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At least 19 recordsLinked to original sources
PubMed1984

Non-bilayer structures in membrane fusion.

Membrane fusion is an ubiquitous event in cell biology. One can distinguish two types of fusion: (i) outside/outside fusion, for example endocytosis, and (ii) inside/inside fusion, for example exocytosis. In spite of this difference in types of fusion in relation to membrane asymmetry and in spite of the large variety of lipid compositions encountered in biological membranes, a universal mechanism can be postulated for the role of lipids in membrane fusion. In this concept the lipids leave the bilayer configuration temporarily and locally. This notion, and the fact that any biological membrane contains a substantial amount of lipids which prefer the non-bilayer hexagonal II phase in physiological conditions, has led to the hypothesis that such hexagonal II phase lipids play a crucial role in membrane fusion. This proposition is strongly supported by model membrane experiments in which it has been demonstrated that factors such as Ca2+ and temperature, which trigger the transition from bilayer to hexagonal II phase, in fact induce membrane fusion.

Animals↗
PubMed1999

Lipid vesicles and membrane fusion.

Membrane fusion is essential for cell survival and has attracted a great deal of both theoretical and experimental interest. Fluorescence (de)quenching measurements were designed to distinguish between bilayermerging and vesicle-mixing. Theoretical studies and various microscopic and diffraction methods have elucidated the mechanism of membrane fusion. These have revealed that membrane proximity and high defect density in the adjacent bilayers are the only prerequisites for fusion. Intermediates, such as stalk or inverse micellar structures can, but need not, be involved in vesicle fusion. Nonlamellar phase creation is accompanied by massive membrane fusion although it is not a requirement for bilayer merging. Propensity for membrane fusion is increased by increasing the local membrane disorder as well by performing manipulations that bring bilayers closer together. Membrane rigidification and enlarged bilayer separation opposes this trend. Membrane fusion is promoted by defects created in the bilayer due to the vicinity of lipid phase transition, lateral phase separation or domain generation, high local membrane curvature, osmotic or electric stress in or on the membrane; the addition of amphiphats or macromolecules which insert themselves into the membrane, freezing or other mechanical membrane perturbation have similar effects. Lowering the water activity by the addition of water soluble polymers or by partial system dehydration invokes membrane aggregation and hence facilitates fusion; as does the membrane charge neutralization after proton or other ion binding to the lipids and intermembrane scaffolding by proteins or other macromolecules. The alignment of defect rich domains and polypeptides or protein binding is pluripotent: not only does it increase the number of proximal defects in the bilayers, it triggers the vesicle aggregation and is fusogenic. Exceptions are the bound molecules that create steric or electrical barriers between the membranes which prevent fusion. Membrane fusion can be non-leaky but it is very common to lose material from the vesicle interior during the later stages of membrane unification, that is, after a few hundred microseconds following the induction of fusion.

Journal Article↗
PubMed2001

Cdc42p functions at the docking stage of yeast vacuole membrane fusion.

Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.

Adenosine Triphosphatases↗
PubMed2000

Insights on membrane fusion.

Membrane fusion is a fundamental cellular process regulating intracellular transport, neurotransmission, enzyme secretion, hormone release, and the entry/exit of viruses, to name a few. Knowledge of how opposing bilayers fuse, besides advancing our understanding of these cellular processes, will provide us with the facts to ameliorate secretory defects and prevent cellular entry or exit of pathogenic viruses. In the last few years, great strides have been made in our understanding of the molecular machinery and mechanism of membrane fusion. In this Special Issue of Cell Biology International, entitled 'Membrane fusion: machinery and mechanism', we have tried to cover several aspects of this vital cellular process, providing insights on the machinery, mechanism and dynamics of the process. Membrane fusion studies reported in this Special Issue have been performed on whole cells, synaptic terminals, viruses, and fusion proteins.

Humans↗
PubMed1999

Role of the N-terminal peptides of viral envelope proteins in membrane fusion.

Membrane fusion is an important biological process that is observed in a wide variety of intra and intercellular events. In this review, work done in the last few years on the molecular mechanism of viral membrane fusion is highlighted, focusing in particular on the role of the fusion peptide and the modification of the lipid bilayer structure. While the Influenza hemagglutinin is currently the best understand fusion protein, there is still much to be learned about the key events in enveloped virus fusion reactions. This review compares our current understanding of the membrane fusion activity of Influenza and retrovirus viruses. We shall be concerned especially with the studies that lead to interpretations at the molecular level, so we shall concentrate on model membrane systems where the molecular components of the membrane and the environment are strictly controlled.

Journal Article↗
PubMed1995

Calorimetric detection of influenza virus induced membrane fusion.

