Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates.

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell-surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell-cell contacts, suggests that such multimeric complexes represent a fusion machine.

Synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines.

At the time of fusion, membranes are packed with fusogenic proteins. Do adjacent individual proteins interact with each other in the plane of the membrane? Or does each of these proteins serve as an independent fusion machine? Here we report that the low pH-triggered transition between the initial and final conformations of a prototype fusogenic protein, influenza hemagglutinin (HA), involves a preserved interaction between individual HAs. Although the HAs of subtypes H3 and H2 show notably different degrees of activation, for both, the percentage of low pH-activated HA increased with higher surface density of HA, indicating positive cooperativity. We propose that a concerted activation of HAs, together with the resultant synchronized release of their conformational energy, is an example of a general strategy of coordination in biological design, crucial for the functioning of multiprotein fusion machines.

An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids.

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus-cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell-cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH-induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone-shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.

The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation.

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Bending membranes to the task: structural intermediates in bilayer fusion.

Merger of lipid bilayers plays a central role in diverse biological fusion reactions. Recent studies suggest that different membrane fusion systems, including fusion of purely lipid bilayers, involve formation of similar stalk-type intermediates--highly bent (net negative curvature) and transient lipidic connections between fusing membranes.

Reversible merger of membranes at the early stage of influenza hemagglutinin-mediated fusion.

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10-20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37 degrees C distinguish the newly identified fusion intermediate from the intermediate arrested at 4 degrees C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.

Membrane rearrangements in fusion mediated by viral proteins.

Diverse enveloped viruses enter host cells by fusing their envelopes with cell membranes. The mechanisms of merger of lipid bilayers of two membranes mediated by influenza hemagglutinin and other viral fusion proteins apparently involve local lipidic connections that evolve into a bilayer septum in which a pore forms and expands.

Reversible large-scale deformations in the membranes of electrically-treated cells: electroinduced bleb formation.

Morphological changes in electrically-treated cells have been investigated by light and scanning electron microscopy. The application of 100-microseconds rectangular pulses of 1.3 kV/cm electric field to different types of cells (FBT, MEF, RAT-1, L-cells) in the physiological medium leads to the formation and growth of spherical and hemispherical protuberances of the cell membrane. The formation of such electroinduced blebs is not associated with the cells' death and is reversible. The electroinduced blebs are mainly formed at those sites of the cell membrane which are subjected to the highest voltage during the electric pulses. Increasing the tonicity of the medium by introducing 20 mM of inulin prevents the bleb formation, indicating the osmotically-dependent nature of the processes involved. When electric pulses are applied to the cells pre-treated with cytochalasin B, the formation of electroinduced blebs occurs independently from cytochalasin-induced ones originally present on such cells. Speculations are presented concerning the nature of the membrane structural changes underlying the electroinduced blebbing and their possible role in some electrically-induced processes.

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.

Electrostimulated uptake of DNA by liposomes.

High molecular mass DNA was efficiently taken up by large unilamellar vesicles exposed to a short pulse of electric field (0.1-1 ms) with an intensity as high as 12.5 kV/cm. The efficiency of uptake increased significantly in presence of Mg2+ ions and was approximately 0.6 and 1.5 micrograms of DNA per mumol of lipid for T7 DNA and plasmid pBR 322, respectively. The results presented indicate that DNA was taken up as a result of the electrostimulated formation of endosome-like vesicles rather than via field-induced membrane pores.

Local deformation of human red blood cells in high frequency electric field.

A method of local and general deformation of single erythrocytes by external forces in high-frequency electric field is described. The method allows the avoidance of any mechanical contact of the cell with electrodes. Under the action of the forces applied human erythrocytes change their shape and produce various membrane structures: long filopodia-like processes, retraction fibers and lamella-like structures. These structures are never formed by erythrocytes under normal conditions, but are typical for fibroblasts, macrophages and epithelium cells. By the method developed the elastic properties of spicules on the membranes of echinocytes were also studied. Deformation of echinocyte in high-frequency electric field leads to the smoothing out of spicules. However, after the electric field is turned off, echinocyte restores its initial forms including the number and localization of all initial spicules on the cell surface.

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores.

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies.

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.

Self-assembly of influenza hemagglutinin: studies of ectodomain aggregation by in situ atomic force microscopy.

