Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Lipid mixing during membrane aggregation and fusion: why fusion assays disagree

The kinetics of lipid mixing during membrane aggregation and fusion was monitored by two assays employing resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N-(lissamine Rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE). For the "probe mixing" assay, NBD-PE and Rh-PE were incorporated into separate populations of phospholipid vesicles. For the "probe dilution" assay, both probes were incorporated into one population of vesicles, and the assay monitored the dilution of the molecules into the membrane of unlabeled vesicles. The former assay was found to be very sensitive to aggregation, even when the internal aqueous contents of the vesicles did not intermix. Examples of this case were large unilamellar vesicles (LUV) composed of phosphatidylserine (PS) in the presence of Mg2+ and small unilamellar vesicles (SUV) composed of phosphatidylserine in the presence of high concentrations of Na+. No lipid mixing was detected in these cases by the probe dilution assay. Under conditions where membrane fusion (defined as the intermixing of aqueous contents with concomitant membrane mixing) was observed, such as LUV (PS) in the presence of Ca2+, the rate of probe mixing was faster than that of probe dilution, which in turn was faster than the rate of contents mixing. Two assays monitoring the intermixing of aqueous contents were also compared. The Tb/dipicolinic acid assay reported slower fusion rates than the 1-aminonaphthalene-3,6,8-trisulfonic acid/N,N'-p-xylylene-bis(pyridinium bromide) assay for PS LUV undergoing fusion in the presence of Ca2+. These observations point to the importance of utilizing contents mixing assays in conjunction with lipid mixing assays to obtain the rates of membrane destabilization and fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

1,2-Dioleoylglycerol promotes calcium-induced fusion in phospholipid vesicles.

The effect of 1,2-dioleoyglycerol (1,2-DOG) on the promotion of Ca(2+)-induced fusion of phosphatidylserine/phosphatidylcholine (PS/PC) vesicles was studied. 1,2-DOG is able to induce the mixing of membrane lipids at concentrations of 10 mol% without mixing of vesicular contents. At concentrations of 20 mol% or higher, 1,2-DOG promotes fusion, lipid and content mixing, of LUV composed of an equimolar mixture of PS and PC, which otherwise are unable to fuse in the presence of Ca2+. Fusion was demonstrated by fluorescence assays monitoring mixing of aqueous vesicular contents and mixing of membrane lipids. Studies by Fourier transform infrared spectroscopy provided evidence for a fusion mechanism different to that of Ca(2+)-induced fusion of pure PS vesicles. Final equilibrium structures were characterized by 31P-NMR and freeze-fracture electron microscopy. Ca(2+)-induced fusion of 1,2-DOG containing vesicles is accompanied by the formation of isotropic structures which are shown to correspond to structures with lipidic particle morphology. The possible fusion mechanisms and implications are discussed.     

Energy transfer measurements of fusion between Sendai virus and vesicles corrected for decreased absorption of acceptor probe

The fusion of Sendai virus at pH 4-7 with artificial lipid vesicles composed of phosphatidylserine or phosphatidylcholine was quantified by measuring fluorescence energy transfer from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine to N-(lissamine-rhodamine-B-sulfonyl)-phosphatidylethanolamine in the target membranes. About 60% of the phosphatidylserine vesicles and virus appeared to fuse at pH 4 and about 100% at pH 5. Fusion was much less under all other conditions. The apparent fusion at pH 4, however, was due to a decrease in absorption of the acceptor probe, instead of dilution of acceptor as a result of fusion of labeled vesicles with unlabeled virus. After correction for this fusion-independent effect of Sendai virus, the extent of fusion was only 4-20% at pH 4 but still 80-100% at pH 5. These findings paralleled the loss of hemagglutinating and hemolytic activities of the virus induced by incubation at pH 4 but not at pH 5. Vesicle-virus hybrids were observed with the electron microscope after incubation at pH 5 but not at pH 7. The assay of membrane fusion by fluorescence energy transfer can be misleading unless correction is made for changes in energy transfer due to fusion-independent effects.     

