fusion pore decreases rapidly as the radius decreases. As a consequence, once the initial barrier to stalk formation is overcome, one which we estimate at 13 k_BT for biological membranes, fusion involving small vesicles should proceed with little or no further input of energy.     

Studies on membrane fusion. 1. Interactions of pure phospholipid membranes and the effect of myristic acid,lysolecithin, proteins and dimethylsulfoxide

The interaction and mixing of membrane components in sonicated unilamellar vesicles and also non-sonicated multilamellar vesicles prepared from highly purified phospholipids suspended in NaCl solutions has been examined. Electron microscopy and differential scanning calorimetry were used to characterize the extent and kinetics of mixing of membrane components between different vesicle populations. No appreciable fusion was detected between populations of non-sonicated phospholipid vesicles incubated in aqueous salt (NaCl) solutions. Mixing of vesicle membrane components via diffusion of phospholipid molecules between vesicles was observed in populations of negatively charged phosphatidylglycerol vesicles but similar exchange diffusion was not detected in populations of neutral phosphatidylcholine vesicles. Incubation of sonicated vesicle populations at temperatures close to or above the phospholipid transition temperature resulted in an increase in vesicle size and mixing of vesicle membrane components as determined by a gradual change in the thermotropic properties of the mixed vesicle population. The interaction of purified phospholipid vesicles was also examined in the presence of myristic acid and lysolecithin. Our results indicate that while these agents enhance mixing of vesicle membrane components, in most cases mixing probably proceeds via diffusion of phospholipid molecules rather than by fusion of entire vesicles. Increased mixing of vesicle membrane components was also produced when vesicles were prepared containing a purified hydrophobic protein (myelin proteolipid apoprotein) or were incubated in the presence of dimethylsulfoxide. In these two systems, however, the evidence suggests that mixing of membrane components results from the fusion of entire vesicles.     

Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay

In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the V_C peptide synaptobrevin-2 (syb(57–92)) increases the docking efficiency by a factor of ∼30, but docking remains rate-limiting. In the presence of V_C peptide, the fusion step occurs on a timescale of ∼10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca²⁺, Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Melittin induces fusion of unilamellar phospholipid vesicles

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26–30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690–692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.     

pH-dependent fusion of liposomes using titratable polycations

Polylysine promoted extensive membrane mixing of liposomes only if the buffer pH was below the pKa of the lysyl residues. This observation suggested that fusion could be regulated in a physiological pH range if the homopolymer of L-histidine was substituted as fusogen. Microgram quantities of polyhistidine were added to liposomes composed of soybean phospholipids, or to defined phospholipid-cholesterol mixtures which simulate the lipid composition of plasma membranes. A quantitative resonance energy transfer assay determined the extent of lipid phase mixing related to fusion. No fusion was detected at pH 7.4, but when the pH was lowered to 6.5 or below, fusion was rapid and substantial. The extent of membrane mixing increased with progressive acidification of the vesicle-fusogen suspension. The charge density of each polyhistidine molecule, not the total cationic charge per vesicle, influenced the extent of fusion. The kinetics of the fusion reaction were rapid, as membrane mixing was completed within 1 min. If the vesicle suspension was acidified before fusogen addition, the rate of membrane mixing slowed 4-fold. This, as well as a slight increase in light scattering noted whenever polyhistidine was added at pH 7.4, suggests an enhancement of fusion kinetics by preaggregation of vesicles at neutral pH. The lipid composition, regulation of membrane mixing by pH in a physiological range, and rapid kinetics suggest that this model of liposome fusion may be pertinent to understanding some biological fusion events.     

Effects of divalent ions on vesicle-vesicle fusion studied by a new luminescence assay for fusion

