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S Nir

Publications and source records attributed to S Nir.

At least 19 recordsLinked to original sources
PubMed1992

Asymmetric fusion between synthetic di-n-dodecylphosphate vesicles and virus membranes.

The interaction between vesicles, prepared from the synthetic amphiphile di-n-dodecylphosphate (DDP), with Sendai virus membranes was investigated. DDP vesicles fuse in the presence of Ca2+ ('symmetric' fusion). However, in the absence of Ca2+, DDP vesicles and Sendai virus, both displaying a high intrinsic fusion capacity with various target membranes, can also readily fuse with each other ('asymmetric' fusion). Under these conditions, fusion was found not to depend on specific viral proteins. Thus fusion occurs over a broad pH range (3.0-9.0) and is not affected by perturbation of viral protein structure. The overall interaction process was further analyzed with a mass action kinetic model. The analysis reveals that the destabilization and reorganization of the synthetic and viral bilayers are as fast as in pure phospholipid systems. Furthermore, the drastic effect of temperature on the overall reaction appears to be related to an effect of this parameter on fusion itself rather than on vesicle-virus aggregation. This could suggest that protein mobility constraints modulate the fusion reaction. The morphology of the fusion products, which consist of a single virus particle and several DDP vesicles, indicates a bilayer stabilization of the fusion product, rather than formation of tubular structures, as observed for symmetric DDP fusion products. The present results further emphasize the high susceptibility of vesicles composed of synthetic amphiphiles to engage in (protein-mediated) membrane fusion. This bears relevance to their potential application as carriers for biomolecules.

Cholesterol↗
PubMed1992

Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.

Animals↗
PubMed1992

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells.

The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes.

Cell Membrane↗
PubMed1991

Fusion of Sendai virus with human HL-60 and CEM cells: different kinetics of fusion for two isolates.

The kinetics of fusion of Sendai virus (Z strain) with the human promyelocytic leukemia cell line HL-60, and the human T lymphocytic leukemia cell line CEM was investigated. Fusion was monitored by fluorescence dequenching of octadecylrhodamine (R-18) incorporated in the viral membrane. For one virus isolate (Z/G), the overall rate of fusion (at 37 degrees C) increased as the pH was lowered, reaching a maximum at about pH 5, the lowest pH tested. For another isolate (Z/SF) the rate and extent of fusion were lower at pH 5 than at neutral pH. Lowering the pH from neutral to 5 after several minutes of incubation of either isolate with HL-60 cells resulted in an enhanced rate of fluorescence dequenching. Nevertheless, experiments utilizing NH4Cl indicated that fusion of the virus with cells was not enhanced by the mildly acidic pH of the endosome lumen. Analysis of the kinetics of fusion by means of a mass action model resulted in good simulation and predictions for the time-course of fusion. For the isolate which showed maximal fusogenic activity at pH 5, the rate constant of fusion (approx. 0.1 s-1) at neutral pH was in the range found previously for virus-liposome fusion, whereas the rate constant of adhesion was close to the upper limit for diffusion-controlled processes (1.4.10(10) M-1 s-1). However, for the other isolate (Z/SF) the rate constant of fusion at neutral pH was very small (less than 0.01 s-1), whereas the rate constant of adhesion was larger (greater than or equal to 2.10(10) M-1 s-1). Lowering the temperature decreased the fusion rate. Experiments involving competition with excess unlabeled virions indicated that not all binding sites for Sendai virus on HL-60 cells are fusion sites. The virus fusion activity towards HL-60 cells at neutral pH was not altered significantly by pre-incubation of the virus at pH 5 or 9, in contrast to earlier observations with liposomes and erythrocyte ghosts, or results based on erythrocyte hemolysis or cell-cell fusion.

Ammonium Chloride↗
PubMed1990

Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA.

