William J. Vail

Cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles

Freeze-fracture electron microscopy was used to study the morphological changes occurring following the addition of Ca-2+ to sonicated preparations of phosphatidylserine in aqueous NaCl buffer. Before the addition of Ca-2+, preparations contained only small (200-500 A diameter) spheroidal vesicles. After the addition of Ca-2+ (10 mM) and incubation for 1 h at 37 degrees C preparations contained only large (2000-10 000 A) apparently multilamellar structures many of which were cylindrical in shape. The lamellae in these cylinders appear to be folded in a spiral configuration. Addition of EDTA to these preparations produced large, closed, spherical, unilamellar vesicles. We suggest the name cochleate lipid cylinders for the spiral structures and propose that they are formed by fusion of unilamellar vesicles into large sheets which fold spirally to form cylinders.

Membrane fusion and molecular segregation in phospholipid vesicles

Abstract Fusion between vesicles prepared from individual or mixed phospholipid species was demonstrated by ultracentrifugation and gel-filtration techniques, electron microscopy and differential scanning calorimetry. Variation of the chemical composition of the vesicles permitted evaluation of the effect of surface charge, Ca2+, fluidity and the presence of cholesterol on the fusion reaction and the segregation of lipid species within fused vesicles. Extensive fusion occurred between negatively charged phosphatidylserine vesicles incubated in the presence of CaCl2 ( > 1 mM ) and in vesicles prepared from greater than 50% phosphatidylserine in phosphatidylcholine in the presence of CaCl2 ( > 4 mM ) and albumin (0.1 mg/ml). Neutral phosphatidylcholine vesicles showed only a limited capacity to fuse. Vesicles containing lipids that were in a liquid-crystalline state were more susceptible to fusion than vesicles composed of lipids that were in the solid phase at experimental temperatures. Incorporation of equimolar amounts of cholesterol into vesicles composed of lipids in a liquid-crystalline state suppressed their ability to fuse. Calorimetric measuremens revealed Ca2+ induced segregation of individual lipids to form separate domains within the vesicle membrane (phase separation). The relationship of fusion between vesicles and fusion occurring in natural membranes was discussed.

Studies on membrane fusion. III. The role of calcium-induced phase changes

The interaction of phosphatidylserine vesicles with Ca2+ and Mg2+ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca2+ and Mg2+ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca2+ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion. The results indicate that at Ca2+ concentrations of 1.0-2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient range of 2.0-5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change. From the effect of variations in pH, temperature, Ca2+ and Mg2+ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.

Studies on membrane fusion. 1. Interactions of pure phospholipid membranes and the effect of myristic acid, lysolecithin, proteins and dimethylsulfoxide

The interaction and mixing of membrane components in sonicated unilamellar vesicles and also non-sonicated multilamellar vesicles prepared from highly purified phospholipids suspended in NaCl solutions has been examined. Electron microscopy and differential scanning calorimetry were used to characterize the extent and kinetics of mixing of membrane components between different vesicle populations. No appreciable fusion was detected between populations of non-sonicated phospholipid vesicles incubated in aqueous salt (NaCl) solutions. Mixing of vesicle membrane components via diffusion of phospholipid molecules between vesicles was observed in populations of negatively charged phosphatidylglycerol vesicles but similar exchange diffusion was not detected in populations of neutral phosphatidylcholine vesicles. Incubation of sonicated vesicle populations at temperatures close to or above the phospholipid transition temperature resulted in an increase in vesicle size and mixing of vesicle membrane components as determined by a gradual change in the thermotropic properties of the mixed vesicle population. The interaction of purified phospholipid vesicles was also examined in the presence of myristic acid and lysolecithin. Our results indicate that while these agents enhance mixing of vesicle membrane components, in most cases mixing probably proceeds via diffusion of phospholipid molecules rather than by fusion of entire vesicles. Increased mixing of vesicle membrane components was also produced when vesicles were prepared containing a purified hydrophobic protein (myelin proteolipid apoprotein) or were incubated in the presence of dimethylsulfoxide. In these two systems, however, the evidence suggests that mixing of membrane components results from the fusion of entire vesicles.

