Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of hemifusion is observed and quantified. In this state, the outer leaflets of both vesicles bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.Received: 26 May 2004, Published online: 3 August 2004PACS: 87.16.Dg Subcellular structure and processes: Membranes, bilayers, and vesicles - 87.15.Vv Biomolecules: structure and physical properties: Diffusion - 64.70.Nd Structural transitions in nanoscale materials     

Phase diagram of a ternary mixture of cholesterol and saturated and unsaturated lipids calculated from a microscopic model

We employ a molecular model to study a ternary mixture of saturated lipid, with tails of 16 carbons, a monounsaturated lipid with tails of 18 carbons, and cholesterol. The model, solved within mean-field theory, produces several forms of phase diagrams depending upon the relative strengths of interactions, but only one that shows the coexistence of two liquid phases observed in experiment. The lipids in the phase rich in cholesterol are more ordered than those in the other. The binary cholesterol, saturated lipid system also exhibits liquid, liquid coexistence.     

Pathway of membrane fusion with two tension-dependent energy barriers

Fusion of bilayer membranes is studied via dissipative particle dynamics (DPD) simulations. A new set of DPD parameters is introduced which leads to an energy barrier for flips of lipid molecules between adhering membranes. A large number of fusion events is monitored for a vesicle in contact with a planar membrane. Several time scales of the fusion process are found to depend exponentially on the membrane tension. This implies an energy barrier of about 10k(B)T for intermembrane flips and a second size-dependent barrier for the nucleation of a small hemifused membrane patch.     

Spontaneous and reversible switch from amphiphilic to oil-like structures

Current theories assume that the amphiphilicity of biological membranes is always preserved. We observed that two hydrogen-bonding lipid layers in contact can spontaneously and reversibly lose their amphiphilic structure and turn into an assembly of oily complexes. This result opens a new angle for understanding the reorganization of lipids during membrane fusion, since similar complexes could fill the troubling hydrophobic voids displayed in the current models. The unique tribological properties described here may also find application in the development of novel nanolubricants.     

Molecular dynamics simulations of the effects of sodium dodecyl sulfate on lipid bilayer

Molecular dynamics simulations have been performed on the fully hydrated lipid bilayer with different concentrations of sodium dodecyl sulfate(SDS). SDS can readily penetrate into the membrane. The insertion of SDS causes a decrease in the bilayer area and increases in the bilayer thickness and lipid tail order, when the fraction of SDS is less than 28%.Through calculating the binding energy, we confirm that the presence of SDS strengthens the interactions among the DPPC lipids, while SDS molecules act as intermedia. Both the strong hydrophilic interactions between sulfate and phosphocholine groups and the hydrophobic interactions between SDS and DPPC hydrocarbon chains contribute to the tight packing and ordered alignment of the lipids. These results are in good agreement with the experimental observations and provide atomic level information that complements the experiments.     

Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This lifetime quenching can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.Abbreviations PEG poly(ethylene glycol) - Na₂EDTA ethyiene-diamine-tetraacedic acid, disodium salt - LUV large, unilamellar vesicles made by rapid extrusion technique - DPH 1,6-diphenyl-trans-1,3,5-hexatriene - DPHpPC 1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine - DPPC 1,2-dipalmitoyl-3-sn-phosphatidylcholine - PA palmitic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE - Rh-PE N-(lissamine Rhodamine B sulfoyl)-PE - R₁₈ octadecyl Rhodamine B chloride - ANTS 1-aminonaphthalene-3,6,8-trisulfonic acid - DPX N,N-p-xylylene-bis(pyradinium bromide)     

A method for quantitative interpretation of fluorescence detection of poly(ethylene glycol)-mediated 1-palmitoyl-2-[[[2-[4-(phenyl-trans-1,3,5-hexatrienyl) phenyl]ethyl]oxyl]carbonyl]3-sn-phosphatidylcholine (DPHpPC) transfer and fusion between phospholipid vesicles in the dehydrated state

