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

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

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca²⁺-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1–2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal H_II phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca²⁺, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca²⁺ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.     

Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures: Structural changes between hexagonal,cubic and bilayer phases

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and ³¹P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (H_II), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic ³¹P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend critially on the homogeneity and the degree unsaturation of the phospholipids.     

The nature of lipidic particles and their roles polymorphic transitions

The structural transition between bilayer (L_α), inverted hexagonal (H_II and inverted cubic (C_II) phases in mixtures of unsaturated phosphatidylethanolamines (PE) and phosphatidylcholines (PC) were investigated. Freeze fracture electron micrographs of intermediate stages of phase transitions showed that C_II was a stable intermediate form between the L_α-H_II transition. The electron microscopic observation was supported by X-ray diffraction and ³¹P-NMR results. Detailed morphology revealed that during the L_α-C_II transition, interlamellae attachment points (conical lipidic particles) connect adjacent bilayers to form arrays of entrapped water pockets (inverted micelles). These water-containg spherical units were packed in a cubic lattice. In the C_II to H_II transition, these spherical units were linearly connected to form tubes. During the L_α-H_II transition, a ripple pattern was observed across the otherwise smooth lamellar. The troughs of the ripples were transformed into linear connections between adjacent bilayers, thereby converting multilayer structures into parallel tubes. No lipidic particles were involved in this type of transition. We show that there are different mechanisms involved in the L_α, H_II, C_II polymorphic transitions, and that different types of ‘lipidic particles’ representing different molecular organizations may be involved in each case. Models of these transitions are proposed.     

Morphology of the intermediate stages in the lamellar to hexagonal lipid phase transition

Summary The addition of calcium to suspensions of egg phosphatidylcholine and cardiolipin converts multiwalled liposomes to the hexagonal (H_II) phase (Rand, R.P., Sengupta, S. (1972)Biochim. Biophys. Acta 255:484–492). We have studied this lamellar to hexagonal phase transition by freeze-fracture, thin-section electron microscopy, and X-ray diffraction and have morphologically characterized the intermediate stages. The first step in the transition involves the invagination and fusion of bilayers, marked by the appearance of lipidic intramembrane particles and crater-like indentations, as the large liposomes are converted to smaller flattened and elongated vesicles. The next step is the formation of tightly packed hexagonal arrays of tubules, each tubule being about 11 to 15 nm in diameter. These tubules are filled with fluid and a lipid bilayer forms the wall of each cylinder. Finally this tubular bilayer phase is converted to the hexagonal (H_II) phase, where the distance between tubes is 5.5 to 7.5 nm.     

Ca2+-induced isotropic motion and phosphatidylcholine flip-flop in phosphatidylcholine-cardiolipin bilayers

Ca²⁺ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by ³¹P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn²⁺ are observed.     

Alterations in phospholipid polymorphism by polyethylene glycol

Summary The fusogen polyethylene glycol is shown to alter the polymorphism of dimyristoyl phosphatidylcholine, soybean phosphatidylethanolamine, bovine phosphatidylserine, egg phosphatidylcholine/cholesterol mixture, dilinoleoylphosphatidylethanolamine/palmitoyl-oleoylphosphatidylcholine mixture, and egg lysolecithin. Suspension of these lipids in 50% polyethylene glycol (mol wt=6000) reduces both the lamellar and the hexagonal II repeat spacings as measured by X-ray diffraction. An increase in the gel to liquid crystalline and bilayer to hexagonal transition temperatures are observed by freeze-fracture, X-ray diffraction, differential scanning calorimetry and³¹P NMR. Freeze-fracture electron micrographs revealed different bilayer defects depending on the physical states of the lipid. Lipidic particles in mixtures containing unsaturated phosphatidylethanolamine is eliminated. Some of the influences of polyethylene glycol on lipids may be explained by its dehydrating effect. However, other nonfusogenic dehydrating agents failed to produce similar results. These findings are consistent with the proposal that close bilayer contact and the formation of bilayer defects are associated with the fusogenic properties of polyethylene glycol.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

Tritrichomonas foetus: Fine Structure of Freeze-Fractured Membranes1

ABSTRACT. Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9–12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/μm² and 468/μm² respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per μm² in the nuclear. membrane.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures. Structural changes between hexagonal, cubic and bilayer phases

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and 31P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (HII), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic 31P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend cirtically on the homogeneity and the degree unsaturation of the phospholipids.     

Rapid transbilayer movement of phosphatidylcholine in unsaturated phosphatidylethanolamine containing model membranes

Aqueous dispersions of egg phosphatidylethanolamine/18 : 1_c, 18 : 1_c-phosphatidylcholine/cholesterol/18 : 1_c, 18 : 1_c-phosphatidic acid (50 : 16 : 30 : 4) undergo a temperature-dependent transition from extended bilayers to structures characterized by isotropic ³¹P-NMR signals and visualized by freeze-fracturing as lipidic particles associated with the bilayer. This transition is accompanied by a 3-fold increase in the phosphatidylcholine pool which can be exchanged by phospholipid exchange protein demonstrating a direct relation between the occurrence of non-bilayer lipid structures and an increased transbilayer movement of phosphatidylcholine.     

An anaesthetic-induced phosphatidylcholine hexagonal phase

The interaction of trichloroethylene, an inhalational general anaesthetic, with phosphatidylcholine model membranes has been studied by ³¹P- and ²H-nuclear magnetic resonance spectroscopy. The incorporation of increasing amounts of trichloroethylene induces a phase transition from a multilamellar bilayer to a hexagonal phase. The effect is enhanced by increasing temperature. No indication of an intermediate cubic (C_II) phase was discovered.     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane 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 - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

The adsorption of divalent cations to phosphatidylcholine bilayer membranes

Electrophoretic mobility and ³¹P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations to phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the ³¹P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the ³¹P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Sr²⁺, Ba²⁺. If we assume that a lipid molecule occupies an area of 60 Ų and that there is a 1: 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy

A new type of lipid organization is observed in mixtures of phosphatidylcholine with cardiolipin (in the presence of Ca²⁺), monoglucosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic ³¹P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.