Membrane fusion induced by the hemagglutinin glycoprotein of influenza virus has been extensively characterized, but the mechanism whereby the protein achieves the merger of the viral and target membrane lipids remains enigmatic. Various lipid intermediate structures have been proposed, and the energies required for their formation predicted. Here, we have analyzed the enthalpies of fusion of influenza with liposomes by titration calorimetry. If a small sample of virus in a weak neutral pH buffer was added to an excess of liposomes at low pH, a two-component reaction was seen, composed of an exothermic reaction and a slower endothermic reaction. The exothermic reaction was the result of acid-base reactions between the neutral pH virus sample and low pH buffer and low-pH-induced changes in the virus. The endothermic reaction was not observed in the absence of liposomes and much reduced if acid-inactivated virus, which had lost its fusion but not its binding activity, was added to liposomes. The endothermic reaction was more temperature dependent than the exothermic reaction; its pH dependence corresponded with that of fusion and its enthalpy was higher if fusion was more extensive. These data indicate that most of the endothermic reaction was due to membrane fusion. The experimentally determined enthalpy of fusion, 0.6-0.7 kcal per mol of viral phospholipids, is much higher than expected on the basis of current theories about the formation of lipid intermediates during membrane fusion.

Buffers↗
PubMed1990

Surface dielectric constant, surface hydrophobicity and membrane fusion.

Membrane fusion induced by ions and its associated membrane property, surface dielectric constant, were studied with the use of acidic and neutral phospholipid vesicles. The fusion of vesicles was monitored by utilizing two fluorescence fusion assays: fluorescence content mixing method and fluorescence labelled membrane component dilution method. For the surface dielectric constant measurements, a fluorescence method was used which detected the environmental effect on the membrane surface upon the addition of various fusogenic cations. Also, the effects of poly-(ethylene glycol) on both fusion and surface dielectric properties were examined. It was found that the extent of fusion correlated well with the degree of lowering in the dielectric constant of the surface membrane, which corresponds to the increase in hydrophobicity of the membrane surface. This agrees with the previously obtained experimental results that the increase in interfacial tension of the membrane, which also corresponds to the increase in surface hydrophobicity, correlates with the extent of membrane fusion.

Calcium↗
PubMed1988

A molecular basis for synexin-driven, calcium-dependent membrane fusion.

Membranes of secretory vesicles fuse with each other and with plasma membranes during exocytosis in many different cell types. The probable role of calcium in the process is now widely accepted, and it is possible that at least one cytosolic mediator of calcium action is synexin. Synexin is a 47,000 Mr calcium-binding protein, initially discovered in the bovine adrenal medulla, which binds to granule membranes and to inner aspects of chromaffin cell plasma membranes. Synexin causes chromaffin granules to aggregate, and such aggregates can be caused to fuse in the additional presence of arachidonic acid. Synexin also mediates the direct fusion of liposomes and chromaffin granule ghosts. To understand better the mechanisms of membrane fusion promoted by synexin we have attempted to define the primary sequence of the protein. Our initial efforts were directed towards purification of bovine synexin in sufficient amounts to allow us to sequence tryptic peptides. However, as the project progressed we also directed our attention to human synexin, preparing peptides from this protein as well. From analysis of bovine peptides we learned that the synexin molecule might be closely related to a class of proteins including lipocortin I, calpactin (p36), endonexin II, protein II and calelectrin 67K. Complete analysis of a human synexin cDNA clone revealed strong homology with bovine synexin. The analysis also showed that synexin contained a unique, long, highly hydrophobic N-terminal leader sequence followed by a characteristic four-fold repeat homologous with those found in other members of the synexin gene family. The highly hydrophobic character of synexin seems consistent with information previously obtained that synexin is able to insert directly into the interior of bilayers prepared not only from purified phosphatidylserine but also from biological membranes. The evidence for such insertions is a dramatic increase in the capacitance of the membrane, formed at the tip of a patch pipette, when calcium-activated synexin is applied to the bilayer. Additional evidence is the fact that synexin also forms calcium-selective channels when the protein is applied to the cytosolic aspect of the plasmalemma when that side is also exposed to calcium at sub-millimolar concentrations. Thus, the synexin molecule not only enters the membrane, but also spans it. From these and other data we have developed the concept that the fusion process may involve synexin forming a 'hydrophobic bridge' between two fusing membranes. Lipid movement across this bridge may then be the material basis for final fusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1980

Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion.

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

Cell Fusion↗
PubMed2001

Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells.

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.

Animals↗
PubMed1984

Lessons for the study of membrane fusion from membrane interactions in phospholipid systems.

