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.     

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.     

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.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10-40 degrees C. The fusogenic activity of the cations decreases in the sequence Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+ for cholesterol concentrations in the range 20-40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca2+ and Ba2+ at 25 degrees C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25 degrees C, Mg2+ is ineffective in causing fusion at all cholesterol concentrations. However, at 30 degrees C, Mg2+-induced fusion is observed with vesicles containing cholesterol. At 40 degrees C, Mg2+ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca2+, Ba2+ and Sr2+. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na+ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (Tm) in Na+ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the Tm upward, with a sequence of effectiveness Ba2+ greater than Sr2+ greater than Mg2+. The Tm of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na+, Sr2+ or Mg2+, the addition of Ba2+ reveals endotherms with Tm progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the Tm, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg2+ concentrations. With Ca2+, saturation is not observed for cholesterol-containing vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.     

Phosphatidylserine Inhibits and Calcium Promotes Model Membrane Fusion

PEG-mediated fusion of SUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, cholesterol, and dioleoylphosphatidylserine was examined to investigate the effects of PS on the fusion mechanism. Lipid mixing, content mixing, and content leakage measurements were carried out with vesicles containing from 0 to 8 mol % PS and similar amounts of phosphatidylglycerol. Fitting these time courses globally to a 3-state (aggregate, intermediate, pore) sequential model established rate constants for each step and probabilities of lipid mixing, content mixing, and leakage in each state. Charged lipids inhibited both the rates of intermediate and pore formation as well as the extents of lipid and contents mixing, although electrostatics were not solely responsible for inhibition. Ca²⁺ counteracted this inhibition and increased the extent of fusion in the presence of PS to well beyond that seen in the absence of charged lipids. The effects of both PS and Ca²⁺ could be interpreted in terms of a previous proposal for the nature of lipid fluctuations that account for transition states for the two steps of the fusion process examined. The results suggest a more significant role for Ca²⁺-lipid interactions than is acknowledged in current thinking about cell membrane fusion.Abbreviations used: SUVs, small unilamellar vesicles; DOPC, 1,2-dioleoyl-3-sn-phosphatidylcholine; DOPE, 1,2-dioleoyl-3-sn-phosphatidylethanolamine; SM, sphingomyelin (bovine brain); CH, Cholesterol; DOPS, 1,2-dioleoyl-3-sn-phosphatidylserine; PS, phosphatidylserine; DOPG, 1,2-dioleoyl-3-sn-phosphatidylglycerol; PG, phosphatidylglycerol; TES, N- tris(hydroxymethyl)methyl}2-2-aminoethane sulfonic acid; PEG, poly(ethylene glycol); CM, contents mixing; LM, lipid mixing     

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.     

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.     

Binding of monovalent cations to phosphatidylserine and modulation of Ca2+- and Mg2+-induced vesicle fusion

The effect of several monovalent cations on the Ca2+-induced aggregation and fusion of sonicated phosphatidylserine (PS) vesicles is studied by monitoring the mixing of internal compartments of the fusing vesicles using the Tb/dipicolinic acid assay. The dissociation of the fluorescent Tb-dipicolinate complex which accompanies Ca2+-induced vesicle fusion is determined directly and is due to leakage of contents and entry of medium into vesicles. PS vesicles do not fuse when the medium contains only monovalent cations (at pH 7.4), regardless of the cation concentration or whether there is aggregation of the vesicles. A mass-action kinetic analysis of the data provides estimates for the rate of aggregation, C11, and for the rate of fusion per se, f11. Values of f11 increase dramatically with reduction in monovalent cation concentration and are primarily determined by binding ratios of Ca2+ or Mg2+ per PS. With 300 mM of monovalent cations, the fusion per se is essentially rate-limiting to the overall fusion process and values of f11 are significantly larger with the monovalent cations which bind the least, i.e., according to the sequence tetramethylammonium greater than K+ greater than Na+ greater than Li+. With monovalent cations in concentrations of 100 mM or less, the aggregation is rate-limiting to the fusion and the overall initial fusion rates are determined by an interplay between aggregation and fusion rates. Under conditions of fast aggregation, the Ca2+-induced fusion of small PS vesicles can occur within milliseconds or less.     

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.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating 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)     

Incorporation of semi-fluorinated alkanes in the bilayer of small unilamellar vesicles of phosphatidylserine: impact on fusion kinetics

Semi-fluorinated alkanes C(n)F(2n+1)C(m)H(2m+1) (FnHm) can be co-dispersed with standard phospholipids to form 'fluorinated' vesicles, i.e. vesicles with an internal fluorinated film within their bilayer membrane. This paper reports the effect of the presence of such FnHm diblocks in phosphatidylserine (PS)-based small unilamellar vesicles (SUVs) on their kinetics of fusion. Fusion was induced by calcium ions and monitored by the terbium/dipicolinic acid assay. The diblocks were composed of a 10-carbon long linear hydrocarbon segment and of a linear fluorocarbon segment of four, six or eight carbon atoms. We found that the incorporation of FnHm in the PS membrane considerably modifies the kinetics of the process of fusion, with Ca(2+) concentration having a much more limited effect on the fluorinated vesicles. Both the rates of fusion and the rates of release of the internal content, as evaluated by the release of 5,6-carboxyfluorescein, were much lower for the fluorinated SUVs than for those based on phosphatidylserine alone, the highest effect being obtained for F6H10 with a 10 times slower rate of fusion and a 40-fold reduction in the release of content. FnHm molecules are proposed to have a dual action: by hindering fusion and release by creating an inert, hydrophobic and lipophobic fluorinated film in the core of the membrane, and by stabilizing the membrane by increasing van der Waals interactions in the hydrocarbon region.     

