Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles

To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.     

Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes

Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry     

Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis

The effects of caffeine, aminophylline, caffeic acid, and calcium deficiency on cytokinesis were studied by light and electron microscopy. All these treatments blocked cell plate formation, resulting in the formation of binucleate cells. The aggregation and organization of membranous vesicles at the ‘presumptive cell plate’ during these treatments appears similar to that of normal cells, but fusion of the vesicles is insufficient to form a complete cell plate. It is suggested that some aspect of membrane recognition and fusion is the process actually interfered with by these treatments. Greater numbers of binucleate cells and fewer partial cell plates were observed in cells treated with caffeine and aminophylline as compared with those exposed to caffeic acid or calcium deficiency, indicating that the latter treatments do not block cell plate formation as efficiently as the former.     

New sterically stabilized vesicles based on nonionic surfactant, cholesterol, and poly(ethylene glycol)-cholesterol conjugates.

Monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-Chol) with polymer chain molecular weights of 1000 (M-PEG1000-Chol) and 2000 (M-PEG2000-Chol) have been newly synthesized and characterized. Their aggregation behavior in mixture with diglycerol hexadecyl ether (C16G2) and cholesterol has been examined by cryotransmission electron microscopy, high-performance gel exclusion chromatography, and quasielastic light scattering. Nonaggregated, stable, unilamellar vesicles were obtained at low polymer levels with optimal shape and size homogeneity at cholesteryl conjugate/ lipids ratios of 10 mol% M-PEG1000-Chol or 5 mol% M-PEG2000-Chol, corresponding to the theoretically predicted brush conformational state of the PEG chains. At 20 mol% M-PEG1000-Chol or 10 mol% M-PEG2000-Chol, the saturation threshold of the C16G2/cholesterol membrane in polymer is exceeded, and open disk-shaped aggregates are seen in coexistence with closed vesicles. Higher levels up to 30 mol% lead to the complete solubilization of the vesicles into disk-like structures of decreasing size with increasing PEG content. This study underlines the bivalent role of M-PEG-Chol derivatives: while behaving as solubilizing surfactants, they provide an efficient steric barrier, preventing the vesicles from aggregation and fusion over a period of at least 2 weeks.     

A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH

The addition of bovine serum albumin (BSA) to 25 ± 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 ± 10 nm diameter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level.     

The role of calcium in fusion of artificial vesicles.

Small phospholipid vesicles (liposomes) fuse upon calcium addition as demonstrated by electron microscopy, light absorbance increases, and mixing of original liposome contents within the boundaries of the fused liposome. The integrity of the fusion event is demonstrated by a novel assay based on the luminescence of firefly extract when mixed with ATP. Subsequent addition of valinomycin or the calcium ionophore A23187 leads to further fusion as shown by electron microscopy, light microscopy, and additional absorbance increase. Concomitant with this second absorbance increase is an increase in the amount of calcium that associates with the liposomes. This increased calcium association is more than can be accounted for by equilibration of 5 mM Ca2+ across the membrane and must indicate exposure of extra calcium binding sites. Binding of calcium to the inner side of the membrane may catalyze the second stage of liposome fusion.     

Mechanisms of leakage

Abstract

Two mechanisms of leakage from liposomes are discussed, (i) Cations such as Ca²⁺ induce graded release whose rate depends mainly on vesicle collisions and is associated in the case of several acidic phospholipids with fusion events. A certain degree of leakage also occurs in between collisions. Consequently, the leakage per fusion is reduced at larger lipid and Ca concentrations, (n) Certain peptides induce leakage by pore formation, which shows 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. A model for final extents and kinetics of leakage due to pore forming peptides is described. This model assumes that pore forming peptides become incorporated into the vesicle bilayer and aggregate to form a pore. Recent developments in the model enable considerations of a reversible or irreversible surface aggregation of peptides. Results of final extents and kinetics of leakage induced by pore forming peptides can be well explained and predicted by this formalism. Studies demonstrate that Ca can play a dual role in affecting leakage. A case is presented where Ca ⁺ inhibits and can even arrest pore formation by a peptide, while promoting vesicle fusion. Conversely, formation of pore structures by a peptide can inhibit vesicle fusion.     

Fusion and fragmentation of phospholipid vesicles by apohemoglobin at low pH.

