Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

Structural studies on phophatidylcholine-cholesterol mixed vesicles.

The homogeneous, single-walled phosphatidylcholine-cholesterol mixed vesicles were prepared by ultrasonic irradiation of egg phosphatidylcholine in the presence of various amounts of cholesterol in solution at 4 degrees under a nitrogen atmosphere followed by molecular sieve chromatography on a Sepharose 4B column. Physicochemical studies performed on these systems invluding sedimentation velocity, diffusion, partial specific volume, intrinsic viscosity, and trapped volume measurements allowed estimation of the weight-average vesicle weight, the vesicle shape, and bilayer membrane thickness of the binary mixture of phosphatidylcholine and cholesterol. Vesicle hydration was calculated using two different methods and the agreement between them was excellent up to cholesterol concentration of 0.32 mole fraction. It was observed that the structural parameters change slowly with increasing cholesterol content up to around 0.3 mole fraction and a relatively abrupt structural alteration occurs above this cholesterol content. This abrupt structural change is consistent with the asymmetrical distribution of lipid composition between the inner and outer bilayer face.     

Localization of barriers to water flow in toad urinary bladder

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct imaging of individual intrinsic hydration layers on lipid bilayers at Angstrom resolution

The interactions between water and biological molecules have the potential to influence the structure, dynamics, and function of biological systems, hence the importance of revealing the nature of these interactions in relation to the local biochemical environment. We have investigated the structuring of water at the interface of supported dipalmitoylphosphatidylcholine bilayers in the gel phase in phosphate buffer solution using frequency modulation atomic force microscopy (FM-AFM). We present experimental results supporting the existence of intrinsic (i.e., surface-induced) hydration layers adjacent to the bilayer. The force versus distance curves measured between the bilayer and the AFM tip show oscillatory force profiles with a peak spacing of 0.28 nm, indicative of the existence of up to two hydration layers next to the membrane surface. These oscillatory force profiles reveal the molecular-scale origin of the hydration force that has been observed between two apposing lipid bilayers. Furthermore, FM-AFM imaging at the water/lipid interface visualizes individual hydration layers in three dimensions, with molecular-scale corrugations corresponding to the lipid headgroups. The results demonstrate that the intrinsic hydration layers are stable enough to present multiple energy barriers to approaching nanoscale objects, such as proteins and solvated ions, and are expected to affect membrane permeability and transport.     

Different effects of di- and triphenyltin compounds on lipid bilayer dithionite permeabilization

Phenyltins are chemicals widely used in industry, hence their occurrence in the human environment is frequent and widespread. Such compounds include hydrophobic phenyl rings bonded to positively charged tin. This molecular structure makes them capable of adsorbing onto and penetrating through biological membranes, hence they are potentially hazardous. Two such compounds, diphenyltin and triphenyltin, show different steric constraints when interacting with the lipid bilayer. It has been demonstrated that these compounds are positioned at different locations within model lipid bilayers, causing dissimilarity in their ability to affect membrane properties. In this paper we present a study regarding the ability of these two phenyltins to facilitate the transport of S2O4(-2) ions across the lipid bilayer, evaluated by a fluorescence quenching assay. In concentration range of up-to 60 microM those compounds do not affect lipid bilayer topology, when evaluated by vesicle size distribution. Both phenyltins facilitate the transfer of S2O4(-2) across the model lipid bilayer, but the dependence of dithionite transport on phenyltin concentration is different for both. In principle, above 20 microM triphenyltin is more efficient in transferring ions across the lipid bilayer than diphenyltin.     

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.     

Active generation and propagation of Ca2+ signals within tunneling membrane nanotubes

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.     

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

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

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.     

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes

The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.     

Molecular dynamics studies of simple membrane — Water interfaces: Structure and functions in the beginnings of cellular life

Molecular dynamics computer simulations of the structure and functions of a simple membrane are performed in order to examine whether membranes provide an environment capable of promoting protobiological evolution. Our model membrane is composed of glycerol 1-monooleate. It is found that the bilayer surface fluctuates in time and space, occasionally creating thinning defects in the membrane. These defects are essential for passive transport of simple ions across membranes because they reduce the Bom barrier to this process by approximately 40%. Negative ions are transferred across the bilayer more readily than positive ions due to favorable interactions with the electric field at the membrane-water interface. Passive transport of neutral molecules is, in general, more complex than predicted by the solubility-diffusion model. In particular, molecules which exhibit sufficient hydrophilicity and lipophilicity concentrate near membrane surfaces and experience interfacial resistance to transport. The membrane-water interface forms an environment suitable for heterogeneous catalysis. Several possible mechanisms leading to an increase of reaction rates at the interface are discussed. We conclude that vesicles have many properties that make them very good candidates for earliest protocells. Some potentially fruitful directions of experimental and theoretical research on this subject are proposed.     

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.     

Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.     