Proton and divalent cations induce synergistic but mechanistically different destabilizations of pH-sensitive liposomes composed of dioleoyl phosphatidylethanolamine and oleic acid

Protons and divalent cations show synergistic effects on the destabilization of liposomes composed of unsaturated phosphatidylethanolamine and oleic acid (Düzgünes et al., Biochemistry (1985) 24, 3091). We have extended these observations and investigated the effects of Ca2+ and Mg2+ on the proton-induced destabilization of dioleoyl phosphatidylethanolamine/oleic acid (DOPE/OA) (4:1 molar ratio) liposomes. Temperature-induced aggregation was measured by 90 degrees light scattering. Lipid mixing was used to monitor vesicle destabilization and freeze-fracture electron microscopy was used to examine the structures formed from DOPE/OA vesicles in the presence of Ca2+ and/or protons. Both Mg2+ and Ca2+ shift the pH required for 50% lipid mixing to higher values. Temperature-induced vesicle aggregation occurs at lower temperatures in the presence of divalent cations and/or protons, indicating that intervesicular repulsions are decreased. Freeze-fracture electron micrographs show that the structures formed from DOPE/OA in the presence of Ca2+ differ significantly from those found in the presence of protons. In general, protons induce the formation of hexagonal phase, while the presence of Ca2+ leads to the formation of extensive regions of lamellar sheets with numerous lipidic particles. The synergistic effect of divalent cations and proton may be important for the maximal biological activity of DOPE/OA liposomes.     

Influence of phospholipid asymmetry on fusion between large unilamellar vesicles.

The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface.     

Interaction of the herbicide atrazine with model membranes. II: Effect of atrazine on fusion of phospholipid vesicles

The effect of atrazine on Ca2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb3+/dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca2(+)-induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca2+. It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca2(+)-induced fusion of vesicles is due to a different localization of the pesticide in the membrane.     

Fusion and phase separation monitored by lifetime changes of a fluorescent phospholipid probe

The sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]- 3-sn-phosphatidylcholine (DPHpPC) to its local concentration in lipid bilayers was used to monitor both lipid mixing and phase separation occurring during membrane vesicle fusion. Vesicles containing 2 mol % DPHpPC were mixed with a 10-fold excess of vesicles devoid of probe. Upon addition of a fusogen, mixing of bilayer lipids associated with fusion was followed as an increase in the fluorescence lifetime of DPHpPC. Ca2+-induced fusion of phosphatidylserine vesicles served to test the method and was shown to have an exponential half-time of 7 s. Phase separation (between the phosphatidylserine head groups of bulk lipid and the phosphatidylcholine head groups of the probe) was monitored by DPHpPC under the same conditions used to follow lipid mixing due to fusion. Phase separation was not significant until 10 min after Ca2+ addition and was completely reversible by disodium ethylenediaminetetraacetate addition. Vesicle aggregation induced by Ca2+ addition to mixed phosphatidylserine/phosphatidylcholine vesicles did not alter the DPHpPC lifetime, indicating that close association of vesicles did not promote intervesicular exchange of the probe. In addition, we have investigated the effects of CA2+ on the fluorescence properties of this probe and of the head-group-labeled fluorescent probes N-(4-nitro-2,1,3-benzoxadiazolyl)phosphatidylethanolamine and N-(lissamine Rhodamine B sulfonyl)dioleoyl-phosphatidylethanolamine, which are used in the fluorescence energy transfer assay of Struck et al.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor

Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3-10-fold without Ca2+. However, with Ca2+ there was an additional 2-5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50?μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.     

Specific interaction of the intermediate filament protein vimentin and its isolated N-terminus with negatively charged phospholipids as determined by vesicle aggregation, fusion, and leakage measurements

The interaction of the intermediate filament protein vimentin and its non-alpha-helical N-terminus with phosphatidylserine and phosphatidylinositol small unilamellar vesicles was investigated by measuring vesicle aggregation, fusion, and leakage. While the N-terminus suppressed Ca2(+)-induced fusion of phosphatidylserine vesicles, it caused their rapid aggregation in the absence of Ca2+; at a molar ratio of lipid to polypeptide of 25:3, the polypeptide/lipid complexes precipitated from the reaction mixture. This aggregation was efficiently diminished by NaCl. The phosphatidylinositol vesicles, on the other hand, became leaky when interacting with the N-terminus of vimentin, even at a molar ratio of lipid to polypeptide of 500:1. The leakage of phosphatidylinositol vesicles was suppressed by the addition of Ca2+ or NaCl to the reaction mixture. Intact vimentin also caused leakage of phosphatidylinositol vesicles, at low and high salt concentration. The results indicate specific and differential interactions of the N-terminus of vimentin with various negatively charged lipid species, although there is an electrostatic component common to these interactions.