Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.     

Permeation of styryl dyes through nanometer-scale pores in membranes

Styryl dyes are widely used to study synaptic vesicle (SV) recycling in neurons; vesicles are loaded with dye during endocytosis, and dye is subsequently released via exocytosis. During putative kiss-and-run exocytosis, efflux of dye from individual SVs has been proposed to occur via two sequential steps: dissociation from the membrane followed by permeation through a small fusion pore. To improve our understanding of the kinetics of efflux of dye from vesicles during kiss-and-run events, we examined the rates of efflux of different dyes through nanometer-scale pores formed in membranes by the toxins melittin and α-hemolysin; these pores approximate the size of fusion pores measured in neuroendocrine cells. We found that the axial diameter of each dye was a crucial determinant for permeation. Moreover, the two dyes with the largest cross-sectional areas were completely unable to pass through pores formed by a mutant α-hemolysin that has a slightly smaller pore than the wild-type toxin. The overall time constant for efflux (seconds) of each dye was orders of magnitude slower than the time constant for dissociation from membranes (milliseconds). Thus, the permeation step is rate-limiting, and this observation was further supported by atomistic molecular dynamics simulations. Together, the data reported here help provide a framework for interpreting dye destaining rates from secretory vesicles.     

Statistical Analysis of Peptide-Induced Graded and All-or-None Fluxes in?Giant Vesicles

Antimicrobial, cytolytic, and cell-penetrating peptides induce pores or perturbations in phospholipid membranes that result in fluxes of dyes into or out of lipid vesicles. Here we examine the fluxes induced by four of these membrane-active peptides in giant unilamellar vesicles. The type of flux is determined from the modality of the distributions of vesicles as a function of their dye content using the statistical Hartigan dip test. Graded and all-or-none fluxes correspond to unimodal and bimodal distributions, respectively. To understand how these distributions arise, we perform Monte Carlo simulations of peptide-induced dye flux into vesicles using a very simple model. The modality of the distributions depends on the rate constants of pore opening and closing, and dye flux. If the rate constants of pore opening and closing are both much smaller than that of dye flux through the pore, all-or-none influx occurs. However, if one of them, especially the rate constant for pore opening, increases significantly relative to the flux rate constant, the process becomes graded. In the experiments, we find that the flux type is the same in giant and large vesicles, for all peptides except one. But this one exception indicates that the flux type cannot be used to unambiguously predict the mechanism of membrane permeabilization by the peptides.     

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles

BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability.MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan.ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes.Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition.General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.     

Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles.

An externally applied electric field across vesicles leads to transient perforation of the membrane. The distribution and lifetime of these pores was examined using 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles using a standard fluorescent microscope. The vesicle membrane was stained with a fluorescent membrane dye, and upon field application, a single membrane pore as large as approximately 7 microm in diameter was observed at the vesicle membrane facing the negative electrode. At the anode-facing hemisphere, large and visible pores are seldom found, but formation of many small pores is implicated by the data. Analysis of pre- and post-field fluorescent vesicle images, as well as images from negatively stained electron micrographs, indicate that pore formation is associated with a partial loss of the phospholipid bilayer from the vesicle membrane. Up to approximately 14% of the membrane surface could be lost due to pore formation. Interestingly, despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres was approximately equal. Ca(2+) influx measurements into perforated vesicles further showed that pores are essentially resealed within approximately 165 ms after the pulse. The pore distribution found in this study is in line with an earlier hypothesis (E. Tekle, R. D. Astumian, and P. B. Chock, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11512--11516) of asymmetric pore distribution based on selective transport of various fluorescent markers across electroporated membranes.     

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).     

Cascades of transient pores in giant vesicles: line tension and transport

Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30-40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore's edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension,. We have studied the effect of the shape of inclusion molecules on. Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces. The effect of some detergents can be dramatic, reducing by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.     

Adventures in membrane protein topology. A study of the membrane-bound state of colicin E1.

The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other.     

YidC-driven membrane insertion of single fluorescent Pf3 coat proteins

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.     

Using giant unilamellar lipid vesicle micro-patterns as ultrasmall reaction containers to observe reversible ATP synthesis/hydrolysis of F0F1-ATPase directly

F(0)F(1)-ATPase within chromatophores, which was labeled with pH-sensitive quantum dots, was encapsulated in large unilamellar lipid vesicles (LUVs) through reverse-phase evaporation. Then a microarray of chromatophore-containing LUVs was created using a micro-contact printing (mu-CP) technique. Through controlled dehydration-rehydration of the lipid patterns, a microarray of single chromatophore-containing giant unilamellar lipid vesicles (GUVs) was formed with desired size and uniform shape. The reversible ATP synthesis/hydrolysis of F(0)F(1)-ATPase in GUVs was directly observed by fluorescence microscopy through the fluorescence intensity increase/decrease in the pH-sensitive quantum dots labeled on the outer surface of the chromatophore. To the best of our knowledge, this is the first direct observation of the reversible behavior of F(0)F(1)-ATPase at the bulk scale.     

Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.     

The dynamics of melittin-induced membrane permeability

The transport of co-encapsulated solutes through the melittin-induced pores in the membrane of giant phospholipid vesicles was studied, and the characteristics of the pore formation process were modeled. Molecules of two different sizes (dextran and the smaller, fluorescent marker Alexa Fluor) were encapsulated inside the vesicles. The chosen individual vesicles were then transferred by micromanipulation from the stock suspension to the environment with the melittin (MLT). The vesicles were observed optically with a phase-contrast microscope and by monitoring the fluorescence signal. Such an experimental setup enabled an analysis of a single vesicle’s response to the MLT on the basis of simultaneous, separate measurements of the outflow of both types of encapsulated molecules through the MLT-induced pores in the membrane. The mechanisms of the MLT’s action were suggested in a model for MLT pore formation, with oligomeric pores continuously assembling and dissociating in the membrane. Based on the model, the results of the experiments were explained as a consequence of the membrane’s permeability dynamics, with a continuously changing distribution of pores in the membrane with regard to their size and number. The relatively stable “average MLT pore” characteristics can be deduced from the proposed model.     

Fluorescence assay for phospholipid membrane asymmetry.

Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanol ami ne was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.     

GM1 asymmetry in the membrane stabilizes pores

Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant unilamellar vesicles with membranes doped with GM1, a ganglioside asymmetrically enriched in the outer leaflet of neuronal cell membranes. Compared with symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes.     

Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel

A critical event during spore germination is the release of Ca‐DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca‐DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E. coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence‐dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E. coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca‐DPA and amino acids during germination of the spore.