We have used in situ tapping mode atomic force microscopy (AFM) to study the structural morphology of two fragments of the influenza hemagglutinin protein bound to supported bilayers. The two proteins that we studied are the bromelain-cleaved hemagglutinin (BHA), corresponding to the full ectodomain of the hemagglutinin protein, and FHA2, the 127 amino acid N-terminal fragment of the HA2 subunit of the hemagglutinin protein. While BHA is water soluble at neutral pH and is known to bind to membranes via specific interactions with a viral receptor, FHA2 can only be solubilized in water with an appropriate detergent. Furthermore, FHA2 is known to readily bind to membranes at neutral pH in the absence of a receptor. Our in situ AFM studies demonstrated that, when bound to supported bilayers at neutral pH, both these proteins are self-assembled as single trimeric molecules. In situ acidification resulted in further lateral association of the FHA2 without a large perturbation of the bilayer. In contrast, BHA remained largely unaffected by acidification, except in areas of exposed mica where it is aggregated. Remarkably, these results are consistent with previous observations that FHA2 promotes membrane fusion while BHA only induces liposome leakage at low pH. The results presented here are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time self-assembly of a membrane fusion protein.

Electro-stimulated transformation of E. coli cells pretreated by EDTA solution.

The phenomenon of transformation of E. coli cells under electric treatment has been studied. The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension. The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA. The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.

Lysolipids reversibly inhibit Ca(2+)-, GTP- and pH-dependent fusion of biological membranes.

Membrane fusion in exocytosis, intracellular trafficking, and enveloped viral infection is thought to be mediated by specialized proteins acting to merge membrane lipid bilayers. We now show that one class of naturally-occurring phospholipids, lysolipids, inhibits fusion between cell membranes, organelles, and between organelles and plasma membrane. Inhibition was reversible, did not correlate with lysis, and could be attributed to the molecular shape of lysolipids rather than to any specific chemical moiety. Fusion was arrested at a stage preceding fusion pore formation. Our results are consistent with the hypothesis that biological fusion, irrespective of trigger, involves the formation of a highly bent intermediate between membranes, the fusion stalk.

[The effect of small concentrations of antibiotics blocking the synthesis of bacterial cell wall on the permeability of Escherichia coli cell wall for plasmid DNA]. / Vliianie malykh kontsentratsii antibiotikov, narushaiushchikh sintez kletochnoi stenki bakterii, na pronitsaemost' kletochnoi stenki Escherichia coli dlia plazmidnykh DNK.

Short treatment of Escherichia coli cells with antibiotics disturbing synthesis of bacterial cell wall in small concentrations renders the cells capable of absorbing foreign plasmid DNA. A novel express-method for transformation of E. coli cells by plasmid DNA has been developed on the basis of the results obtained. The whole procedure can be performed at room temperature. Depending on cell strain and the plasmid size, the efficiency of transformation can vary from 1.10(4) to 5.10(5) transformants per 1 mkg of DNA. The method suggested improves significantly the every-day work aimed at constructing plasmids.

[Cation selectivity of a BLM in a state of "stress"]. / Kationnaia selektivnost' BLM v "stressovom" sostoianii.

In asymmetric conditions (by electrolyte concentration) cyclic voltage current curves of BLM were measured in a stress state initiated after a short -- time effect of a strong electric field. BLM from negatively charged lipids were shown to exhibit notable cation -- anion selectivity under these conditions. The result obtained agrees with the assumption to the effect that stress and preclamping fluctuations are related to the formation of through pores the inner face of which is lined with the polar heads of lipids.

[Reversible electrical breakdown of cholesterol-containing lipid bilayer membranes modified with holothurin A]. / Obratimyi élektricheskii proboi kholesterinsoderzhashchikh bisloinykh lipidnykh membran, modifitsirovannykh goloturinom A.

Reversible electrical breakdown of cholesterol-containing BLM modified with holothurin A, i. e. a significant reversible increase of membrane conductivity under the effect of the electrical field was described. So when the voltage approximately 0.3 V was applied to the BLM for 10 ms its conductivity was reversibly increased by 4 orders as compared to the initial one. Reproducibility of current oscillograms, non-linear current breakdown--potential relationship and non-linear spasmodic decrease of conductivity at step-like reduction of the potential on the membrane show similarity of this phenomenon with the reversible breakdown of the membranes of oxidized cholesterol and of asolectin BLM in the presence of UO22+ ions. The mechanism of reversible electrical breakdown of BLM is discussed in terms of the development of a great number of local conductive defects in the membrane under the effect of the electrical field.

[Liposome fusion with planar lipid membranes]. / Sliianie liposom s ploskoi lipidnoi membranoi.

Potentiodynamic method was used for investigating bilayer lipid membrane (BLM)- liposomes (LS) interaction. BLM was formed from egg lecithin and its mixture with phosphatidylserine (PS); LS-from PS. It is shown that in the presence of calcium in the aqueous phase (1-10 mM) and charged phospholipids in both LS and BLM, fusion of LS with BLM is observed. The fusion event was registered by measuring the increase of negative charge density on BLM surface opposite to that facing the LS.