A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.     

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 μm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. “Short” DNAs (oligonucleotide 21b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (C _endo) of d-erythro-sphingosine (Sph⁺) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below C _endo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. C _endo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph⁺ GUVs and about 20 mol% for SOPC/Sph⁺ or eggPC/Sph⁺ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes. Received: 18 April 1998 / Revised version: 6 August 1998 / Accepted: 7 August 1998     

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.     

Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.     

Bakterielle Membranvesikel

Bacterial membrane vesicles Cells of all three domains of our life (eukaryotes, bacteria and archaea) produce and segregate membrane vesicles surrounded by a lipid double membrane. Most of them are spherical with a diameter of 20–500 nm and can contain in their interior, the lumen, different types of molecules called cargo. In most cases they contain different proteins, polysaccharides and metabolites and sometimes nucleic acids (DNA, RNA) as well as misfolded proteins. Membrane vesicles play an important role in the horizontal gene transfer and in pathogenesis. Furthermore, it has been shown quite recently that membrane vesicles can transfer phage receptors to phage resistant cells and even closely related species. Worldwide several companies investigate their application as vaccines. In addition, investigations are going on to find out whether membrane vesicles can be used in genomic engineering.     

Giant Unilamellar Vesicles Formed by Hybrid Films of Agarose and Lipids Display Altered Mechanical Properties

Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.     

Interactions of DNA with giant liposomes

DNA interactions with the bilayers of cationic liposomes were studied using a novel model experiment: DNAs were locally injected by a micropipette to a part of a giant unilamellar vesicle. The resulting phenomena were directly observed in optical microscope. Giant unilamellar vesicles (GUVs), about 100 microm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. The effects of DNAs of different length were tested: (i) 'short' DNAs-oligonucleotide 21b, and calf thymus 250 bp; (ii) 'long' DNAs-plasmid DNAs in super coil or liner form (between 2.7 and 8.0 kbp). DNAs were injected native, as well as marked with the fluorescent dye Hoechst. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis was observed due to the DNA/lipid membrane local interactions for all DNAs tested. Some of the DNA in the formed complex was associated with the induced endosomes, and some of it remained spread over the 'mother' GUV membrane for all DNAs tested, except for the longest one--the linear plasmid of 8 kbp. The last remained at the 'mother' GUV membrane and was not transported with the induced endosomes to the internal GUV space. Possible mechanisms for DNA/lipid membrane interaction were suggested. One of them involves DNA encapsulation within an inverted micelle included in the lipid membrane. The model observations could help in understanding events associated with interaction of DNA with biological membranes, as well as cationic liposomes/DNA complexes formation in gene transfer processes.     

Giant Unilamellar Vesicles Formed by Hybrid Films of Agarose and Lipids Display Altered Mechanical Properties

Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

Single-molecule manipulation of macromolecules on GUV or SUV membranes using optical tweezers

Despite their wide applications in soluble macromolecules, optical tweezers have rarely been used to characterize the dynamics of membrane proteins, mainly due to the lack of model membranes compatible with optical trapping. Here, we examined optical trapping and mechanical properties of two potential model membranes, giant and small unilamellar vesicles (GUVs and SUVs, respectively) for studies of membrane protein dynamics. We found that optical tweezers can stably trap GUVs containing iodixanol with controlled membrane tension. The trapped GUVs with high membrane tension can serve as a force sensor to accurately detect reversible folding of a DNA hairpin or membrane binding of synaptotagmin-1 C2AB domain attached to the GUV. We also observed that SUVs are rigid enough to resist large pulling forces and are suitable for detecting protein conformational changes induced by force. Our methodologies may facilitate single-molecule manipulation studies of membrane proteins using optical tweezers.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

Biological and molecular characteristics of the premalignant mouse mammary gland

We tested the ability of saturated n-monocarboxylic acids ranging from eight to 12 carbons in length to self-assemble into vesicles, and determined the minimal concentrations and chain lengths necessary to form stable bilayer membranes. Under defined conditions of pH and concentrations exceeding 150 mM, an unbranched monocarboxylic acid as short as eight carbons in length (n-octanoic acid) assembled into vesicular structures. Nonanoic acid (85 mM) formed stable vesicles at pH 7.0, the pK of the acid in bilayers, and was chosen for further testing. At pH 6 and below, the vesicles were unstable and the acid was present as droplets. At pH ranges of 8 and above clear solutions of micelles formed. However, addition of small amounts of an alcohol (nonanol) markedly stabilized the bilayers, and vesicles were present at significantly lower concentrations (approximately 20 mM) at pH ranges up to 11. The formation of vesicles near the pK(a) of the acids can be explained by the formation of stable RCOO(-)...HOOCR hydrogen bond networks in the presence of both ionized and neutral acid functions. Similarly, the effects of alcohols at high pH suggests the formation of stable RCOO(-)...HOR hydrogen bond networks when neutral RCOOH groups are absent. The vesicles provided a selective permeability barrier, as indicated by osmotic activity and ionic dye capture, and could encapsulate macromolecules such as DNA and a protein. When catalase was encapsulated in vesicles of decanoic acid and decanol, the enzyme was protected from degradation by protease, and could act as a catalyst for its substrate, hydrogen peroxide, which readily diffused across the membrane. We conclude that membranous vesicles produced by mixed short chain monocarboxylic acids and alcohols are useful models for testing the limits of stabilizing hydrophobic effects in membranes and for prebiotic membrane formation.     

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.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Freezing of nonwoody plant tissues: IV. Nucleation sites for freezing and refreezing of onion bulb epidermal cells

A freezing microscope and videotape recorder were used to study ice formation in onion bulb epidermal cells during repeated freezing. Behavior during refreezing after freezing and thawing indicated that the barrier to ice crystal propagation previously reported in living cells was not damaged by freezing. Refreezing in many cells began at a site other than that responsible for the initial freezing.     

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes to form protocellular structures under prebiotic conditions.     

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.