Human apohemoglobin in acidic media was found to induce fusion of phosphatidylcholine/phosphatidylserine (1:1) vesicles at low protein concentration but to fragment the same vesicles to form micellar complex at high protein concentration. The fusion was demonstrated by size increase, vesicle content mixing, lipid mixing, and electron microscopy. The micellization of phospholipid vesicles was observed by light scattering, gel filtration, and electron microscopy. The hydrophobic labeling of the apohemoglobin/vesicle complex followed by CNBr cleavage of apohemoglobin showed that an N-terminal segment of the beta subunit with a molecular weight of approximately 6,000 seems to be mainly involved in the fusion process, but the whole sequences of both alpha and beta chains participate in the micellization process.     

Aggregation of hapten-bearing liposomes mediated by specific antibodies.

We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.     

Biophysical characterization of the non-fusogenic interaction between liposomes and the shrimp bifunctional lipoprotein-beta-glucan binding protein HDL/BGBP

Shrimp High Density Lipoprotein-beta-Glucan Binding Protein (HDL/BGBP) has been studied by its role in nutrition and innate defense. Although the mechanisms of lipid loading are still unknown, HDL-BGBP binds and aggregates phospholipids vesicles in vitro. To gain insights into the HDL-BGBP mechanism of interaction with membranes, we have used fluorescence spectroscopy and electron microscopy. Data show that HDL-BGBP does not induce membrane fusion, leakage nor lipid exchange, although microstructural changes are clearly observed. This work supports a model where protein aggregation leads to liposome clustering. Such interaction may be a critical factor for the activation of the shrimp blood cell in vivo.     

Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues

Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.     

Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze fracture study

The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Calcium-dependent aggregation and fusion of phosphatidylcholine liposomes induced by complexes of flavonoids with divalent iron

It was found that complexes of the flavonoids quercetin, taxifolin, catechin and morin with divalent iron initiated an increase in light scattering in a suspension of unilamellar 100nm liposomes. The concentration of divalent iron in the suspension was 10μM. Liposomes were prepared from 1-palmitoyl-2-oleoylglycero-3-phoshpatidylcholine. The fluorescent resonance energy transfer (FRET) analysis of liposomes labeled with NBD-PE and lissamine rhodamine B dyes detected a slow lipid exchange in liposomes treated with flavonoid-iron complexes and calcium, while photon correlation spectroscopy and freeze-fracture electron microscopy revealed the aggregation and fusion of liposomes to yield gigantic vesicles. Such processes were not found in liposomes treated with phloretin because this flavonoid is unable to interact with iron. Rutin was also unable to initiate any marked changes because this water-soluble flavonoid cannot interact with the lipid bilayer. The experimental data and computer calculations of lipophilicity (cLogP) as well as the charge distribution on flavonoid-iron complexes indicate that the adhesion of liposomes is provided by an iron link between flavonoid molecules integrated in adjacent bilayers. It is supposed that calcium cations facilitate the aggregation and fusion of liposomes because they interact with the phosphate moieties of lipids.     

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.     

Rapid kinetics of Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. The effect of bilayer curvature on leakage

We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion. Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation. Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation. Thus fusion takes place in the microsecond time domain. Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse. On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size. Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made. NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals. Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion

Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.     

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.     

Diffusion and redistribution of lipid-like molecules between membranes in virus-cell and cell-cell fusion systems.

The kinetics of redistribution of lipid-like molecules between the membranes of two fused spherical vesicles is studied by solving the time-dependent diffusion equation of the system. The effects on the probe redistribution rate of pore size at the fusion junction and the relative sizes of the vesicles are examined. It is found that the redistribution rate constant decreases significantly, but not drastically, as the relative size of the pore to that of the vesicles decreases (the bottleneck effect). In general, the time scale of the probe redistribution rate is determined by the size of the vesicles that is loaded with the probe before the activation of the fusion. For a pore size 50 A in diameter and a typical diffusion coefficient of 10(-8) cm2/s for lipids, the mixing half times for typical virus-cell and cell-cell fusion systems are less than 30 ms and above 200 s, respectively. Thus, although the redistribution of lipid-like probes by diffusion is not rate limiting in virus-cell fusion, redistribution by diffusion is close to rate limiting in spike-protein mediated cell-cell fusion.