Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

H+- and Ca2+-induced fusion and destabilization of liposomes

A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.     

The role of calcium in fusion of artificial vesicles.

Small phospholipid vesicles (liposomes) fuse upon calcium addition as demonstrated by electron microscopy, light absorbance increases, and mixing of original liposome contents within the boundaries of the fused liposome. The integrity of the fusion event is demonstrated by a novel assay based on the luminescence of firefly extract when mixed with ATP. Subsequent addition of valinomycin or the calcium ionophore A23187 leads to further fusion as shown by electron microscopy, light microscopy, and additional absorbance increase. Concomitant with this second absorbance increase is an increase in the amount of calcium that associates with the liposomes. This increased calcium association is more than can be accounted for by equilibration of 5 mM Ca2+ across the membrane and must indicate exposure of extra calcium binding sites. Binding of calcium to the inner side of the membrane may catalyze the second stage of liposome fusion.     

Ionic strength of the intermembrane space of intact mitochondria as estimated with fluorescein-BSA delivered by low pH fusion

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.     

Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components

Divalent cation-induced fusion of large unilamellar vesicles (approx. 0.1 micron diameter) made of phosphatidylserine (PS) or phosphatidylglycerol (PG) has been studied. Intermixing of aqueous contents during fusion was followed by the Tb/dipicolinic acid fluorescence assay, and intermixing of membrane components by resonance energy transfer between fluorescent lipid probes. Both assays gave identical threshold concentrations for Ca2+, which were 2 mM for PS and 15 mM for PG. The dependencies of the initial rate of fusion on the concentration of PG vesicles determined by either assay were identical, the order of this dependence being 1.2 in the concentration range of 5-200 microM lipid. For PS liposomes, this order was found to be 1.5 in the fluorescent lipid assay. No leakage of contents was detected during the fusion of PG vesicles. Mg2+ inhibited the Ca2+-induced fusion of PS vesicles, but did not cause any fusion by itself, consistent with previous results with the Tb/dipicolinic acid assay.     

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.     

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.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

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.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

Kinetics of calcium-induced mixing of lipids and aqueous contents of large unilamellar phosphatidylserine vesicles

Calcium ion-induced fusion events in suspensions of large unilamellar phosphatidylserine (PS) liposomes were monitored by fluorescence methods. Mixing of vesicle contents was studied by measuring the increase in terbium emission intensity due to formation of a complex between Tb³⁺ ions and dipicolinic acid trapped in the liposomes. Lipid redistribution was determined with the aid of the resonance transfer of excitaton energy using dipalmitoylphosphatidylethanolamine labelled with the donor N-(7-nitro-2,1,3-benzoxadiazol-4-yl) or the acceptor tetramethylrhodamine at the free amino group. The two methods yielded significantly different results. While recombination of contents could not be detected at Ca²⁺ concentrations below 2.5 mM the threshold concentration for lipid mixing was 1 mM. For saturating Ca²⁺ concentrations (>5 mM Ca²⁺) initial rates were higher by almost an order of magnitude for lipid mixing than for recombination of liposome contents. These observations indicate that the observation of rapid lipid mixing phenomena does not allow one to draw conclusions as to the fate of the enclosed volumes.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca(2+)-induced fusion of PA liposomes and a large decrease in the threshold Ca(2+) concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca(2+) resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization.     

Parameters affecting fusion between liposomes and synaptosomes. Role of proteins,lipid peroxidation,pH and temperature

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe²⁺ (2.5 m)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.This work was supported by Junta National de Investigação Científica e Tecnológica (JNICT) and the Calouste Gulbenkian Foundation, Portugal.     

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.     

Free fatty acid enhancement of cation-induced fusion of liposomes: synergism with synexin and other promoters of vesicle aggregation

The effect of free fatty acids on the cation-induced fusion of large unilamellar vesicles (liposomes) was investigated by using fluorescent assays which monitor the mixing of aqueous contents of liposomes. Overall fusion was modeled as a two-step process involving aggregation of vesicles followed by actual fusion. Different experimental conditions were used which favored either aggregation or fusion as the rate-limiting step in the overall process. When phosphatidylserine liposomes were induced to fuse by 4 mM Ca2+ plus 5 mM Mg2+, preincubation with arachidonic acid showed a dramatically increased overall rate of fusion compared to the same liposomes not treated with fatty acid. When fusion was induced by 3 mM Ca2+, arachidonic acid had little effect. These results were interpreted in terms of the action of arachidonic acid only at the fusion step per se and not the aggregation step. Therefore, the enhancement of the overall fusion rate would be observed solely under conditions where the actual fusion of liposomes was rate limiting (Ca/Mg) rather than the aggregation of liposomes (Ca alone). When other liposome systems were tested, the effect of arachidonic acid was observed only under fusion rate-limiting conditions. Arachidonic acid was found to act synergistically with promoters of liposomal aggregation, such as Mg2+, spermine, and synexin, to enhance the overall rate of liposome fusion, as would be expected from action at separate kinetic steps. The dependence of the fusion rates on arachidonic acid concentration demonstrated an apparently cooperative effect. The structure of the fatty acid is of critical importance in determining its effects, as shown by the fact that 16-doxylstearic acid always increased the rate of fusion while 5-doxylstearic acid always decreased the rate of fusion under all conditions tested. A number of different fatty acids, including oleic acid, elaidic acid, 16-doxylstearic acid, myristic acid, and stearic acid, were effective at increasing the fusion rate to varying extents. In general, unsaturated fatty acids were more effective than saturated ones, either due to partitioning into the membrane or because of structural requirements for promotion of fusion.     

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)     

Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.     

The electrophoretic properties and aggregation of mouse lymphoma cells, chinese-hamster fibroblasts and a somatic-cell hybrid.

1. The electrophoretic mobilities of a mouse lymphoma cell, a Chinese-hamster fibroblast and a somatic-cell hybrid (also fibroblastic), produced by fusion of the hamster cell and a mouse lymphoma cell, were measured at 25 degrees C over a range of pH, concentration of Ca2+ ions and concentration of La3+ ions. 2. All the cells have pI at pH3.5. 3. Ca2+ ions decrease the mobilities and zeta potentials of the cells to zero in the range 1-100mM. 4. La3+ ions lower the mobilities and zeta potentials in the range 10 muM-1 mM, and the cells become positively charged above 1 mM. 5. The data are consistent with specific adsorption of La3+ ions on approx. 2 X 10(14) sites/m2 of cell surface with a free energy of approx. -37kJ/mol. 6. The effects of Ca2+, La3+ and ionic strength on the extent of aggregation of the cells and of neuraminidase-treated cells were studied. 7. Ca2+ ions do not markedly increase aggregation, whereas La3+ ions gave rise to extensive aggregation in the range 10 muM-1 mM, corresponding to the region of La3+ adsorption. 8. Both fibroblastic cell lines are aggregated at high ionic strength. 9. The fibroblastic cells have larger amounts of trypsin-sensitive carbohydrate than does the lymphoma cell; the possible role of this material in cellular aggregation is discussed.