Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.     

Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density

We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent of inner membrane folding. The data reveal that neither the extent of mitochondrial matrix density nor the complexity of the inner membrane folding have a significant effect on the mobility of inner membrane redox components. Diffusion coefficients for Complex I (NADH:ubiquinone oxidoreductase), Complex III (ubiquinol: cytochrome c oxidoreductase), Complex IV (cytochrome oxidase), ubiquinone, and phospholipid were found to be effectively invariant with the matrix density and/or membrane folding and essentially the same as values we reported previously for spherical, fused, ultralarge, matrix-free, inner membranes. Diffusion of proton-transporting Complex V (ATP synthase) appeared to be 2-3-fold slower at the greatest matrix density and degree of membrane folding. Consistent with a diffusion-coupled mechanism of electron transport, comparison of electron transport frequencies (productive collisions) with the theoretical, diffusion-controlled, collision frequencies (maximum collisions possible) revealed that there were consistently more calculated than productive collisions for all redox partners. Theoretical analyses of parameters for submicron fluorescence recovery after photobleaching measurements in intact mitoplasts support the finding of highly mobile redox components diffusing at the same rates as determined in conventional fluorescence recovery after photobleaching measurements in fused, ultralarge inner membranes. These findings support the Random Collision Model of Mitochondrial Electron Transport at the level of the intact mitoplast and suggest a similar conclusion for the intact mitochondrion.     

Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy

The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane.     

Nuclear fusion during yeast mating occurs by a three-step pathway

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in ~80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.     

Fluorescence measurements of fusion between human erythrocytes induced by poly(ethylene glycol).

The kinetics of poly(ethylene glycol) (PEG)-induced fusion between intact human erythrocytes was continuously monitored by a fluorescence lipid mixing method, utilizing the dequenching of the fluorescence probe, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl ] phosphatidylcholine (C12-NBD-PC). The steady-state fluorescence intensity was detected from the surface of cells in a monolayer on an alcian blue-coated glass coverslip. The relief of fluorescence self-quenching after fusion between C12-NBD-PC labeled and unlabeled intact erythrocytes was measured. The extent of fluorescence dequenching was normalized based on the measured concentration of probes in membranes, the projected partial dequenching due both to dilution by intercellular fusion, and the dilution between the inner and outer leaflets of membranes (flip-flop). There was no significant increase in fluorescence intensity during PEG treatment of 5 min, at 4 degrees C. Intensity increased immediately after the dilution of PEG, and reached saturation in 30 min. The efficiency of fusion increased with the increasing of PEG concentrations. Only 4% enhancement of saturated relative fluorescence intensity was detected in 25 wt% PEG-induced cell fusion; 23% enhancement in 30 wt%; and 66% enhancement in 35 wt%. The transfer of fluorescent probes between membrane bilayer leaflets (flip-flop) was also monitored during the fusion process. Flip-flop was monitored in confluent monolayers as well as in isolated cells. There was no significant spontaneous flip-flop within 30 min of dilution. The relative fluorescence intensity enhancement contributed by the dilution of probes between fused labeled and unlabeled cells (at a 1:1 ratio) was found to account for only 39% of the observed final dequenching, whereas the contribution by flip-flop associated with cell fusion was found to account for 9%, and flip-flop without fusion contributed approximately 18%. A portion of the flip-flop is a consequence of hemolysis. Therefore, fluorescence dequenching measurements of fusion of whole cells must be interpreted with caution.     

Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.     

Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases—OPA1 and the mitofusins Mfn1 and Mfn2—are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.     

Fusion of bacteriorhodopsin with submitochondrial particles yields a new system with retention of energy coupling and acquisition of photophosphorylation activity

Submitochondrial particles were fused with purple membranes of Halobacterium halobium cells by means of a freeze-thawe sonication procedure. It is reported that fusion of inner mitochondrial membranes with a bacterial membrane yields a new particle which shows not only retention of redox- and photon-linked energy-coupling activities, but also creation of an additional energy-coupling process, light-driven ATP synthesis.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

The mitochondrial inner membrane protein mitofilin controls cristae morphology

Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.     

Functional incorporation of beef-heart cytochrome c oxidase into membranes of Streptococcus cremoris

Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome-c-oxidase-mediated oxidation of the electron donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems. The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton-motive-force-dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton-motive-force-generating system.