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.     

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures

Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered–liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.     

Preparation of unilamellar lipid vesicles at 37°C by vaporization methods

We have developed a method utilizing low boiling solvents to prepare large, unilamellar vesicles at physiologic temperatures. Solutions of ethyl methyl ether or dichlorofluoromethane (Freon-21) at 4°C containing solubilized lipids were injected into a column of a aqueous buffer at 37°C. Vesicles prepared in this manner have been examined by freeze-fracture, negative stain electron microscopy, and fluorescence microscopy. The principal advantages of this technique are: (1) heat labile substances may be more readily entrapped in the internal vesicle volume without thermal denaturation, and (2) the range of lipids which are soluble in dichlorofluoromethane is greater than that of many other solvents, e.g. diethyl ether.     

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a F?rster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.     

Extrusion of electroformed giant unilamellar vesicles through track-etched membranes

Unilamellar vesicle populations having a narrow size distribution and mean radius below 100 nm are preferred for drug delivery applications. In the present work, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used to prepare giant unilamellar vesicles (GUVs) by electroformation and multilamellar vesicles (MLVs) by thin film hydration. Our experiments show that in contrast to MLVs, a single-pass extrusion of GUVs through track-etched polycarbonate membranes at moderate pressure differences is sufficient to produce small liposomes having low polydispersity index. Moreover, we observe that the drug encapsulating potential of extruded liposomes obtained from GUVs is significantly higher compared to liposomes prepared by extrusion of MLVs. Furthermore, our experiments carried out for varying membrane pore diameters and extrusion pressures suggest that the size of extruded liposomes is a function of the velocity of GUV suspensions in the membrane pore.     

A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10–30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.     

Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures

Images of giant unilamellar vesicles (GUVs) formed by different phospholipid mixtures (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1, 2-dilauroyl-sn-glycero-3-phosphocholine (DPPC/DLPC) 1:1 (mol/mol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE/DPPC), 7:3 and 3:7 (mol/mol) at different temperatures were obtained by exploiting the sectioning capability of a two-photon excitation fluorescence microscope. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN), 6-propionyl-2-dimethylamino-naphthalene (PRODAN), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE) were used as fluorescent probes to reveal domain coexistence in the GUVs. We report the first characterization of the morphology of lipid domains in unsupported lipid bilayers. From the LAURDAN intensity images the excitation generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domain. On the basis of the phase diagram of each lipid mixture, we found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region in all lipid mixtures. At temperatures corresponding to the phase coexistence region we observed lipid domains of different sizes and shapes, depending on the lipid sample composition. In the case of GUVs formed by DPPE/DPPC mixture, the gel DPPE domains present different shapes, such as hexagonal, rhombic, six-cornered star, dumbbell, or dendritic. At the phase coexistence region, the gel DPPE domains are moving and growing as the temperature decreases. Separated domains remain in the GUVs at temperatures corresponding to the solid region, showing solid-solid immiscibility. A different morphology was found in GUVs composed of DLPC/DPPC 1:1 (mol/mol) mixtures. At temperatures corresponding to the phase coexistence, we observed the gel domains as line defects in the GUV surface. These lines move and become thicker as the temperature decreases. As judged by the LAURDAN GP histogram, we concluded that the lipid phase characteristics at the phase coexistence region are different between the DPPE/DPPC and DLPC/DPPC mixtures. In the DPPE/DPPC mixture the coexistence is between pure gel and pure liquid domains, while in the DLPC/DPPC 1:1 (mol/mol) mixture we observed a strong influence of one phase on the other. In all cases the domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This observation is also novel for unsupported lipid bilayers.     

Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles

Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid-lipid and lipid-protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains.     

Lipid lateral organization on giant unilamellar vesicles containing lipopolysaccharides

We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ∼15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (∼380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Coexisting stripe- and patch-shaped domains in giant unilamellar vesicles

We report a new type of gel-liquid phase segregation in giant unilamellar vesicles (GUVs) of mixed lipids. Coexisting patch- and stripe-shaped gel domains in GUV bilayers composed of DOPC/DPPC or DLPC/DPPC are observed by confocal fluorescence microscopy. The lipids in stripe domains are shown to be tilted according to the DiIC18 fluorescence intensity dependence on the excitation polarization. The patch domains are found to be mainly composed of DPPC-d62 according to the coherent anti-Stokes Raman scattering (CARS) images of DOPC/DPPC-d62 bilayers. When cooling GUVs from above the miscibility temperature, the patch domains start to appear between the chain melting and the pretransition temperature of DPPC. In GUVs containing a high molar percentage of DPPC, the stripe domains form below the pretransition temperature. Our observations suggest that the patch and stripe domains are in the Pbeta' and Lbeta' gel phases, respectively. According to the thermoelastic properties of GUVs described by Needham and Evans [(1988) Biochemistry 27, 8261-8269], the Pbeta' and Lbeta' phases are formed at relatively low and high membrane tensions, respectively. GUVs with high DPPC percentage have high membrane surface tension and thus mainly exhibit Lbeta' domains, while GUVs with low DPPC percentage have low membrane surface tension and form Pbeta' domains accordingly. Adding negatively charged lipid to the lipid mixtures or applying an osmotic pressure to GUVs using sucrose solutions releases the surface tension and leads to the disappearance of the Lbeta' gel phase. The relationship between the observed domains in free-standing GUV bilayers and those in supported bilayers is discussed.     

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.     

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

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     

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.     

The lateral diffusion of selectively aggregated peptides in giant unilamellar vesicles

We have systematically investigated the effect of aggregation of a transmembrane peptide on its diffusion in dimyristoylphosphatidylcholine and in palmitoyloleoylphosphatidylcholine model membranes. The hydrophobic segment of the b subunit from E. coli F(1)F(0)-ATP synthase was modified with a histidine tag at the carbonyl terminus and was aggregated selectively by using a series of multivalent, dendritic chelating agents with nitrilotriacetic acid functional groups. Peptide complexes ranging from monomers to hexamers were formed and studied in giant unilamellar vesicles. The rate of diffusion for the transmembrane peptide complexes were found to depend on the size of the complex. The results agree with predictions from the free area model for monomers and dimers, and the hydrodynamic continuum model for tetramers, pentamers, and hexamers. Comparisons with diffusion of lipids confirm that the diffusion of a transmembrane peptide is enhanced by coupling of density fluctuations between the two monolayers.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.