A method has been developed for calculating the expected fluorescence lifetime of the DPH _p PC probe distributed between different membrane environments. We show how this method can be used to distinguish between lipid transfer and fusion between large unilamellar vesicles occurring in the presence of poly(ethylene glycol) (PEG). This application of the calculation took into consideration the heterogeneity of microenvironments experienced by the probe in a sample containing vesicle aggregates of different sizes. Assuming that the aggregate size distribution was a delta function of the aggregate size, comparison of the calculated and observed lifetimes yielded an estimate of the vesicle aggregate size. For vesicles of varying compositions in the presence of dehydrating concentrations of PEG, this method suggested that only small aggreggates formed. For vesicles that could be demonstrated by other means not to have fused, the data were consistent with lipid transfer occurring only between the outer leaflets of two to four vesicles, even at high PEG concentrations. For vesicles that could be demonstrated to fuse by contents mixing and size changes, the fluorescence lifetime data were consistent with lipid transfer between both the inner and the outer leaflets of two to four fused vesicles. At very high PEG concentrations, where extensive rupture and large, multilamellar products were previously observed, the lifetime data were consistent with much more extensive lipid transfer within larger aggregates. The agreement of predictions made on the basis of lifetime measurements with other observations attests to the validity of the fluorescence lifetime method. In addition, the model and data presented here provide evidence that fusion occurs between small numbers of PEG-aggregated vesicles before the removal of PEG.     

Role of non-lamellar-forming lipid in promotion of liposomal fusion

Membrane fusion is an important process in a wide range of cellular and sub-cellular activities. It is evident that during the intermediate stages of fusion some transitory non-bilayer configurations must appear within the lipid moiety. Using fluorescence techniques, we have studied here the process of aggregation and fusion of liposomes made of lipids, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). When mixed together, the complete fusion between these two liposomes took around 44 h as both DPPC and DMPC favour lamellar configuration. When the mixture was incubated at 42°C the fusion process was completed after 23 h. But, when 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was added in the liposomal matrix the time for fusion was reduced to 21 h for mixture without incubation and 17 h when the mixture was incubated. This indicates that DPPE having a tendency to assume non-lamellar conformation, promoted destabilisation of the lamellar conformation within the liposome which facilitated the fusion between two apposing bilayers.     

电子自旋标记于两种共存的磷脂质相态中的不对称分布

该文探讨自旋标记于不同磷脂质相中不对称分布的分子机制. 透过电子自旋共振（ESR）波谱技术以及理论波谱分析,探究两种在磷脂质不同烷基位点上的自旋标记（16-PC & 5-PC)于两种明显不同物理性质的磷脂质 (DPPC & DLPC) 所构成的细胞膜两相共存区域具有不对称分布情形. 根据实验结果,如果自旋探针平均分布在两相之间,则其波谱将能根据杠杆法则（lever rule) 来拟合波谱的结果,而自旋探针的分布反映了该探针周围环境的脂质所给予的立体障碍,而这个不对称的情形可以合理的解释成自旋标记的表面自由面积所造成. 根据结果,16-PC会平均分布在两个不同的相区域,而5-PC则会有不对称分布的情形.     

An exactly solvable two-dimensional biomembrane model

A model for a two-dimensional lipid bilayer, in which both short-range repulsive forces and long-range attractive forces piay a role, is solved exactly. First, in the absence of attractive forces, the configurational entropy is calculated by restricting the lipid hydrocarbon tails to a lattice and solving the resulting counting problem exactly. When long-range attractive forces which decrease exponentially with distance are acting between the polar head groups of the lipids, the partition function can still be calculated exactly, using integration over Gaussian random fields. The thermodynamic functions show a singularity which reflects the completion of the process of self-assemblage of the lipid bilayer. Finally, the model is used to test an approximation method for lipid bilayers.     

Coarse-grained simulations on interactions between spectrins and phase-separated lipid bilayers

Spectrin, the principal protein of the cytoskeleton of erythrocyte, plays a crucial role in the stability and flexibility of the plasma membrane of erythrocyte. In this work, we investigate the interactions between spectrins and phase-separated lipid bilayers using coarse-grained molecular dynamics simulation. We focus on the preference of spectrins with different lipids, the effects of the anionic lipids and the residue mutation on the interactions between spectrins and the lipid bilayers. The results indicate that spectrins prefer to contact with phosphatidylethanolamine (PE) lipids rather than with phosphatidylcholine (PC) lipids, and tend to contact with the liquid-disordered (Ld) domains enriched in unsaturated PE. Additionally, the anionic lipids, which show specific interaction with the positively charged or polar amino acids on the surface of the spectrins, can enhance the attraction between the spectrins and lipid domains. The mutation leads to the decrease of the structural stability of spectrins and increases the curvature of the lipid bilayer. This work provides some theoretical insights into understanding the erythrocyte structure and the mechanism of some blood diseases.     