The synthetic, amphipathic peptide GALA undergoes a pH-dependent conformational change and induces leakage of contents from large unilamellar phosphatidylcholine vesicles when in a helical conformation. The kinetics of this process have been investigated over a wide range of pH and lipid and peptide concentrations. Leakage from lipid vesicles is rapidly initiated (within 2 s) when the pH is lowered below 6 and is rapidly terminated when the pH is raised to 7.5. The leakage shows a selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. Using this experimental data, we have developed a mathematical description of the kinetics of leakage induced by GALA. This model assumes that GALA becomes incorporated into the vesicle bilayer and aggregates to form a pore. Leakage occurs when a critical number of peptides assemble into a supramolecular aggregate in the bilayer. Leakage curves generated at lipid/peptide ratios ranging from 500/1 to 30000/1 can be well described by this formalism. On the basis of the results and the model, we suggest that GALA forms a transbilayer channel composed of 8-12 monomers. The channel diameter ranges from 5 to 10 A. To the best of our knowledge, this is the first model that can predict the leakage kinetics of solutes entrapped in lipid vesicles induced by a pore-forming peptide. The analysis may be of general use in defining the kinetics and state of aggregation of similarly acting peptides and proteins which form multimeric assemblies in membranes.

Amino Acid Sequence↗
PubMed1990

Molecular mechanisms of calcium-induced membrane fusion.

We have reviewed studies on calcium-induced fusion of lipid bilayer membranes and the role of synexin and other calcium-binding proteins (annexins) in membrane fusion. We have also discussed the roles of other cations, lipid phase transitions, long chain fatty acids and other fusogenic molecules. Finally, we have presented a simple molecular model for the mechanism of lipid membrane fusion, consistent with the experimental evidence and incorporating various elements proposed previously.

Animals↗
PubMed1990

Fusion of enveloped viruses with cells and liposomes. Activity and inactivation.

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a "fresh" batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via "inactive" sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.

Cell Fusion↗
PubMed1989

The kinetic mechanism of cation-catalyzed phosphatidylglycerol transbilayer migration implies close contact between vesicles as an intermediate state.

We have investigated variations in the rate of Mn2+-catalyzed phosphatidylglycerol transbilayer migration [Lentz, Madden, & Alford (1982) Biochemistry 21, 6799] with changes in phospholipid and cation concentration over more than a 100-fold range of both parameters. The slope of a double logarithmic plot of the rate of transbilayer lipid migration versus lipid concentration was 1.7, suggesting that lipid redistribution was dependent on vesicle aggregation or collision. A model involving transitory dimerization of vesicles was able to account for the concentration dependence of the transbilayer redistribution rate. The observed variation in rate with the logarithm of Mn2+ concentration was complex: linear above 0.4 microM (corresponding to roughly 2.5 Mn2+ per vesicle) but involving a steeper dependence on Mn2+ below 0.04 microM (roughly four vesicles per Mn2+). The rate of transbilayer redistribution increased substantially between 37 and 56 degrees C, yielding a nonlinear Arrhenius plot. There was no evidence of either fusion or lipid exchange between vesicles at the low concentrations of Mn2+ needed for transbilayer redistribution. The data are consistent with a model suggesting transitory "micro-domains" of a dehydrated, interbilayer complex as involved in the transition state and are inconsistent with a model involving an inverted micelle-type structure for the transition state.

Biological Transport↗
PubMed1989

Mechanism of fusion of Sendai virus: role of hydrophobic interactions and mobility constraints of viral membrane proteins. Effects of polyethylene glycol.

The mechanism of Sendai virus fusion was investigated by studying the effect of the dehydrating agent polyethylene glycol (PEG) on the interaction of the virus with erythrocyte membranes. The initial rate of virus fusion, monitored continuously by a fluorescence membrane fusion assay, increases approximately 5-fold in the presence of small amounts (4%, w/v) of PEG. The polymer did not trigger a massive nonspecific fusion event, as the limited number of virus particles that fuse per erythrocyte ghost remains unaltered. A mass action kinetic analysis reveals that the binding rate constant increases approximately 1.5-fold; however, the fusion rate constant is enhanced by about an order of magnitude. The results demonstrate that hydrophobic interaction forces dominate the actual fusion step of the virus. Below about 22 degrees C, the viral membrane proteins appear to be clustered, as revealed by temperature-dependent fluorescence measurements of fluorescently tagged viral proteins. Clustering is not modulated by the presence of PEG, and fusion at those conditions is not observed. It is concluded that in addition to hydrophobic interactions, constraints in the mobility of the viral membrane proteins codetermine the fusogenic capacity of the virus. Such constraints have to be relieved in order to allow the occurrence of the hydrophobic interactions. PEG primarily affects the surface properties of the viral membrane, including the properties of the membrane glycoproteins. We hypothesize that during virus-target membrane interaction but prior to the actual fusion reaction, the fusion protein may undergo a conformational change, triggered by an enhancement in hydrophobic environment, which accounts for the need to establish close, i.e. fusion-susceptible intermembrane contact between virus and target membrane.