Physical properties of phosphatidylcholine-phosphatidylinositol liposomes in relation to a calcium effect

Physical properties of binary mixtures of dipalmitoylphosphatidylcholine and yeast phosphatidylinositol were studied by ESR analysis using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and lipid spin probes, freeze-fracture electronmicroscopy and particle microelectrophoresis, and they were compared with those of phosphatidylcholine/bovine brain phosphatidylserine mixtures. The phase diagram of the binary mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol was obtained from the thermal features of TEMPO spectral parameter in the lipid mixtures. The phase diagram provided evidence that these two phospholipids in various combinations were miscible in the crystalline state. The addition of 10 mM Ca2+ slightly shifted the phase diagram upward. TEMPO titration of the binary mixture of dipalmitoylphosphatidylcholine and bovine brain phosphatidylserine revealed that 10 mM Ca2+ caused the complete phase separation of this lipid mixture. Studies of phase separations using phosphatidylcholine spin probe manifested that 10 mM Ca2+ induced almost complete phase separation in egg yolk phosphatidylcholine/bovine brain phosphatidylserine mixtures but only slight phase separation in egg yolk phosphatidylcholine/yeast phosphatidylinositol mixtures. However, some phase changes around the fluidus and the solidus curves were visualized by the freeze-fracture electronmicroscopy. The molecular motion of lipid spin probe was decreased by the addition of Ca2+ in the liposomes containing phosphatidylinositol. The temperature dependence of electrophoretic mobility was also examined in the absence and presence of 1 mM Ca2+. Liposomes of dipalmitoylphosphatidylcholine-phosphatidylinositol (90 : 10, mol/mol) exhibited a clear transition in the thermal features of electrophoretic mobilities. Raising the phosphatidylinositol content up to 25 mol% rendered the transition broad and unclear. The addition of 1 mM Ca2+ decreased the electrophoretic mobility but did not change its general profile of the thermal dependence. These results suggest that the addition of calcium ions induced a small phase change in the binary mixture of phosphatidylcholine and phosphatidylinositol while Ca2+ causes a remarkable phase separation in phosphatidylcholine/phosphatidylserine mixture. The physical role of phosphatidylinositol is discussed related to the formation of diacylglycerol.     

Aluminum-induced lipid phase separation and membrane fusion does not require the presence of negatively charged phospholipids

The interaction of Aluminum with phosphatidyl serine lipid vesicles containing variable amounts of phosphatidyl ethanolamine, phosphatidyl choline and cholesterol has been studied by lipid phase separation monitored by fluorescence quenching. The interaction of Al3+ with neutral phospholipid membranes has also been investigated. Maximal lipid phase separation can be demonstrated in mixed phosphatidyl ethanolamine-cholesterol vesicles when using concentrations of aluminum between 87.5 and 125 microM. Millimolar concentrations of Ca2+, Mn2+, Cd2+ and Zn2+ were without any effect. Aluminum also induced fusion of phospholipid membranes monitored by resonance energy transfer between N-(7-nitro-2,1,3, benzoxadiazol-4 yl) phosphatidyl ethanolamine and N-(lissamine Rhodamine B-sulfonyl) phosphatidyl ethanolamine, either when containing low amounts of phosphatidyl serine (12.5%) or without any negatively charged phospholipid. Aluminum-induced fusion of liposomes was also monitored by the fluorescence of the terbium-dipicolinic acid complex (Tb-DPA3-) formed during fusion of vesicles containing either Tb-(citrate)6- complex or sodium salt of dipicolinic acid.     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

Phosphatidylethanol counteracts calcium-induced membrane fusion but promotes proton-induced fusion

