Light scattering study of phosphatidylcholine vesicles at the pretransition

Laser light scattering has been used to investigate the thermal pretransition of dipalmitoylglycerophosphocholine vesicles with variable radius as obtained by the mild sonication method. Intensity changes in 90° scattered light are observed at the pretransition for larger vesicles and actually increase with increasing vesicle size, reaching a constant value.This constant value is in good agreement with the value calculated from the refractive index data.The intensity ratio of scattered light at temperatures of 30°C and 40°C (I₄₀/I₃₀) approaches unity at a radius of small single-bilayer vesicle. This result is interpreted as no pretransition for small vesicles in agreement with the calorimetric results. An expression of the particle scattering factor is also presented for multilayered shells composed of anisotropic elements. It is shown numerically, using this expression, that changes in the lipid layer thickness and the tilting angles at the pretransition have no effects on the scattering factor. Therefore it is concluded that the intensity changes in scattered light reflect the changes in the refractive index of the vesicle originating in the polar head groups.     

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca²⁺-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca²⁺ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10^?6–10^?5 M and Ca²⁺ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

A theory of osmotic lysis of lipid vesicles

Osmotic lysis of vesicles is shown to begin when the membrane expansion due to osmotic pressure exceeds its critical value, delta S, at which a membrane ruptures to form a pore. The dependence of delta S on the vesicle radius and respective osmotic pressures are obtained. It is found that osmotic pressure necessary for small (100 A) vesicles to rupture should exceed 30 atm, for large (10 000 A) vesicles it being as small as 10(-3) atm. In the case of large (greater than or approximately 1000 A) vesicles the value of relative expansion of the membrane at which its rupture occurs in a reasonable time only depends slightly on the vesicle radius. For instance, for 10 000 A vesicles it amounts to 3%. The tension of membrane rupture is about 8 dyn/cm for large vesicles. Membrane tension, although it decreases considerably as a result of rupture and pore formation, does not vanish completely. It supports the residual intravesicular pressure causing the efflux of vesicle (cell) contents. Simultaneously, osmotic influx of water through the membrane occurs that results in either complete rupture of the membrane with the efflux of the whole of the contents, or its gradual washout in either of two, quasi-steady or pulse-wise regimes. In the first case a pore is steadily open, whereas in the second case it alternately opens and closes, ejecting about 5% of internal solution each time. Lysis kinetics is analyzed. Pulse-wise regime of lysis is shown to be the most likely one.     

Effects of osmotic stress on mast cell vesicles of the beige mouse

The large size of the vesicles of beige mouse peritoneal mast cells (4 microns in diameter) facilitated the direct observation of the individual osmotic behavior of vesicles. The vesicle diameter increased as much as 73% when intact cells were perfused with a 10 mM pH buffer solution; the swelling of the vesicle membranes exceeded that of the insoluble vesicle gel matrix, which resulted in the formation of a clear space between the optically dense gel matrix and the vesicle membrane. Hypertonic solutions shrank intact vesicles of lysed cells in a nonideal manner, suggesting a limit to the compressibility of the gel matrix. The nonideality at high osmotic strengths can be adequately explained as the consequence of an excluded volume and/or a three-dimensional gel-matrix spring. The observed osmotic activity of the vesicles implies that the great majority of the histamine known to be present is reversibly bound to the gel matrix. This binding allows vesicles to store a large quantity of transmitter without doing osmotic work. The large size of the vesicles also facilitated the measurement of the kinetics of release as a collection of individual fusion events. Capacitance measurements in beige mast cells revealed little difference in the kinetics of release in hypotonic, isotonic, and hypertonic solutions, thus eliminating certain classes of models based on the osmotic theory of exocytosis for mast cells.     

Elasticity of synthetic phospholipid vesicles and submitochondrial particles during osmotic swelling

A rapid and accurate method has been developed for measuring the elastic response of vesicle bilayer membranes to an applied osmotic pressure. The technique of dynamic light scattering is used to measure both the elastic constant and the elastic limit of dioleoylphosphatidic acid (DOPA) and DOPA-cholesterol vesicles and of submitochondrial particles derived from the inner membrane of bovine heart mitochondria. The vesicles prepared by the pH-adjustment method are unilamellar and of uniform size between 240 and 460 nm in diameter. The vesicles swell uniformly upon dilution. The observed change in size is not due to any change in the shape of the vesicles. The data also indicate that the vesicles are spherical and not flaccid. The total vesicle swelling in these studies resulted in a 3-4% increase in surface area for vesicles swollen in 0.15 M KCl and a 5-10% increase in surface area for vesicles swollen in 0.25 M sucrose. This maximum represents the elastic limit of the vesicles. Evidence is presented to show that the vesicles release contents after swelling to this maximum, reseal immediately, and reswell according to the osmotic pressure. For DOPA vesicles in a 0.15 M KCl-tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.55), the observed membrane modulus is found to be in the range of 10(8) dyn/cm2. The modulus was found to be in the order of 10(7) dyn/cm2 for DOPA vesicles in a 0.25 M sucrose-Tris-HCl buffer (pH 7.55). This is comparable to that of submitochondrial particles in the same sucrose-Tris-HCl buffer. The observed membrane modulus also decreases with vesicle size. Its magnitude and its variation with ionic strength indicate that the major component of bilayer elasticity is neither the inherent elasticity of the bilayer nor the bending modulus. The variation of the membrane modulus with respect to curvature suggests that its principal component may be related to surface tension effects including the negative charges on the vesicle surface. There is considerable variation between vesicles swollen in sucrose and those swollen in KCl in the membrane modulus, in the elastic limit at which the vesicles burst, and in the transbilayer pressure difference at bursting. The latter was found to be 4-6 mosM (10(5) dyn/cm2) in sucrose solution and 20-4 mosM (10(6) dyn/cm2) in KCl solution.     