'Fusion' in model systems usually refers to the decay of membrane configurations that are inherently unstable because of the method of preparation. Natural fusion is a controlled event during which the underlying forces and instabilities are subject to the additional effects of biochemical reactions. To understand biological fusion one must be able first to assess the interplay among these physical and chemical factors. This paper reviews traditional measurements of electrostatic double layer and electrodynamic van der Waals forces acting between bilayer membranes. It also describes the much stronger hydration forces that have now been systematically studied. An essential part of any fusion event is the ability of membrane surfaces to overcome or circumvent the hydration barrier in order to make contact. This may be accomplished through applied force, through bridging substances that displace water from the membrane surface, or through biochemical modification of surfaces. In model systems, destruction of the hydration layer can cause violent adhesion, membrane deformation, and rupture. Natural fusion proceeds by more subtle processes whereby interfacial forces are harnessed in ways not yet understood.

Animals↗
PubMed1985

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles.

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Retinal and retinol promote membrane fusion.

Disk membranes from the bovine retinal rod outer segments (ROS) were found to fuse with vesicles made of lipids extracted from unbleached ROS disk membranes, using a lipid mixing assay for membrane fusion (relief of self-quenching of R18, octadecylrhodamine B chloride). If the retinal chromophore of rhodopsin was reductively linked to opsin before lipid extraction, the vesicles made of the extracted lipids were not suitable targets for fusion of the disk membranes. The addition of retinal and retinol to these vesicles restored their ability to fuse. Therefore, the presence of all-trans retinal was implicated in promoting membrane fusion in this system. To test this possibility, the ability of retinal and retinol to influence the phase behavior and the fusion capability of large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl-DOPE) was examined. Both retinal and retinol stimulated the fusion of vesicles of N-methyl-DOPE (contents mixing with ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX, p-xylylene bis(pyridinium bromide)). Both compounds reduced the onset temperature for isotropic resonances in the 31P-NMR spectra of N-methyl-DOPE dispersions and the onset temperature, TH, for formation of hexagonal II phase. These results were consistent with previous studies in which the onset temperature for the 31P-NMR isotropic resonances were correlated with stimulation of membrane fusion. These data suggested that both retinal and retinol may stimulate membrane fusion by destabilizing the bilayers of membranes.

Animals↗
PubMed1994

Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adopt an HII phase with its ability to promote membrane fusion.

A critical step in membrane fusion involves the formation of a lipid intermediate which shares a conformational similarity with an inverted hexagonal phase (HII). Since plasmenylethanolamines possess a marked propensity for hexagonal phase formation and represent a major lipid constituent of several membrane systems which undergo rapid membrane fusion (e.g., plasma membranes and synaptic vesicle membranes), we compared the relative fusogenicity of lipid vesicles containing plasmenylethanolamine to that of vesicles containing their diacyl phospholipid counterpart (i.e., phosphatidylethanolamine). Vesicles comprised of equimolar mixtures of phosphatidylcholine and phosphatidylethanolamine fused slowly with phosphatidylserine vesicles in the presence of 10 mM CaCl2, as assessed either by lipid mixing (dequenching of octadecyl rhodamine fluorescence, 7.4 Fmax% s-1) or internal contents mixing (fluorescence enhancement from the resultant Tb/dipicolinic acid charge transfer complex, 8.7Fmax% s-1). In stark contrast, vesicles comprised of equimolar mixtures of phosphatidylcholine and plasmenylethanolamine fused three times more rapidly, as assessed by both lipid mixing (22.1 Fmax% s-1) and internal contents mixing (21.4Fmax% s-1) assays. The importance of an HII-like intermediate in membrane fusion was further substantiated by demonstration that plasmenylethanolamines containing arachidonic acid at the sn-2 position (which demonstrate a greater propensity for HII phase formation) exhibited the most rapid rate of membrane fusion (five times greater than phosphatidylethanolamine containing oleic acid at the sn-2 position). Furthermore, vesicles containing plasmenylethanolamines in physiologic ratios with other phospholipids (i.e., PC/PE/PS, 45:45:10, mol/mol) underwent fusion six times more rapidly (4.4Fmax% min-1) than corresponding vesicles in which plasmenylethanolamine was replaced with phosphatidylethanolamine (0.7Fmax% min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes.

The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.

Adenosine Triphosphatases↗
PubMed1996

The fusion pore and mechanisms of biological membrane fusion.

Membrane fusion occurs as part of processes as different as synaptic neurotransmitter transmission and infection with influenza virus. Recent evidence paints a picture in which the organization of proteins into a macromolecular scaffold brings the two fusing membranes together and induces hemifusion, that is, the fusion of the apposing leaflets of the two membranes to form a common bilayer. A small dynamic fusion pore forms in the common bilayer and usually expands to allow complete membrane merging. The mechanisms of fusion appear to be remarkably similar in exocytosis and virus-induced fusion. During exocytotic fusion, there is an additional twist to the mechanism, as sometimes the fusion pores close after release of small non-quantal amounts of secretory products.

Cell Fusion↗
PubMed2000

SNAREs contribute to the specificity of membrane fusion.

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.

Amino Acid Sequence↗