The susceptibility of phosphatidylethanol-containing lipid vesicles towards Ca2+- and proton-induced fusion has been investigated, using a system of interacting vesicles. The results show that phosphatidylethanol-rich vesicles are quite resistant to Ca2+-induced fusion while being highly sensitive to proton-induced fusion. Inclusion of phosphatidylethanol was also found to promote and inhibit, respectively, the proton-induced and Ca2+-induced fusion of bilayer vesicles containing also phosphatidylethanolamine and either phosphatidylserine or phosphatidic acid. Thus, phosphatidylethanol affected Ca2+- and proton-induced fusion in opposite directions, in contrast to the naturally occurring anionic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol, which affect the sensitivity to Ca2+- and H+-induced fusion in the same direction. However, the fusion competence of phosphatidylethanol vesicles in response to both Ca2+ and H+ was inversely related to the apparent thickness of the polar headgroup layer, determined by using lectin-glycolipid interaction as a steric probe, as previously found for vesicles containing naturally occurring anionic phospholipids.     

Micromolar concentrations of Zn2+ potentiates Ca2+-induced phase separation of phosphatidyl serine containing liposomes

Fluorescence quenching of 1-acyl-2-[6[(7 nitro-2,1,3-benzoxadiazol-4yl) amino]caproyl] phosphatidyl choline in small unilamellar vesicles consisting of phosphatidyl serine has been used to monitor the lipid phase separation induced by Zn2+ and Ca2+. Phase separation of vesicle membranes was observed with Zn2+ at concentrations as low as 125 microM. Low concentrations of Zn2+ required long incubation times to reach maximal quenching (120 minutes at 375 microM). When low concentrations of Ca2+ were added to the preparation during the developing phase of Zn2+-induced quenching, an explosive increase in fluorescence quenching was instantenously observed. Phase separation induced by sub-millimolar concentrations of Ca2+ could be increased at least 4 times when vesicles were pre-incubated with 250 microM of Zn2+.     

The interaction of spectrin-actin and synthetic phospholipids. II. The interaction with phosphatidylserine.

Sonicated vesicles of phosphatidylserine and phosphatidylserine/phosphatidylcholine mixtures were recombined with spectrin-actin from human erythrocyte ghosts. Morphological properties and physicochemical characteristics of the recombinates were studied with freeze etch electron microscopy, 31P NMR and differential scanning calorimetry. Sonicated dimyristoyl phosphatidylserine vesicles show a decrease in enthalpy change of the lipid phase transition upon addition of spectrin-actin. These vesicles collapse and fuse, into multilamellar structures in the presence of spectrin-actin, as demonstrated by freeze fracturing and NMR. Spectrin-actin cannot prevent the salt formation between phosphatidylserine and Ca2+, all phosphatidylserine is withdrawn from the lipid phase transition. In contrast a protection against the action of Mg2+ could be observed. Mixed bilayers of dimyristoyl phosphatidylserine/dimyristoyl phosphatidylcholine show phase separations at molar ratios above 1/1 (van Dijck, P.W.M., de Kruijff, B., Verkleij, A.J., van Deenen, L.L.M. and de Gier, J. (1978) Biochim. Biophys. Acta 512, 84--96). These phase spearations can be prevented by spectrin-actin. Ca2+-induced lateral phase separations in cocrystallizing phosphatidylserine/phosphatidylcholine mixtures, can be reduced by spectrin-actin. Formation of the Ca2+-phosphatidylserine salt, occurring in addition to lateral phase separation when mixtures contain more than 30 mol % phosphatidylserine, cannot be prevented by spectrin-actin.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles. Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca2+-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca2+ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca2+ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca2+ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca2+ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca2+, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca2+ concentration, and identical to that with pure phosphatidylserine in excess Ca2+. The results imply that Ca2+- induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.     