Delta-endotoxin-induced leakage of 86Rb+-K+ and H2O from phospholipid vesicles is catalyzed by reconstituted midgut membrane

Brush border membrane from Heliothis virescens catalyzed delta-endotoxin-induced leakage of ⁸⁶Rb⁺-K⁺ and H₂O from phospholipid vesicles. Activated delta-endotoxin [CrylA(c)-55 kDa] from Bacillus thuringiensis kurstaki strain EG2244 producing a single CrylA(c) toxin, when incorporated into phospholipid vesicles, made these vesicles more leaky to ⁸⁶Rb⁺-K⁺ than phospholipid vesicles without toxin. This effect was assayed by following the movement of ⁸⁶Rb⁺ into the vesicles in response to a KCl gradient. When toxin was added to the outside of phospholipid vesicles, ⁸⁶Rb⁺ uptake was impeded. Vesicles prepared with H. virescens brush border membrane catalyzed the association of the toxin with the vesicle, and stimulated KCl gradient-induced ⁸⁶Rb⁺ uptake. Toxin did not catalyze the leakage of ³⁶Cl⁻, suggesting that the toxin created a cation-selective leak. Toxin enhanced the permeability of phospholipid vesicles to H₂O, demonstrated by the enhanced rate of vesicle shrinking under increased osmotic pressure. This was analyzed spectrophotometrically by following the rate of vesicle shrinking in response to a 10 mM KCl gradient. In the presence of concentrated phosphatidylcholine vesicles, toxin spontaneously associated with the vesicles so as to enhance the rate of vesicle shrinking in an osmotic gradient. The rate of vesicle shrinking the presence of KCl and toxin was catalyzed by the presence of brush border reconstituted into the vesicles, reducing the effective toxin concentration 1000-fold.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

Structure of the wall of Halobacterium halobium gas vesicles

The gas vesicles of Halobacterium halobium have been studied by recording X-ray diffraction patterns from both intact and collapsed vesicles. The wall is found to be remarkably thin; the average thickness is no more than 20 Å. Electron microscopy indicates that the wall consists of ribs, and the X-ray data confirm this. The thickness is therefore greater than 20 Å at some points and less at others. The X-ray data also indicate that the ribs on the two sides of the collapsed vesicle are intermeshed.Our data indicate a large amount of β-sheet in the wall. The β-sheet consists of parallel (or anti-parallel) polypeptide chains which are regularly hydrogen-bonded to one another. This bonding locks the presumed subunit proteins into the wall, which is important for its function at the gas-liquid interface. The β-sheet is in two layers, one on top of the other. The two layers together can stiffen the wall and hence strengthen the vesicle against collapse.     

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 μm to 3.0 μm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.     

Preparation and characteristics of lipid vesicles

Summary As determined by electron microscopy, lipid sonicated in buffer initially forms large vesicles which may be multilamellar. Prolonged sonication results in a population of vesicles of smaller, but not uniform diameters. These vesicles are bounded by only one bilayer. The lipid suspension can be partially fractionated according to size by column chromatography. A fraction of the eluate has been selected for further study. The weight-average vesicle weight and average radius of gyration are obtained by lightscattering measurements. The volume of buffer enclosed by the vesicles is determined using¹⁴C- or³H-labelled sugars as a marker. These values are in reasonable agreement with the corresponding values calculated from the size distribution of the vesicle fraction obtained by electron microscopy.     

Mechanical properties of vesicles. II. A model for osmotic swelling and lysis.

Vesicle polydispersity and leakage of solutes from the vesicle lumen influence the measurement and analysis of osmotically induced vesicle swelling and lysis, but their effects have not been considered in previous studies of these processes. In this study, a model is developed which expressly includes polydispersity and leakage effects. The companion paper demonstrated the preparation and characterization of large unilamellar lipid vesicles. A dye release technique was employed to indicate the leakage of solutes from the vesicles during osmotic swelling. Changes in vesicle size were monitored by dynamic light scattering (DLS). In explaining the results, the model identifies three stages. The first phase involves differential increases in membrane tension with strain increasing in larger vesicles before smaller ones. In the second phase, the yield point for lysis (leakage) is reached sequentially from large sizes to small sizes. In the final phase, the lumen contents and the external medium partially equilibrate under conditions of constant membrane tension. When fit to the data, the model yields information on polydispersity-corrected values for membrane area compressibility, Young's modulus, and yield point for lysis.     

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 microm to 3.0 microm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.     

On the structural and functional components of coated vesicles.

Despite the diversity of their known functions, coated vesicles from different tissues contain a rather similar spectrum of proteins, in addition to their major coat protein, clathrin. In particular, each coated vesicle preparation shows a doublet of polypeptide species, on sodium dodecyl sulphate-containing gel electrophoresis, of apparent molecular weight in the region 30,000 to 36,000. Using bullock brain as a source, these molecules are found in association with possible trimers or higher multiples of clathrin, obtained by dissolving coated vesicles in cholate. They may play a structural role relating to the vertices or edges of the lattices of pentagons and hexagons of the polyhedral coats.Purified coated vesicles (e.g. from chicken oocytes) seem to contain relatively small amounts of specific proteins in terms of “contents”. This suggests that the bulk of the isolated particles, especially those in the small size range (500 to 800 Å diam.), may be “empty” of contents, although many still retain a lipid vesicle. These empty structures could represent a pool of recycling coated vesicle components formed after release (possibly from larger vesicles (800 to 1500 Å diam.)) of the specific contents, at their particular destination.     

Analysis of the source of heterogeneity in the osmotic response of plant membrane vesicles

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity.     

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.