Dynamic light scattering study of calcium-induced fusion in phospholipid vesicles

Acidic sonicated phospholipid vesicles can undergo dramatic morphological changes due to fusion in the presence of divalent metal ions. For example, small spherical phosphatidylserine vesicles can form scroll-like cylinders which precipitate in the presence of Ca²⁺ above a threshold concentration. Subsequent addition of EDTA will yield large, unilamellar vesicles. These events have previously been established through the combined use of differential scanning calorimetry and freeze-fracture electron microscopy. We have applied the technique of dynamic light scattering to follow these fusion events rapidly, accurately, and non-perturbatively as they occur in solution at calcium concentrations slightly below threshold for precipitation.     

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.     

Lectins facilitate calcium-induced fusion of phospholipid vesicles containing glycosphingolipids

Ca2+-induced fusion of phospholipid vesicles containing globoside (GL-4) or disialoganglioside (GDla) is several-fold slower than the fusion of the pure phospholipid vesicles. Lectins specific for these glycosphingolipids, soybean agglutinin and wheat germ agglutinin, respectively, enhance the rate of fusion when added to the vesicle suspension before the introduction of Ca2+. The enhancement depends on the lectin concentration and the time of preincubation with the lectin. We propose that lectins facilitate membrane fusion by inducing intermembrane contact, which is the first step in the overall process of membrane fusion, or by laterally phase separating the inhibitory glycolipids.     

Dynamic morphology of calcium-induced interactions between phosphatidylserine vesicles.

Structural changes in phosphatidylserine vesicles exposed to calcium chloride for various times have been observed by means of video-enhanced light microscopy and freeze-fracture electron microscopy. Large flat double-bilayer diaphragms form at the contacts between aggregated vesicles within milliseconds. Bilayers at and outside of diaphragms rupture and allow vesicles to collapse completely by flattening against each other within seconds. Collapse through intermediate states to a stable multilamellar phase is complete within minutes. The Ca-induced attraction energy and the resultant flattening at contacts between vesicles is far beyond that needed to stress bilayers to the point of rupture. Although the destabilizing response to this stress is preferential to the diaphragm region, 40% of adhering pairs rupture outside of the diaphragm region rather than fuse with each other. In this respect the mechanism of fusion between these vesicles may be fundamentally different from the controlled fusion process in cells.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Role of synexin in membrane fusion. Enhancement of calcium-dependent fusion of phospholipid vesicles

Synexin, a soluble adrenal medullary and liver protein which causes calcium-dependent aggregation of isolated chromaffin granules, was isolated and purified according to published procedures. The effects of synexin on the kinetics of membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Synexin lowers the threshold of CA2+ concentration required for fusion of large unilamellar vesicles of phosphatidylserine and a mixture of phosphatidylserine with phosphatidylethanolamine. synexin also increases drastically the initial rate of fusion. the initial rate of fusion increases with the quantity of synexin present in the reaction mixture. In the presence of 1-2 mM Ca2+ and 50 microM phospholipid, synexin at 20 to 40 micrograms/ml increases the rate of fusion by two orders of magnitude. Mg2+ does not support synexin-induced fusion. With vesicles containing a mixture of phosphatidylserine with phosphatidylcholine, synexin enhances aggregation in the presence of CA2+, without promoting fusion. Synexin may play a role in exocytosis by promoting fusion of membranes containing specific phospholipids in the presence of Ca2+.     

Melittin induces fusion of unilamellar phospholipid vesicles

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26–30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690–692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.     

Quantitation of phospholipid vesicles and their cholesterol content in human bile by quasi-elastic light scattering

The proportion of biliary cholesterol carried by phospholipid vesicles may be an important determinant of the lithogenicity of bile. The distribution of biliary cholesterol between vesicles and other aggregational forms is often determined by gel filtration under standard conditions. The aim of this study was to measure the proportion of biliary cholesterol in vesicles in native unprocessed bile and to compare it with values obtained by chromatography. A modified quasi-elastic light-scattering method was used to measure vesicular cholesterol in whole bile. It was suitable only for lightly pigmented biles with a relatively monodisperse population of vesicles. In ten human biles examined, the proportion of cholesterol in vesicles by gel filtration was 40 +/- 8.1% (mean +/- S.D.) by chemical measurement, and 38 +/- 7.2% by [3H]cholesterol estimation. Quasi-elastic light-scattering measurements of these biles produced vesicular cholesterol values of 36 +/- 9.4%. Chromatography may affect lipid particles in bile. Nevertheless, it provides a relatively accurate measurement of biliary cholesterol in vesicles.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Dynamic light scattering study of muscle F-actin

By use of digital autocorrelation and fast Fourier methods, dynamic light-scattering studies of in vitro reconstituted muscle F-actin were made over a wide range of concentrations, 0.01-2 mg/ml F-actin. Measurements of correlation function [g1(t)]2 showed that a transition from a dilute to a semidilute regime for the Brownian motions of filaments occurred at around 0.3 mg/ml F-actin. Beyond this concentration, profiles of successively measured [g1(t)]2 showed very poor reproducibility. This resulted from the existence of very slow components, which could not be measured with a high statistical accuracy even for a measuring time of 3600 s/run. On the other hand, subtraction of these components automatically by an electronic circuit, [g-1(t)]2, or by computer processing, [g1(t)]2, resulted in a fairly good reproducibility of the profiles. The decay characteristics of [g1(t)]2 (and [g-1(t)]2) were very similar to those of [g1(t)]2 for dilute solutions. A theoretical model will be discussed which could account for the above situation. The time sequence [n(t,T)] of photoelectron counts at a sampling time T of light scattered from semidilute solutions of F-actin was stored on magnetic tapes, and both power spectra S(f) and correlation functions [g-1(t)]2 were computed by taking the ensemble average over many short records with duration 1024T. Since both S(f) and [g-1(t)]2 lacked frequency components lower than 1/(2048T) Hz, their profiles were highly reproducible. An analysis of S(f) confirmed our earlier results which had shown an apparent contradiction to later results by a correlation method. A comparison of S(f) and [g-1(t)]2 based on the same [n(t,T)] clarified the reasons why the bandwidth gamma of S(f) largely differed from the bandwidth gamma of [g1(t)]2 and [g-1(t)]2. The temperature dependence of gamma suggested that F-actin would be flexible and that the flexibility parameter would change with temperature.     

Penetration and fusion of phospholipid vesicles by lysozyme

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine ([125I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [125I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.     

Lysozyme induced fusion of negatively charged phospholipid vesicles

Lysozyme promotes fusion of negatively charged phospholipid vesicles prepared by ethanolic injection. Vesicle fusion was a leaky process as revealed by the release of encapsulated carboxyfluorescein or Tb-DPA complex. Extensive proteolysis of lysozyme inhibited the fusion process. The fusion process was critically dependent on the medium ionic strength; 100 mM of any salt was sufficient to inhibit totally the fusion activity of the protein. The high efficiency of lysozyme (80% RET) was almost constant in the pH range from 4.0 to 9.0, but it was sharply diminished when the pH of the medium was at the isoelectric point of the protein (pI 11.0). Fusion induced by chemically modified lysozyme, showed that the pH profile changed according to the isoelectric point of the protein derivative. These observations stress the importance of electrostatic interactions in the process of fusion induced by lysozyme.     

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.     

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.     

Effect of membrane phase transition on long-time calcium-induced fusion of phosphatidylserine vesicles

Dynamic light scattering has been used to study the temperature dependence of the extent of long-time calcium-induced fusion of sonicated vesicles composed of various natural and synthetic phosphatidylserine with different acyl chains. The vesicles of each composition are found to exhibit a peak temperature in the vicinity of which the extent of fusion shows a distinct maximum. The fusion peak temperature increases as the bilayer gel-to-liquid-crystal phase transition temperature increases. The results suggest a role played by membrane fluidity in determining fusion efficiency.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.     

Ovalbumin-induced fusion of acidic phospholipid vesicles at low pH

The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidylserine (PS) and PS/phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phospholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2,500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.