Cell-Free System↗
PubMed1989

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes.

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry.

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.

Calcium↗
PubMed1988

Synexin enhances the aggregation rate but not the fusion rate of liposomes.

The effect of synexin on the calcium-induced fusion of large unilamellar liposomes was studied by using two assays for the mixing of aqueous contents. The results were analyzed in terms of the mass action kinetic model, which describes the overall fusion reaction as a two-step sequence consisting of a second-order process of liposome aggregation followed by a first-order fusion reaction. By using several different lipid compositions and varying the electrolyte composition, it was possible to select the rate-limiting step of the overall fusion process. When aggregation was the rate-limiting step, as in the case of Ca2+-induced fusion of phosphatidylserine (PS), phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3), and PS/PE (1:3) liposomes, synexin increased the overall fusion kinetics by increasing the aggregation rate constant (up to 100-fold). When aggregation was rapid compared to destabilization of apposed membranes, i.e., fusion was rate limiting, synexin either had no effect or reduced the overall fusion kinetics. In one such case involving liposomes composed of PA/PS/PE/phosphatidylcholine (PC) (10:15:65:10), synexin reduced the fusion rate constant by 50%. The effect of calcium-induced synexin polymerization was investigated by preincubation of synexin with calcium prior to addition of liposomes. Prepolymerization by Ca2+ always decreased the activity of synexin such that it was less than the activity of an equal amount of untreated monomers. However, it was found that the activity of synexin monomers polymerized to an average hexameric size was greater than that of one-sixth as many untreated monomers, with respect to the liposome aggregation rate constant. Neither polymers nor monomers increased the fusion rate constant.

Animals↗
PubMed1988

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide.

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.

Amino Acid Sequence↗
PubMed1988

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin.

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.

Bile Acids and Salts↗
PubMed1988

Precipitation of calcium palmitate from bile salt-containing dispersions.

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.

Bile Acids and Salts↗
PubMed1988

Mass action kinetics of virus-cell aggregation and fusion.

A simple approximate solution for the mass action kinetics of small particles (viruses or vesicles) binding to large particles (cells) and their subsequent fusion has been derived. The solution is evaluated in terms of the measurable fluorescence changes expected when the virus or vesicles are labeled with fluorescent probes, which are diluted into the cellular membrane by fusion. Comparison with numerical integrations shows that the approximate solution is extremely accurate. Analytic simplifications for a variety of special cases of this general problem are also shown.

Cell Membrane↗
PubMed1987

Effect of synexin on aggregation and fusion of chromaffin granule ghosts at pH 6.

Fusion of chromaffin granule ghosts was induced by synexin at pH 6, 37 degrees C, in the presence of 10(-7) M Ca2+. To study the kinetics and extent of this fusion process we employed two assays that monitored continuously mixing of aqueous contents or membrane mixing by fluorescence intensity increases. In both assays chromaffin granule ghosts were either labeled on the membrane or in the included aqueous phase. The ratios of blank to labeled chromaffin granule ghosts were varied from 1 to 10. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of aggregation followed by a first-order fusion reaction. The model calculations gave fare simulations and predictions of the experimental results. The rate constants describing membrane mixing are more than 2-fold larger than those for volume mixing. The analysis also indicated that the initial aggregation and fusion processes, up to dimer formation, were extremely fast. The rate constant of aggregation was close to the limit in diffusion-controlled processes, whereas the fusion rate constant was about the same as found in fastest virus-liposome fusion events at pH 5. A small increase in volume was found to accompany the fusion between chromaffin granule ghosts. Using ratios of synexin to chromaffin granule ghost protein of 0.13, 0.41 and 1.15 indicated that the overall fusion rate was larger for the intermediate (0.41) case. The analysis showed that the main activity of synexin was an enhancement of the rate of aggregation. At intermediate or excessive synexin concentrations it, respectively, promoted moderately, or inhibited the actual fusion step.

Animals↗