The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions

Small unilamellar vesicles comprised of a mixture of phosphatidylethanolamine/phosphatidylcholine/cholesterol (3 : 1 : 2) fuse to form large multilamellar vesicles on increasing the temperature from 0 to 50°C. This event is associated with the appearance of lipidic particles at the fusion sites, consistent with a role as intermediary structures during the fusion process. Further, for phosphatidylcholine/cardiolipin (1 : 1) liposomes in the presence of Mn²⁺ a direct relationship between lipidic particles and the hexagonal (H_II) phase is demonstrated which suggests that lipidic particles can also occur as intermediaries between bilayer and hexagonal (H_II) structures.     

Ca-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

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     

Ca2+-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

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.     

Fusion of phospholipid vesicles in association with the appearance of lipidic particles as visualized by freeze fracturing

The addition of Ca2+ to small unilamellar vesicles of an equimolar mixture of egg phosphatidylcholine and cardiolipin induces fusion of these vesicles in association with the appearance of lipidic particles on the fusion sites.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

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.     

Membrane Bending Energy and Fusion Pore Kinetics in Ca-Triggered Exocytosis

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca²⁺-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca²⁺-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca²⁺-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.     

Polyamines Biological modulators of membrane fusion

The effect of polyamines on the kinetics of Ca²⁺- and Mg²⁺-mediated membrane fusion was studied by following the intermixing of the contents of vesicles composed of phosphatidate/phosphatidylserine/ phosphatidylethanolamine/cholesterol (1:2:3:2). Addition of polyamines at specific concentration ranging from 40 to 400 μM promoted aggregation of the vesicles. In addition, low levels of spermine (50–100 μM) enhanced both Ca²⁺ - and Mg²⁺-mediated fusion. The initial fusion rate of this membrane system increased more than 200-fold when fusion was initiated by Ca²⁺ after 5 min pre-incubation of vesicles with 50 μM spermine. These results indicate that in addition to their other known effects on cellular metabolism, polyamines may be involved in modulating intracellular membrane fusion.     

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.     

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca²⁺-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca²⁺-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.     

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

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

Control of membrane fusion by phospholid head groups I. Phosphatidate/phosphatidylinositol specificity

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca²⁺ or Mg²⁺ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90° light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca²⁺ and Mg²⁺ at threshold concentration ranges of 0.03–0.1 mM (Ca²⁺) and 0.07–0.15 mM (Mg²⁺) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca²⁺ or Mg²⁺ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5–10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.     

Specific lipids supply critical negative spontaneous curvature--an essential component of native Ca2+-triggered membrane fusion

The Ca²⁺-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, αT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary “spontaneous” lipid rearrangements that must occur during native membrane fusion—Ca²⁺-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.     

Studies on membrane fusion. III. The role of calcium-induced phase changes

The interaction of phosphatidylserine vesicles with Ca²⁺ and Mg²⁺ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca²⁺ and Mg²⁺ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca²⁺ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion.The results indicate that at Ca²⁺ concentrations of 1.0–2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient in itself to induce fusion without a concomitant phase change. Mg²⁺ in the range of 2.0–5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change.From the effect of variations in pH, temperature, Ca²⁺ and Mg²⁺ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.     

Reaction characteristics and mechanisms of lipid bilayer vesicle fusion

Small unilamellar lipid bilayer vesicles were prepared from brain phosphatidylserine, egg phosphatidylcholine, and synthetic dipalmitoylphosphatidylcholine, and were fused into larger structures by freezing and thawing, addition of calcium chloride, and passage through the lipid phase transition temperature. Fusion reactions were studied by electron microscopy, light scattering, and use of fluorescent probes. Fusion was accompanied by leakage of lipid vesicle constituents and of water-soluble solutes in the inner vesicle compartments, and by uptake of these types of components from the external solution. Such leakage was greater during fusion by freezing than by Ca²⁺. Passage through the transition temperature produced a moderate degree of fusion, without loss of membrane components. It is concluded that each fusion method gives rise to a characteristic size or narrow range of sizes of fusion products. The fraction of small vesicles fused into larger structure depends on the method of vesicle preparation, composition of the lipid bilayer, and composition of the external solution. Fusion is induced by creation of a discontinuity in the bilayer or by removal of water associated with the bilayer. The amount of water removed controls the extent of fusion. This is maximized in bilayers when in the liquid-crystal phase, as against the gel phase, in vesicles made by ethanol injection, as against sonication, and in charged bilayers, as against neutral ones.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals

The effect of divalent metals on the interaction and mixing of membrane components in vesicles prepared from acidic phospholipids has been examined using freeze-fracture electron microscopy and differential scanning calorimetry. Ca²⁺, and to a certain extent Mg²⁺, induce extensive mixing of vesicle membrane components and drastic structural rearrangements to form new membranous structures. In contrast to the mixing of vesicle membrane components in the absence of Ca²⁺ described in the accompanying paper which occurs via diffusion of lipid molecules between vesicles, mixing of membrane components induced by Ca²⁺ or Mg²⁺ results from true fusion of entire vesicles. There appears to be a “threshold” concentration at which Ca²⁺ and Mg²⁺ become effective in inducing vesicle fusion and the threshold concentration varies for different acidic phospholipid species. Different phospholipids also vary markedly in their relative responsiveness to Ca²⁺ and Mg²⁺, with certain phospholipids being much more susceptible to fusion by Ca²⁺ than Mg²⁺. Vesicle fusion induced by divalent cations also requires that the lipids of the interacting membranes be in a “fluid” state ($\text{T > T}{}_{\mathrm{c}}$). Fusion of vesicle membranes by Ca²⁺ and Mg²⁺ does not appear to be due to simple electrostatic charge neutralization. Rather the action of these cations in inducing fusion is related to their ability to induce isothermal phase transitions and phase separations in phospholipid membranes. It is suggested that under these conditions membranes become transiently susceptible to fusion as a result of changes in molecular packing and creation of new phase boundaries induced by Ca²⁺ (or Mg²⁺).     

Fusion of secretory vesicles isolated from rat liver

Summary Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca²⁺. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca²⁺-concentrations and is half maximal around 10^–6 m. Other divalent cations (Ba²⁺, Sr²⁺, and Mg²⁺) were ineffective. Mg²⁺ inhibits Ca²⁺-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca²⁺ specific and exhibits properties similar to the exocytotic process of various secretory cells.Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system.The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca²⁺, and in its Ca²⁺-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences.