Direct observation of lipid domains in free standing bilayers: from simple to complex lipid mixtures

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this short presentation, I will address the most relevant findings reported from our laboratory, regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylamino-naphthalene (LAURDAN) under the two-photon excitation fluorescence microscopy will be particularly addressed, especially, the possibility to obtain information about the phase-state of different lipid domains directly from the fluorescent images.     

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.     

Membrane composition of jetted lipid vesicles: a Raman spectroscopy study

Microfluidic jetting is a promising method to produce giant unilamellar phospholipid vesicles for mimicking living cells in biomedical studies. We have investigated the chemical composition of membranes of vesicles prepared using this approach by means of Raman scattering spectroscopy. The membranes of all jetted vesicles are found to contain residuals of the organic solvent decane used in the preparation of the initial planar membrane. The decane inclusions are randomly distributed over the vesicle surface area and vary in thickness from a few to several tens of nanometers. Our findings point out that the membrane properties of jetted vesicles may differ considerably from those of vesicles prepared by other methods and from those of living cells. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide,asialo-GM1 and GM1 with POPC

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC₁₈ show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide > asialo-GM1 > GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.     

Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles.

Using the sectioning effect of the two-photon fluorescence microscope, we studied the behavior of phospholipid giant unilamellar vesicles (GUVs) composed of pure diacylphosphatidylcholine phospholipids during the gel-to-liquid crystalline phase transition. We used the well-characterized excitation generalized polarization function (GP(ex)) of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN), which is sensitive to the changes in water content in the lipid vesicles, to monitor the phase transition in the GUVs. Even though the vesicles do not show temperature hysteresis at the main phase transition, we observed different behaviors of the vesicle shape, depending on how the GUV sample reaches the main phase transition. During the cooling cycles, we observed an increase in the vesicle diameter at the phase transition ( approximately 0.5-1%), followed by a decrease in the diameter when the vesicle reached the gel phase. During the heating cycles and close to the phase transition temperature, a surprising behavior is observed, showing a sequence of different vesicle shapes as follows: spherical-polygonal-ellipsoidal. We attribute these changes to the effect of lipid domain coexistence on the macroscopic structure of the GUVs. The "shape hysteresis" in the GUVs is reversible and largely independent of the temperature scan rate. In the presence of 30 mol% of cholesterol the events observed at the phase transition in the GUVs formed by pure phospholipids were absent.     

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.     

Simultaneous monitoring of electroformation of phospholipid vesicles by quartz crystal microbalance and optical microscopy

The electroformation of giant vesicles from 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) and optical microscopy, simultaneously using a novel sample cell design. A gold-coated QCM crystal was used as one of the electrodes and an Indium–tin-oxide (ITO)-coated glass slide was used as the second electrode for electroformation. Increases in the frequency and decreases in the dissipation were observed immediately upon voltage application between the two electrodes, indicating the loss of lipid from the QCM surface. Concurrently, we observed vesicles on the QCM electrode surface by differential interference contrast (DIC)-optical microscopy. The lipid-coated substrates were measured with AFM at various stages in the electroformation, and a significant change in the morphology of the lipid film was observed. Ellipsometry was used to find the average thickness of lipid film. The QCM data were fitted to a viscoelastic model to determine the viscoelastic properties and time dependence of the film thickness. All methods used to determine film thickness give values in reasonable quantitative agreement. Differences between the methods are consistent with what one might expect due to what is actually measured in the individual techniques. The comparison between mass loss and observed vesicles suggest that the vesicles formed are first localized to the substrate and then slowly released into the solution. By comparing the mass lost from the lipid film, to the total surface area of lipid vesicles observed, it is apparent that only a relatively small fraction of the lipid goes into the production of unilamellar vesicles with sizes detectable with optical microscopy.     

Hierarchical Unilamellar Vesicles of Controlled Compositional Heterogeneity

Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles) that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.     

Electron microscopic investigation of the morphology and calcium-induced fusion of lipid vesicles with an oligomerised inner leaflet

The lipid head groups in the inner leaflet of unilamellar bilayer vesicles of the synthetic lipids DHPBNS and DDPBNS can be selectively oligomerised. Earlier studies have established that these vesicles fuse much slower and less extensively upon oligomerisation of the lipid head groups. The morphology and calcium-induced fusion of vesicles of DHPBNS and DDPBNS were investigated using cryo-electron microscopy. DHPBNS vesicles are not spherical but flattened, ellipsoidal structures. Upon addition of CaCl(2), DHPBNS vesicles with an oligomerised inner leaflet were occasionally observed in an arrested hemifused state. However, the evidence for hemifusion is not equivocal due to potential artefacts of sample preparation. DDPBNS vesicles show the expected spherical morphology. Upon addition of excess CaCl(2), DDPBNS vesicles fuse into dense aggregates that show a regular spacing corresponding to the bilayer width. Upon addition of EDTA, the aggregates readily disperse into large unilamellar vesicles. At low concentration of calcium ion, DDPBNS vesicles with an oligomerised inner leaflet form small multilamellar aggregates, in which a spacing corresponding to the bilayer width appears. Addition of excess EDTA results in slow dispersal of the Ca2+-lipid aggregates into a heterogeneous mixture of bilamellar, spherical vesicles and networks of thread-like vesicles. These lipid bilayer rearrangements are discussed within the context of shape transformations and fusion of lipid membranes.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms

We describe the interaction of various phospholipases A₂ (PLA₂) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA₂s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA₂ with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA₂, S49 PLA₂ and K49 PLA₂) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA₂ compared with D49 PLA₂, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.     

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: a comparative study of the affinity constants and conformational changes

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP(2)-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP(2)-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.     

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.     

Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.     

Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV)

Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a number of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a functional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imaging, we find that vesicles prepared significantly above and below the melting temperature T(m) have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid-ordered (L(o)) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (L(o)) and (L(d)) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organization within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures.     

Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683-1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.     

Accelerated interleaflet transport of phosphatidylcholine molecules in membranes under deformation.

Biological membranes are lamellar structures composed of two leaflets capable of supporting different mechanical stresses. Stress differences between leaflets were generated during micromechanical experiments in which long thin tubes of lipid (tethers) were formed from the surfaces of giant phospholipid vesicles. A recent dynamic analysis of this experiment predicts the relaxation of local differences in leaflet stress by lateral slip between the leaflets. Differential stress may also relax by interleaflet transport of lipid molecules ("flip-flop"). In this report, we extend the former analysis to include interleaflet lipid transport. We show that transmembrane lipid flux will evidence itself as a linear increase in tether length with time after a step reduction in membrane tension. Multiple measurements were performed on 24 different vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 3% dinitrophenol-linked di-oleoyl-phosphatidylethanolamine. These tethers all exhibited a linear phase of growth with a mean value of the rate of interlayer permeation, cp = 0.009 s-1. This corresponds to a half-time of approximately 8 min for mechanically driven interleaflet transport. This value is found to be consistent with longer times obtained for chemically driven transport if the lipids cross the membrane via transient, localized defects in the bilayer.