Protein-mediated intermembrane contact facilitates fusion of lipid vesicles with planar bilayers

Fusion of phospholipid vesicles with planar bilayer membranes occurs provided there is an intermembrane contact, which can be mediated by phospholipid-binding proteins, even in the absence of calcium. The firm attachment phase is then followed by the osmotically-driven fusion. These results show that hydrophobic proteins (not necessarily Ca²⁺-binding proteins) may enhance fusion by promoting contact of membranes. Such proteins may operate synergistically with Ca²⁺ to reduce the threshold concentration of Ca²⁺ needed for fusion of biological membranes. Protein-mediated intermembrane contact resulting in fusion may play a crucial role in the regulation and catalysis of biological fusion events.     

Auto-oxidation-induced fusion of lipid vesicles

Spontaneous size changes of small unilamellar vesicles with initial mean diameters of 25 nm measured by quasi-elastic light scattering (QELS) and electron microscopy are reported. After the size conversion the vesicles have mean diameters of about 70 nm and are of the unilamellar and multilamellar type. The fact that auto-oxidation initiates this process is established by the comparison of the results for vesicles which differ only in the degree of auto-oxidation. The role of phosphatidylcholine hydroperoxides as fusogens is discussed.     

Current-voltage characteristics of planar lipid membranes with attached Halobacterium cell-envelope vesicles

We have studied the photoactivity of a system consisting of large, planar, essentially solvent free bilayers bearing adsorbed cell-envelope vesicles prepared from Halobacterium halobium (strain L 33). The system was made conductive by addition of a proton carrier (SF-6847). We observed photocurrents which were linearly dependent upon transmembrane voltage. Current-voltage curves were found to be well described by an equivalent circuit with the following significant parameters: planar bilayer conductance, planar bilayer-vesicle contact area conductance, cell-envelope vesicle conductance, and chloride pump equivalent voltage-generator potential. These parameters are uniquely obtained as a result of a few independent current measurements. The stationary photovoltage was dependent upon chloride concentration, and from this dependence an active transport (pump) affinity of the system for chloride was calculated to be about 50 mM.     

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

A new method for membrane reconstitution: fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).     

Presence of two types of flowers with respect to nectar sugar in two gregariously flowering species

Many species of animal-pollinated flowers are known to vary widely in the nectar content of flowers. Some proportion of flowers in many species is apparently nectarless, and such flowers are believed to be ‘cheaters’. Cheating may explain a part of the variability in nectar content. If cheating exists as a qualitatively different strategy then we expect bimodality in the distribution of nectar content of flowers. It has been shown in a multispecies study that gregarious species have a higher proportion of cheater flowers. We studied the frequency distribution of total nectar sugar in two gregariously flowering species Lantana camara and Utricularia purpurascens, which differed in other floral and ecological characters. At the population level, both the species showed significant bimodality in the total sugar content of flowers. The obvious sources of heterogeneity in the data did not explain bimodality. In Lantana camara, bimodality was observed within flowers of some of the individual plants sampled. In Utricularia purpurascens the proportion of nectarless flowers was more in high-density patches, suggesting that the gregariousness hypothesis may work within a species as well. The results support the hypothesis of cheating as a distinct strategy since two distinct types of flowers were observed in both the species. The effect of density in Utricularia purpurascens also supports the gregariousness hypothesis.     

Role of channels in the fusion of vesicles with a planar bilayer.

Fluorescence microscopy combined with electrical conductance measurements were used to assess fusion of phospholipid vesicles with a planar bilayer. Large unilamellar vesicles (0.5-3 microns diam.) filled with the fluorescent dye, calcein, were made both with or without porin channels. Vesicle-bilayer fusion was induced by increasing the osmolarity of the solution on the side of the bilayer to which the vesicles were added. Fusion was detected optically by the fluorescent flash due to release of vesicular contents. Although both porin-containing and porin-free vesicles give the same kind of flash upon content release, the conditions necessary to induce release are very different. Only 4% of the porin-free vesicles fuse (release their contents) when subjected to 3 M urea. However, the same conditions induce 53% of the porin-containing vesicles to fuse and most of these fusions occur at a lower osmolarity ([urea] less than 400 mM). Thus channels greatly enhance fusion in this model system. A physical model based on the postulate that fusion is induced by an increase in surface tension, predicts that three conditions are necessary for fusion in this system: (a) an open channel in the vesicle membrane, (b) an osmotic gradient across the bilayer, and (c) the vesicle in contact with the planar membrane. These are the conditions that experimentally produce fusion in the model system.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calciuminduced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes

Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca(2+)-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle-membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Stability and fusion of lipid vesicles containing headgroup-modified analogues of phosphatidylethanolamine

We have used lipid mixing, contents mixing and contents-leakage assays to characterize the divalent cation-mediated interactions of vesicles composed of various headgroup-modified analogues of phosphatidylethanolamine, PE (N- and C-2-alkylated derivatives, and analogues with increased separations of the phosphoryl and amino groups) together with a low mole percentage of phosphatidylserine (PS). Vesicles containing different structural analogues of PE exhibit marked differences, both in the threshold divalent cation concentrations that are required to initiate vesicle-vesicle interactions and in the rates of contents mixing and leakage observed at suprathresholds divalent cation concentrations. The efficiencies of divalent cation-promoted contents leakage, and to a slightly lesser extent those of contents mixing, for PS/PE (analogue) vesicles show a marked inverse correlation with the lamellar-to-hexagonal II transition temperature (TH) of the PE (analogue) component. However, the destabilization kinetics for such vesicles show no abrupt changes over the temperature range around the equilibrium TH value measured for the vesicle lipids. Vesicles combining PS with different PE analogues exhibit divalent cation thresholds for aggregation that are not correlated with the TH values of the PE (analogue) components but appear instead to be correlated with the equilibrium interbilayer separations measured in multilamellar dispersions of these species. We have identified headgroup-modified analogues of PE that can be used to prepare vesicles that fuse more rapidly under a given set of conditions, or that show a bette ratio of fusion-to-contents-leakage rates, than do PE-containing vesicles. These results may be useful both for understanding better the bases for the high fusion-supporting ability of PE and for the preparation of lipid vesicles 'tailored' for particular practical applications.     

A comparison between microsatellite and single-nucleotide polymorphism markers with respect to two measures of information content

Using the Genetic Analysis Workshop 14 (GAW14) simulated dataset, we compare microsatellite and single-nucleotide polymorphism (SNP) markers in terms of two measures of information content, the traditional entropy-based information content measure, and a new "relative information" measure. Both attempt to measure the amount of information contained in the markers about the identity-by-descent (IBD) sharing among relatives. The performance of the two information measures are compared based on their variability and ability to predict change in the LOD score (Delta LOD) as map density increases for SNP markers. Although in a linked region, LOD scores are correlated with measures of information, we observe that none of the measures predict the LOD score itself very well. In an unlinked region, the LOD score is not related to either measures of information. The information content of microsatellite markers with 7.5-cM spacing is slightly higher than that of SNP markers with 3-cM spacing. At these map densities, microsatellites are found to be uniformly more informative than SNPs irrespective of their level of heterozygosity. For SNPs, we found that as the level of heterozygosity increases, the information content increases. As reported in all other previous studies, we also found that high-density SNPs have higher information content compared to low-density microsatellites. Performance of both the two information measures considered here are similar, but the relative information measure predicts Delta LOD as marker density increases better than the traditional entropy-based information measure.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Isolation and characterization of two types of sarcoplasmic reticulum vesicles.

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain "light" and "heavy" vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca-2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca-2+ pump protein (55-65% of the protein) two other major protein components, the Ca-2+ binding and M55 proteins which accounted for 20-25 and 5-7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32-P-labelled phosphoenzyme levels proportional to their Ca-2+ pump protein content and contained similar Ca-2+-stimulated ATPase activities. They were capable of accumulating Ca-2+ in the presence of ATP and of releasing the accumulated Ca-2+ when placed into a medium with a low Ca-2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca-2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca-2+ per mg protein), in accordance with their high content of Ca-2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca-2+ binding and M55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca-2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca-2+ binding and/or M55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.