Binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of F-glycoprotein of HVJ (Sendai virus): possible implication in the fusion reaction

Specificity of the binding of sterols and related compounds with purified F-protein (fusion protein) of the HVJ (Sendai virus) was studied by binding competition with [3H]cholesterol. Requirement for cholesterol or the A/B ring trans structure and nonrequirement for the 3-hydroxyl group were found in this binding. Binding of 125I-labeled Z-Phe-Tyr, an inhibitory peptide of viral membrane-cell membrane fusion, was studied by using purified proteins and virions. F-Protein and virions showed a specific binding with the peptide, whereas the result was negative with hemagglutinin and neuraminidase protein. Thermolysin-truncated F-protein (an F-protein derivative deprived of a 2.5-kDa fragment from the N-terminal of the F1 subunit and without fusogenic activity) exhibited a considerably diminished binding ability both to cholesterol and to inhibitory peptides. Therefore, the N-terminal hydrophobic sequence that was previously assigned as fusogenic seems to be the binding site of these molecules. In support of this, the binding of cholesterol with F-protein was inhibited by Z-Phe-Tyr and other fusion inhibitory peptides, whereas it was not affected with non-fusion-inhibitory Z-Gly-Phe. These results are discussed in relation to the notion that the binding of the N-terminal portion of the F1 subunit of F-protein with cholesterol in the target cell membranes facilitates the fusion reaction.     

Reconstitution and fusogenic properties of Sendai virus envelopes

Sendai virus membranes were reconstituted by detergent dialysis, using the non-ionic detergents Triton X-100 and octyl glucoside. Membrane reassembly was determined by measuring the surface-density-dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co-reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X-100 with the reconstituted membrane was revealed by an artificial detergent-effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer. Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

Some characteristics of fusogenic activity of cytoplasmic latrotoxin-like brain protein

We studied fusion of negatively charged artificial phospholipid vesicles (liposomes) in the presence of two electrophoretic fractions (molecular mass of about 90 and 50 kdalton) of latrotoxin-like (L) protein. It was shown that both fractions are capable of causing liposome fusion in acidic media. Treatment of native preparations of L protein with NEM depressed their fusogenic activity. Some common characteristics of L protein and well-known fusogenic proteins allow us to account for the possibility of participation of L protein in fusion of the membranes in the cell.     

The preparation of cell fusion-inducing proteoliposomes from purified glycoproteins of HVJ (Sendai virus) and chemically defined lipids

Cell fusion-inducing (fusogenic) proteoliposomes of defined chemical composition were reconstituted from purified glycoproteins of hemagglutinating virus of Japan (Sendai virus) either with lipids extracted from the virus particles or with a chemically defined lipid mixture. Cell fusion reactions induced by the reconstituted system have several important characteristics similar to the virus-induced fusion reaction: fusogenic activity of the proteoliposomes depends on the presence of active fusion protein in the vesicles and, in the case of Ehrlich tumor cells, the fusion is almost completely inhibited by adding cytochalasin D to a final concentration of 4 microgram/ml. The only known difference between the original and reconstituted systems is that a greater amount of the latter is necessary for the same degree of fusogenic activity. Thus, the reconstituted system can be used as a model for the Sendai virus-induced fusion reaction. A lipid mixture (phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine:sphingomyelin = 1:2:1:1, by weight, and cholesterol equimolar to the total phospholipids) similar to that of the virion was active for reconstitution, whereas a mixture containing the same composition of phospholipids but no cholesterol, and ones containing cholesterol with only a single species of phospholipid were not reconstitutively active.     

Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure

The transmembrane protein bovine rhodopsin was reconstituted with egg phosphatidylcholine (PC) by using a modified detergent dilution technique employing the nonionic detergent octyl-beta-D-glucoside (octyl glucoside). Using this technique, reconstituted membranes having molar phospholipid/protein ratios between 60:1 and 255:1 were prepared. This is in contrast to the results obtained when an octyl glucoside dialysis technique was employed (Jackson, M.L. and Litman, B.J. (1982) Biochemistry 21, 5601-5608). In the latter case, the highest molar phospholipid/protein ratio that could be obtained when reconstituting rhodopsin with egg PC was approximately 50:1. Reconstituted vesicles prepared by the octyl glucoside dilution technique were examined by negative stain and freeze-fracture electron microscopy, and it was found that the vesicles were unilamellar providing the molar PC/protein ratio was below about 200:1, whereas in preparations having ratios higher than this, a significant number of the vesicles were multilamellar. The mean vesicle diameter showed no trend based on the molar PC/protein ratio within the range of 82:1 to 186:1. The mean diameters of the preparations were between 520 and 850 A. Approximately equal numbers of protein particles were observed on the concave and convex fracture faces of the freeze-fracture micrographs of the reconstituted membranes which is indicative of a symmetric distribution of the protein across the bilayer.     

Phospholipid diversity: Correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Tobacco mosaic virus protein induces fusion of liposome membranes

The fusogenic properties of tobacco mosaic virus (TMV) coat protein were investigated. Tobacco mosaic virus protein induces membrane fusion of a population of L-alpha-dimyristoylphosphatidylcholine (DMPC) and DL-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles giving rise to larger particles as seen by a drastic absorbance increase of the liposomal solution. Differential scanning calorimetry spectra demonstrate complete mixing of the acyl chains of the lipids during fusion. Electron micrographs indicate that the fused entities are multilamellar.     

Triggering and visualizing the aggregation and fusion of lipid membranes in microfluidic chambers

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an approximately 6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.     

Agents inducing high Mg2+-ATPase activity of isolated coupling factor 1 from spinach chloroplasts

Conditions are reported under which purified coupling factor 1 (CF1) from spinach chloroplasts exhibits Mg2+-dependent ATPase activity of about 120 mumoles/min/mg protein. It is shown that CF1, partially activated by treatment with heat and dithiothreitol (DTT), can be further activated by octyl glucoside. The Mg2+-dependent ATPase activity increases linearly as a function of the concentration of octyl glucoside from about 20 mumoles/min/mg protein in the absence of detergent to 120 mumoles/min/mg protein in the presence 15 mM octyl glucoside. This concentration is below the critical micellar concentration (CMC) of the detergent, indicating that the monomeric form is responsible for the activation. Without treatment with heat and DTT, the Mg2+-dependent ATPase activity of CF1 is virtually zero, but can be stimulated by octyl glucoside. In this case, however, only concentrations around CMC give a substantial increase in activity (about 50 mumoles/min/mg at 28 mM octyl glucoside). Concentrations higher than CMC inhibit both latent and heat-activated CF1.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.     

Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions

Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.     

Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes

A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.     

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.     

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Early steps of supported bilayer formation probed by single vesicle fluorescence assays

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.