Fusion Intermediates Research Articles

Intermediates in membrane fusion.

Intermediates in membrane fusion.

Stages leading to and following fusion of sperm and egg plasma membranes.

The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (OsO4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-OsO4 mixture induced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.

Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms

To understand the mechanism of membrane fusion, we have to infer the sequence of structural transformations that occurs during the process. Here, it is shown how one can estimate the lipid composition-dependent free energies of intermediate structures of different geometries. One can then infer which fusion mechanism is the best explanation of observed behavior in different systems by selecting the mechanism that requires the least energy. The treatment involves no adjustable parameters. It includes contributions to the intermediate energy resulting from the presence of hydrophobic interstices within structures formed between apposed bilayers. Results of these calculations show that a modified form of the stalk mechanism proposed by others is a likely fusion mechanism in a wide range of lipid compositions, but a mechanism based on inverted micellar intermediates (IMIs) is not. This should be true even in the vicinity of the lamellar/inverted hexagonal phase transition, where IMI formation would be most facile. Another prediction of the calculations is that traces of apolar lipids (e.g., long-chain alkanes) in membranes should have a substantial influence on fusion rates in general. The same theoretical methods can be used to generate and refine mechanisms for protein-mediated fusion.

Lipid vesicle fusion induced by phospholipase C activity in model bile.

Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. Vesicle growth over 2 h could be detected with phospholipase activity as little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been generated as a result of the initial phospholipase C treatment. The presence of fusion intermediates was confirmed using transmission electron microscopy. In addition, kinetically slow vesicle fusion with intravesicle content mixing and minimal leakage was also confirmed by fluorescence spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50 mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was obtained at 30 min at 25 degrees C with phospholipase C activity. This level of enzyme activity approximates that found in human gallbladder bile (1.2 nmol/min per ml). We conclude that the hydrolysis products of phospholipase C activity can, in very small amounts (3-7 mol% diacylglycerol), lead to destabilization and fusion of cholesterol-saturated biliary vesicles. A reappraisal of the importance of phospholipase C hydrolysis products in the pathogenesis of cholesterol gallstones is warranted based on these observations.

Intermediates and kinetics of membrane fusion

Recently, it has become clear that the influenza virus fusion protein, hemagglutinin (HA), produces membrane destabilization and fusion by a multistep process, which involves the aggregation of the HAs to form a fusion site. While the details of this process are under debate, it is important to recognize that proposing any sequence of "microscopic" fusion intermediates encumbers general "macroscopic" kinetic consequences, i.e., with respect to membrane mixing rates. Using a kinetic scheme which incorporates the essential elements of several recently proposed models, some of these measurable properties have been elucidated. First, a rigorous mathematical relationship between fusion intermediates and the fusion event itself is defined. Second, it is shown that what is measured as the macroscopic "fusion rate constant" is a simple function of all of the rate constants governing the transitions between intermediates, whether or not one of the microscopic steps is rate limiting. Third, while this kinetic scheme predicts a delay (or lag) time for fusion, as has been observed, it will be very difficult to extract reliable microscopic information from these data. Furthermore, it is predicted that the delay time can depend upon HA surface density even when the HA aggregation step is very rapid compared with fusion, i.e., the delay time need not be due to HA aggregation. Fourth, the inactivation process observed for influenza virions at low pH can be described within this kinetic scheme simply, yet rigorously, via the loss of the fusion intermediates. Fifth, predicted Arrhenius plots of fusion rates can be linear for this multistep scheme, even though there is no single rate-determining step and even when a branched step is introduced, i.e., where one pathway predominates at low temperature and the other pathway predominates at high temperature. Furthermore, the apparent activation energies obtained from these plots bear little or no quantitative resemblance to the microscopic activation energies used to simulate the data. Overall, these results clearly show that the intermediates of protein mediated fusion can be studied only by using assays sensitive to the formation of each proposed intermediate.

Inhibition of sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine ( N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R 18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy- l-Phe- l-Phe (Z- l-Phe- l-Phe), Z- l-Phe, Z-D-Phe, and Z-GIy- l-Phe- l-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z- d-Phe- l-PheGly and Z-GIy- l-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31 P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z- d-Phe- l-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-GIy- l-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures. These data taken collectively suggest that the peptides that inhibit the fusion of enveloped viruses could act, at least in part, by interfering with the formation of intermediates in membrane fusion (possibly punctate membrane defects induced by the virus) that force lipids into structures with small radii of curvature.