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.

Studies on Membrane Fusion with Natural and Model Membranes

Fusion of membranes is a common and highly important event in the biology of eukaryotic cells. Membrane fusion is required for the uptake by endocytosis and the intracellular digestion of extracellular material and also for the transport of intracellular materials to the extracellular space by exocytosis. The formation of endocytotic vesicles at the cell surface involves invagination of a segment of the plasma membrane, which must then fuse with itself in order to form a closed vesicle. Subsequently, the digestion of the contents of the endocytotic vesicles involves a series of membrane fusion sequences between these vesicles and lysosomes and Golgi vesicles (review, Edelson and Cohn, 1978). Membrane fusion also plays a prominent role in the reverse process of exocytosis in which fusion takes place at the cell surface between the membrane of the exocytotic vesicle and the plasma membrane. Exocytotic discharge is basic to the processes of cell excretion and secretion and is involved in the release of a wide variety of enzymes, hormones, and neurotransmitter substances from such cells as the newly fertilized egg, blood platelets, leukocytes, mast cells, nerve cells, cells participating in the formation of kinins, angiotensin, and erythropoietin, and hormone-producing cells in the adrenal medulla, neurohypophysis, anterior pituitary, thyroid, and pancreas (reviews, Ceccarelli et al, 1974; Douglas, 1975; Carafoli et al., 1975).

Interaction of a hydrophobic protein with liposomes evidence for particles seen in freeze fracture as being proteins

Abstract Freeze fractures of liposome membranes showed a smooth fracture surface while liposomes into which a hydrophobic protein from human myelin had been incorporated showed a particulate fracture surface. The evidence suggests that at least some of the analogous particles present on fracture surfaces of biological membranes could be proteins, embedded in a phospholipid bilayer.

Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid. A spin label study.

Lipophilin, a hydrophobic protein purified from the proteolipid of normal hupid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electreter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component waicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged.

Site of biosynthesis of mammalian cytochrome c oxidase subunits

In Saccharomyces cerevisiae [l-3] and in Neurospora crassa [ 1,4] it has been shown that the three largest subunits of cytochrome c oxidase (EC 1.9.3.1) are synthesized in mitochondria, while the remaining subunits are synthesized on cytosolic ribosomes. In Locusta migratora, at least one of the subunits of insect cytochrome c oxidase is synthesized in mitochondria [S] . Previous work with mammalian cells has suggested a mitochondrial origin for at least some of the subunits of cytochrome c oxidase because formation of the enzyme is inhibited by chloramphenicol or its sulfamoyl analog, Tevenel [ 1,6,7] . The present work provides immunological evidence that at least two of the subunits of mammalian cytochrome c oxidase are synthesized in mitochondria.

The structure of cytochrome oxidase membranes

Two preparations of oxidized membranous cytochrome oxidase were examined using both the negative stain and freeze fracture techniques of electron microscopy. The cholate preparation, containing 27% phospholipid by weight, appeared as membrane sheets. With the freeze fracture technique, cross fractures of the sheets showed particles abutted one against another. The Triton preparation, containing 42% phospholipid by weight, was decidedly vesicular and only particulate membrane fracture surfaces were usually seen. In the case of membranous cytochrome oxidase it is proposed that fractures follow the surface rather than a centre plane through the hydrophobic interior of the membrane. The observed particles appeared as prolate spheroids of 135 A X 68 A with the long axis extended through the membrane. From the size shape relationships, a molecular weight of 244 000 was calculated which is in agreement with values reported for dispersed cytochrome oxidase using other physical techniques. At least 50% of the protein is exposed to the aqueous environment. Cytochrome oxidase is a lipoprotein that spontaneously forms membranes under conditions of low ionic strength and the absence of detergents. Sun et al. [ 1 ] showed that these membranes were trilaminate structures 55 A thick when examined by conventional thin section techniques. Negatively stained preparations showed a membrane surface covered with 50 A particles. Seki and Oda [2] have shown