Deployment of membrane fusion protein domains during fusion

It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion proteins SV5 F1 and HIV gp120/41, and for the intracellular SNARE fusion system. By comparing these domains, we have constructed a minimal set which appears to be adequate to explain how the conformational changes can produce a successful fusion event, i.e. communication of aqueous compartments.     

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues.     

Transbilayer phosphatidylcholine distributions in small unilamellar sphingomyelin-phosphatidylcholine vesicles: effect of altered polar head group

The effect of the altered polar head group of phosphatidylcholine (PC) on its transbilayer distributions in small unilamellar vesicles containing sphingomyelin (SM) was ascertained with phospholipase A2 as the external membrane probe. These vesicles were formed by sonication and fractionated by centrifugation. The vesicle size was determined by gel-permeation chromatography and solute entrapment. Experiments were done to confirm that phospholipase A2 treatments did not induce fusion, lyse the vesicles, or cause PC to migrate across the vesicle bilayer. The complete degradation of external PC in intact vesicles was assured by carrying out the enzyme reactions in the absence as well as in the presence of 9.2 X 10(-5) M bovine serum albumin. In small vesicles comprised of SM and 30 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC preferentially distributed in the inner monolayer. This preference of DPPC in these vesicles disappeared upon introducing one C2H5 group at the carbon atom adjacent to the quaternary ammonium residue in its polar head group and was reversed when the C2H5 group was replaced by C6H5 and C6H5CH2 substituents or when the P-N distance was increased. These results indicate that the effective polar head-group volume is an important factor in determining the phospholipid distributions across the small vesicle bilayer.     

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

Membrane fusion during secretion. A hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment

Interpretation of freeze-fracture and thin-section results shows that fusion of the peripheral vesicle with the plasmalemma of a Phytophthora palmivora zoospore occurs at several discrete sites and results in the formation and expansion of a particle-free bilayer membrane diaphragm and in the appearance of a polymorphic network of membrane-bounded tunnels, the lumina of which are continuous with the cytoplasm. The outer half of the bilayer membrane diaphragm appears continuous with the outer half of the plasma membrane; the inner half of the bilayer membrane diaphragm with the inner half of the peripheral vesicle membrane; and the inner half of the plasmalemma with the outer half of the peripheral vesicle membrane. Interpretation of our results leads us to formulate a hypothesis for a sequence of several intermediate stages involved in membrane fusion. The initial fusion event is viewed as a local catastrophe (Thom, R. 1972. Stabilite Structurelle et Morphogenese. W. A. Benjamin Inc., Reading, Mass.) involving the sudden reorganization of apposed elements of the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Fusion of apposed components at the rim of the perimeter of fusion results in the formation of a toroid hemi-micelle which provides continuity between the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Simultaneously, apposed components at the site of fusion may reorganize into an inverted membrane micelle. A bilayer membrane diaphragm is then formed by apposition and flowing of components form the outer half of the plasmalemma and the inner (exoplasmic) half of the peripheral vesicle membrane. The existence of large areas of membrane contact before fusion may lead to several fusion events and the formation of a polymorphic network of membrane- bound tunnels.     

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.     

Effect of phase transition on the kinetics of dye transport in phospholipid bilater structures.

Binding of 8-anilino-1-naphthalenesulfonate to dimyristoyl-L-alpha-lecithin bilayers enhances the fluorescence quantum yield of the dye molecule by 100-fold. By following the generation of fluorescence after a rapid mixing in a stopped-flow apparatus (mixing time 2 msec), kinetics of the binding of the fluorescence probe to the phospholipid vesicles has been investigated in the temperature range where the crystal-liquid crystal phase transition of the bilayer structures occurs. No reactions depending on the dye or the vesicle concentrations were detected. This suggests that the initial adsorption of the dye was very rapid. Two kinetic phases which appear in the 50 msec and the second time ranges are unimolecular. The faster one has a small amplitude and is observable in the entire temperature range studied. In the phase transition region the slower reaction becomes the major kinetic phase. It also increases the apparent concentration of bound dye by a factor of 2. These observations suggest that the 50-msec reaction has detected a reorientation of the probe molecule after the initial binding, and that the slow reaction represents a transport of the dye molecule into the inner layer of the lipid vesicle. The transport reaction is extremely temperature sensitive and exhibits a maximum rate at the midpoint of the bilayer phase transition (Tm = 24.1 degrees). the Arrhenius plot of the transport reaction shows a maximum at the Tm. the same temperature dependence was also observed for the bromothymol blue transport reaction. However, no such effects were detected for less amphiphilic molecules such as tetracycline, chlortetracycline, and pyrene. In the latter systems only a slight bending of the Arrhenius plots were seen at the phase transition temperature. Since the kinetics of the transport of 8-anilino-1-naphthalenesulfonate is sensitive to the physical state of the phospholipid bilayers this reaction may be used for probing membrane structures.