Variable‐temperature Raman spectro‐microscopy for a comprehensive analysis of the conformational order in PEGylated lipids

The investigation of phase transitions and associated changes in the conformational order of lipids is of importance in various research areas dealing with phenomena such as the formation and fusion of vesicles, transmembrane diffusion and membrane interactions with drugs and proteins. In this article, we have focused on the study of thermotropic phase behaviors and associated changes in the conformational order of two newly developed synthetic PEGylated lipids trademarked as QuSomes. In contrast to conventional phospholipids, this new kind of lipid forms liposomes spontaneously upon hydration, without the supply of external activation energy. Variable‐temperature Raman spectro‐microscopy has been employed in order to plot the transition temperature profiles showing the phase behavior of these new lipids composed of 1,2‐dimyristoyl‐rac‐glycerol‐3‐dodecaethylene glycol (GDM‐12) and 1,2‐distearoyl‐rac‐glycerol‐3‐triicosaethylene glycol (GDS‐23). Furthermore, several spectral indicators were calculated and correlated which allowed for the deduction of various aspects of molecular structure as well as intramolecular motion and intermolecular interactions. To confirm the observations, differential scanning calorimetry (DSC) was applied and revealed a good agreement with the Raman spectroscopy results. Finally, this information may find application in various studies including the development of lipid‐based novel substances and drug delivery systems. Copyright © 2008 John Wiley & Sons, Ltd.     

A simulation study on multicomponent lipid bilayer

Simulation of a multicomponent lipid bilayer having a fixed percentage of cholesterol is done to study phase transition leading to domain formation. The concept of random lattice has been used in simulation to account for the coupling between the internal and translational degrees of freedom of lipid molecules. Considering a canonical ensemble, dissimilar lipid molecules are allowed to exchange their positions in the lattice subject to standard metropolis algorithm. The steps involved in the process effectively takes into account for the movement of sphingolipids and cholesterol molecules helping formation of cholesterol rich domains of saturated lipids as found in natural membranes.     

Anomalous phase sequences in lyotropic liquid crystals

We present a coarse-grained model in order to describe the unusual sequence of mesophases observed in aqueous solutions of nonionic lipids, such as monoolein. The lipid molecules are modeled as a rigid head and a flexible Gaussian tail, and water is treated explicitly. A key component of the model is thermally reversible hydrogen bonding between the lipid head and water resulting in changes in both head volume and the interactions of the hydrated head with its surroundings. Phase diagrams obtained from unit-cell self-consistent field simulations capture the qualitative thermotropic and lyotropic phase behavior of the monoolein-water system. The unusual phase sequences result from a competition between hydrogen bond formation, changes in head volume and interactions, lipid tail entropy, and the hydrophobic effect.     

Decaying two-dimensional turbulence in a circular container

We present direct numerical simulations of two-dimensional decaying turbulence at initial Reynolds number 5 x 10(4) in a circular container with no-slip boundary conditions. Starting with random initial conditions the flow rapidly exhibits self-organization into coherent vortices. We study their formation and the role of the viscous boundary layer on the production and decay of integral quantities. The no-slip wall produces vortices which are injected into the bulk flow and tend to compensate the enstrophy dissipation. The self-organization of the flow is reflected by the transition of the initially Gaussian vorticity probability density function (PDF) towards a distribution with exponential tails. Because of the presence of coherent vortices the pressure PDF become strongly skewed with exponential tails for negative values.     