An architecture for the fusion site of Influenza hemagglutinin

The recent finding that more than one Influenza hemagglutinin (HA) is required at the fusion site for HA-expressing fibroblasts [1], together with the crystal structure of HA at neutral pH [2], provide the basic elements of a plausible model for this fusion site. Within an aggregate of HA trimers at low pH, we propose fusion intermediates which are based upon a minimal alteration to the known neutral pH structure of HA and which should have reasonable activation energies. This is the first model of a glycoprotein-mediated fusion site which explicitly accounts for the disposition of the lipids within these intermediates. While the fusion site created by HA will not be the same as that of eukaryotic fusion complexes [3], general characteristics could be shared.

Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.

Intermediary structures during membrane fusion as observed by cryo-electron microscopy

Lipidic phases, containing ‘lipidic particles’ (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcholine and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca 2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed ‘directly’ by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles.

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantified from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.

Membrane fusion: Kinetics and mechanisms

Membrane fusion is an essential process of biological cell function and is presumably mediated by specific membrane proteins. Nevertheless, the final step is the rearrangement of the molecular packing of the lipid molecules in the two apposed membranes to form a single, unified membrane. Recent work on the fusion of phospholipid vesicles, or liposomes, has provided both molecular descriptions of their fusion mechanisms and, of equal importance, a methodology for obtaining the same depth of knowledge for biological membranes. In this review, we describe the current status of liposomal fusion mechanisms, the effect of cellular cytoplasmic agents (proteins, polypeptides and polyamines) on their fusion and (as the best understood biological system) the fusion of viruses with membranes. To the extent possible, we describe both the aggregation kinetics (where the membranes overcome the physical forces opposing their close approach, e.g., electrostatic and hydration) and the fusion kinetics (where the destabilization of the apposed membranes can lead to either fusion or lysis). We also discuss the thermodynamic phases of lipids which sometimes govern the interaction between two liposomes. For phospholipid vesicles we have been able to identify two canonical fusion mechanisms and examples of both mechanisms where the intermembrane intermediates which cause fusion appear to be sufficiently long lived to be examined structurally. We believe that accounting for both the kinetic stages of fusion (i.e., the rate limiting step) and the structural analysis of fusion intermediates will be crucial for elucidating the molecular mechanisms of biological membrane fusion.

DEVELOPMENT AND EVALUATION OF A FREEZE‐ETCH, FREEZE‐FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM

A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze‐etch, freeze‐fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen‐cooled Freon‐22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze‐fracture was carried out under standard conditions. Evaluation of replicas from glycerol‐imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol‐treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol‐treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.

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 Ca 2+-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 Ca 2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca 2+ 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.

Molecular and electronic structure of a mixed-spin diiron "wedged" metallacarborane, (Me2C2B4H4)2[FeII(low-spin)][FeII(high-spin)]L2 [L2 = (THF)2 or (MeO)2C2H4], an oxidative fusion intermediate

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMolecular and electronic structure of a mixed-spin diiron "wedged" metallacarborane, (Me2C2B4H4)2[FeII(low-spin)][FeII(high-spin)]L2 [L2 = (THF)2 or (MeO)2C2H4], an oxidative fusion intermediateRussell N. Grimes, Richard B. Maynard, Ekk Sinn, Greg A. Brewer, and Gary J. LongCite this: J. Am. Chem. Soc. 1982, 104, 22, 5987–5992Publication Date (Print):November 1, 1982Publication History Published online1 May 2002Published inissue 1 November 1982https://doi.org/10.1021/ja00386a026RIGHTS & PERMISSIONSArticle Views73Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (717 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Ion specificity in fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles

Fusion of phosphatidylcholine-phosphatidic acid mixed lipid vesicles has been studied under the influence of the divalent Ca 2+, Mg 2+, Cd 2+, and Ba 2+, which range in size from much smaller to much larger, than Ca 2+. Fusion has also been studied under the influence of Eu 3+, which has a similar radius but different charge. The effects of these ions are reflected in the different degrees of fusion determined by changes in vesicle size, and in the varying fusion rates monitored by 1H-NMR spectroscopy. Cd 2+, which has an ionic radius similar to Ca 2+, exhibits the same effect on fusion as Ca 2+, while other ions show lower efficiencies. This suggests that the vesicle fusion intermediate has a geometry ideally suited to the binding of Ca 2+ or Cd 2+.