Summary A new assay has been developed for vesicle-vesicle fusion based upon the mixing of intravesicular contents of two sets of vesicles. Purified firefly luciferase and MgCl₂ were incorporated into one set of vesicles (LV) and ATP into the other (AV). Vesicles were prepared from soybean phospholipids. The luminescence that resulted from hydrolysis of ATP by luciferase was measured to determine the extent of mixing of the intravesicular contents. In the absence of divalent ions, incubation of a mixture of LV and AV did not produce luminescence. However, if Ca⁺⁺ or other divalent ions were present at millimolar concentrations, luminescence occurred. The luminescence did not result from extravesicular reaction of vesicle contents that had leaked into the medium. Instead, luminescence resulted from the mixing of intravesicular spaces of AV and LV in fused vesicles. Optical density changes and negative stain electron microscopy indicated that Ca⁺⁺ induced extensive aggregation of vesicles. However, quantitation of the maximum possible luminescence indicates that only a small percentage (less than 1%) of the vesicles actually fused in a fusion experiment.Addition of EDTA to chelate Ca⁺⁺ after luminescence had been induced resulted in a two-to threefoldincrease in light emission which then rapidly decayed. These results suggest that the sudden removal of Ca⁺⁺ caused a transient increase in fusion after which subsequent fusion was inhibited. It was also found that the vesicles were relatively stable to hypotonic solutions.     

Interaction of phospholipid vesicles with cultured myogenic cells : Effects on cell fusion

The effects of defined acyl chain, unilamellar phosphatidylcholine vesicles on the development of cultured embryonic chick muscle was studied. An inhibition of myoblast fusion was observed when vesicles were incubated with cells below the vesicle gel-liquid crystalline phase transition temperature (T_c). This inhibition could be at least partially reversed by culturing the vesicletreated cells above the T_c of vesicles. Evidence supporting adhesion as the mechanism of vesiclecell interaction mediating inhibition of myoblast fusion was derived from scanning electron microscopy (SEM) which demonstrated the presence of vesicle-like particles on the cell membrane under conditions in which myoblast fusion was inhibited. Pretrypsinization of myoblasts before their incubation with vesicles prevented this fusion inhibition, suggesting that vesicles may interact with cell membrane proteins which are involved in the myoblast fusion and/or recognition process.     

Real-time Observation of Lipoplex Formation and Interaction with Anionic Bilayer Vesicles

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.     

Proton-Induced Permeability and Fusion of Large Unilamellar Vesicles by Covalently Conjugated Poly(2-Ethylacrylic Acid)

Abstract

Proton sensitive large unilamellar vesicles (LUV) were constructed by immobilization of the pH sensitive synthetic polymer poly(2-ethylacrylic acid) onto the outer monolayer. Thiolated poly(2-ethylacrylic acid) (PEAA-SH) was covalently conjugated to the surface of LUVs composed of egg phosphatidylcholine (EPC) and cholesterol (Choi) through the thiol-reactive maleimide lipid MPB-DSPE (N-(4-(p-maleimidophenyl)butyryl)-1,2-distearoyl-sn-glyc-ero-3-phosphoethanolamine). The resulting PEAA- LUVs were shown to be stable at neutral pH (pH 7.0 to 8.0). Under acidic conditions, however, proton-ation of PEAA resulted in interaction with both the membrane it was linked to and the membrane of target vesicles, causing membrane destabilization and release of vesicle contents. Moreover, conjugated PEAA is shown to mediate fusion with target membranes in a pH dependent manner. PEAA-mediated permeabilization and vesicle-vesicle fusion occurred only when the polymer was covalently linked to the LUV surface. Proton dependent fusion of PEAA-LUVs was also observed with erythrocyte ghosts. This pH-dependent release of vesicle contents and fusion of PEAA-LUVs occurred below pH 6.8, which is well within the pH range expected to be encountered inside the endosomes in the endocytic pathway, indicating the potential of PEAA-LUVs as a drug carrier system for intracellular drug delivery.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR2, a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR₂ is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR₂ when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR₂ cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR₂ on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR₂-induced liposome aggregation, as long as Ca²⁺ is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis < vesicle aggregation < total lipid mixing < inner lipid mixing < contents mixing. Concomitant measurements of the threshold diacylglyceride + ceramide concentrations in the bilayer show that late events, e.g. lipid mixing, require a higher concentration of PlcHR₂ products than early ones, e.g. aggregation. When the above results are examined in the context of the membrane effects of other phospholipid phosphocholine hydrolases it can be concluded that aggregation is necessary, but not sufficient for membrane fusion to occur, that diacylglycerol is far more fusogenic than ceramide, and that vesicle membrane permeabilization occurs independently from vesicle fusion.     

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca²⁺-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1–2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal H_II phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca²⁺, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca²⁺ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.