Transient ordered domains in single-component phospholipid bilayers

We report evidence of dense, ordered nanodomains in single-component fluid lipid bilayers. Our atomic-scale molecular dynamics simulations suggest that the area available to a lipid acyl chain exhibits large fluctuations, resulting in denser and sparser domains. The sizes of the dense domains can be up to approximately 10 nm, and their lifetimes are of the order of approximately 10 ns. In addition, our simulations suggest that domains of lipids with highly ordered acyl chains form predominantly within the dense regions, their sizes ranging from a few chains up to a few nanometers, and with lifetimes between approximately 10 ps-10 ns. These observations shed light on the origin of experimentally observed fluctuations, as well as on the mechanisms of phase transitions in lipid membranes.     

Molecular dynamics study of the infiltration of lipid-wrapping C60 and polyhydroxylated single-walled nanotubes into lipid bilayers

Because of the many potential medical applications of nanoparticles, considerable research has been conducted on the interactions between nanoparticles and biomembranes. We employed coarsegrained molecular dynamics simulations to study the infiltration of lipid-wrapping C₆₀ and polyhydroxylated single-walled nanotubes. Diffusion coefficients and scaling factors are adopted to quantify the diffusivity of the biomembranes, and the rupture tension is used to measure the lateral strength of the lipid bilayer. According to our simulations, all wrapped nanoparticles, except those wrapped by dipalmitoyl-glycero-phosphoglycerol, can be inserted into the bilayers. Our simulations also reveal that the bilayers remain in free diffusion after the nanoparticle insertions while their diffusion coefficient can be altered significantly. The polyhydroxylated single-walled nanotubes lead to significant changes to the lateral strength of biomembranes and this effect depends on the quantity of the inserted nanoparticles. The simulations demonstrate the feasibility of using these methods to deliver nanoparticles while some suggestions are given for choosing the appropriate lipids for wrapping. The results also suggest that the functionalized nanoparticles could be applied in strengthening or weakening the lateral strength of biomembranes for specific purposes.     

Pore formation phase diagrams for lipid membranes

Critical lateral pressure for a pore formation and phase diagram of porous membrane are derived analytically as functions of the microscopic parameters of the lipid chains. The derivation exploits path-integral calculation of the free energy of the ensembles of semi-flexible strings and rigid rods that mimic the hydrophobic tails of lipids in the lipid bilayers and bolalipid membranes respectively. Analytical expressions for the area stretch/compressibility moduli of the membranes are derived in both models.     

Two-dimensional dye crystals with controllable optical properties

The structure, molecular ordering and optical properties of single crystals of cyanine dyes grown by adsorption from a water subphase to a positively charged lipid monolayer are discussed. These crystals are one monolayer thick, of uniform dimensions between 10 and 100μm (depending on nucleation conditions) and of rectangular shape. Single crystals were studied by transmission electron diffraction and by polarized absorption and emission spectroscopy.

We show that the crystals consist of two rows of densely stacked molecules with two different orientations of the long molecular axes. This leads to two perpendicularly polarized absorption bands. The measured splitting is in accordance with results of extended dipole calculations. The latter were performed for crystals of three slightly different molecules where the angles between the long axes varied between 70° and 100°. The aliphatic tails form a lattice which is epitaxially related to the centered rectangular one of the dyes. It is incommensurate, and the tails are tilted with tilt azimuth either parallel to the a or to the b axis (depending on the type of dye) of the dye lattice.

The procedure allows formation of mixed crystals with structural parameters, depending on composition, between those of the pure compounds. Hence also the optical spectra can be tuned via composition maintaining sharp band edges and emission bands.     

Semiempirical law for the dipole moments of low-molecular-weight gelators

Some small chiral organic molecules are capable of forming gel in low-concentration solutions. This phenomenon is of great theoretical interest, but its molecular details are still unclear. High chemical diversity of such gelators impedes the investigation of their common structural properties important for gel formation. Here, we calculated the total dipole moment for a set of gelators with sufficiently different chemical structures by means of molecular dynamics simulations. It was found that all considered molecules have a dipole moment of ～3 D or more. This means that the energy of the dipole-dipole interaction between two point dipoles at a distance of 6–8 Å is ～kT. This distance roughly corresponds to molecules in close contact. This makes it possible to conclude that the dipole moment can orient gelator molecules before aggregation, thereby playing a key role in the process of gelation. This orientation effect determines the anisotropy of aggregates and the gelation of the solution.