Rate and extent of poly(ethylene glycol)-induced large vesicle fusion monitored by bilayer and internal contents mixing

Poly(ethylene glycol) (PEG) of average molecular weight 8000 was used to mediate the fusion of large unilamellar vesicles composed of dipalmitoylphosphatidylcholine. Fusion was monitored by fluorescence assays of lipid mixing and aqueous contents mixing. The extent of lipid mixing, as monitored by DPHpPC fluorescence lifetime, indicated that large unilamellar vesicles underwent a single fusion cycle when incubated with PEG and subsequently diluted into buffer. The ANTS/DPX assays for contents mixing and leakage indicated that, while addition and dilution of PEG were accompanied by extensive contents leakage, this occurred on a much different time scale as compared to contents mixing. Both the lipid-mixing and contents-mixing assays gave comparable estimates for the number of rounds of fusion that occurred in a given time following PEG addition, although the contents-mixing assay always yielded an estimate 10-15% larger than the lipid-mixing assay. These assays were used to evaluate several factors purported to influence PEG-induced fusion. First, the initial rate of fusion was found to be dependent on PEG concentration in the range of 0-35 wt %, while the extent of fusion was not. In addition, a substantial rate enhancement occurred when vesicles were incubated with greater than 26% PEG. Second, the creation of an osmotic gradient upon dilution of vesicle-PEG mixtures was shown to have no effect on either the extent or the initial rate of fusion. Consistent with this observation, both contents and lipid mixing were found to occur prior to and independent of the dilution of the PEG-vesicle suspension. Third, impurities, either present in our commercially available PEG or added to vesicle-PEG mixtures, also had no effect on the rate or extent of fusion. Fourth, another dehydrating polymer, dextran (average mol wt 9000), was capable of promoting fusion, though at a much lower rate than PEG. These results suggest that even partial bilayer dehydration accompanied by vesicle collapse and close interbilayer contact may be sufficient to induce vesicle fusion.     

Ca2+-induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles.

The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3-sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl2 were studied by mixing membrane lipids based on an assay of fluorescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with increasing molar concentration of sulfatide. This inhibitory effect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 - 9.0. Similar inhibitory effect was also observed in Ca2+-induced fusion of DOPE/ganglioside GM1 vesicles but at a lower concentration of the glycosphingolipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca2+-induced lipid mixing and the pH sensitivity of this system was not affected.These results are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was retained when they were allowed to fuse with synaptosomes in the absence of Ca2+ by a mechanism involving protein mediation.     

Role of synexin in membrane fusion. Enhancement of calcium-dependent fusion of phospholipid vesicles

Synexin, a soluble adrenal medullary and liver protein which causes calcium-dependent aggregation of isolated chromaffin granules, was isolated and purified according to published procedures. The effects of synexin on the kinetics of membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Synexin lowers the threshold of CA2+ concentration required for fusion of large unilamellar vesicles of phosphatidylserine and a mixture of phosphatidylserine with phosphatidylethanolamine. synexin also increases drastically the initial rate of fusion. the initial rate of fusion increases with the quantity of synexin present in the reaction mixture. In the presence of 1-2 mM Ca2+ and 50 microM phospholipid, synexin at 20 to 40 micrograms/ml increases the rate of fusion by two orders of magnitude. Mg2+ does not support synexin-induced fusion. With vesicles containing a mixture of phosphatidylserine with phosphatidylcholine, synexin enhances aggregation in the presence of CA2+, without promoting fusion. Synexin may play a role in exocytosis by promoting fusion of membranes containing specific phospholipids in the presence of Ca2+.     

Effects of phorbol ester and teleocidin on Ca2+-induced fusion of liposomes

The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the fusion. This latter phenomenon was also observed by measuring the excitation energy transfer. The promotion of membrane fusion by teleocidin was ascribed to dehydration of the membrane surface, the suppressive effect of TPA to desorption of Ca2+ from the membrane surface. Thus, Ca2+-induced fusion of PS vesicles was shown to be sensitive to defects of the membrane surface, but insensitive to defects of the hydrophobic core of the lipid membrane.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.