Change of line tension in phase-separated vesicles upon protein binding

We measured the effect of a model membrane-binding protein on line tension and morphology of phase-separated lipid-bilayer vesicles. We studied giant unilamellar vesicles composed of a cholesterol/dioleoylphosphatidylcholine/palmitoylsphingomyelin mixture and a controlled mole fraction of a Ni-chelating lipid. These vesicles exhibited two coexisting fluid-phase domains at room temperature. Owing to the line tension, σ, between the two phases, the boundary between them was pulled like a purse string so that the smaller domain formed a bud. While observing the vesicles in a microscope, histidine-tagged green fluorescent protein was added, which bound to the Ni-chelating lipid. As protein bound, the vesicle shape changed and the length of the phase boundary increased. The change in morphology was attributed to a reduction of σ between the two phases because of preferential accumulation of histidine-tagged green fluorescent protein-Ni-chelating lipid clusters at the domain boundary. Greater reductions of σ were found in samples with higher concentrations of Ni-chelating lipid; this trend provided an estimate of the binding energy at the boundary, approximately k(B)T. The results show how domain boundaries can lead to an accumulation of membrane-binding proteins at their boundaries and, in turn, how proteins can alter line tension and vesicle morphology.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Mechanical stability of micropipet-aspirated giant vesicles with fluid phase coexistence

Micropipet aspiration of phase-separated lipid bilayer vesicles can elucidate physicochemical aspects of membrane fluid phase coexistence. Recently, we investigated the composition dependence of line tension at the boundary between liquid-ordered and liquid-disordered phases of giant unilamellar vesicles obtained from ternary lipid mixtures using this approach. Here we examine mechanical equilibria and stability of dumbbell-shaped vesicles deformed by line tension. We present a relationship between the pipet aspiration pressure and the aspiration length in vesicles with two coexisting phases. Using a strikingly simple mechanical model for the free energy of the vesicle, we predict a relation that is in almost quantitative agreement with experiment. The model considers the vesicle free energy to be proportional to line tension and assumes that the vesicle volume, domain area fraction, and total area are conserved during aspiration. We also examine a mechanical instability encountered when releasing a vesicle from the pipet. We find that this releasing instability is observed within the framework of our model that predicts a change of the compressibility of a pipet-aspirated membrane cylinder from positive (i.e., stable) to negative (unstable) values, at the experimental instability. The model furthermore includes an aspiration instability that has also previously been experimentally described. Our method of studying micropipet-induced shape transitions in giant vesicles with fluid domains could be useful for investigating vesicle shape transitions modulated by bending stiffness and line tension.     

Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.     

Simulations of lipid transfer between a supported lipid bilayer and adsorbing vesicles

Recent experiments demonstrate transfer of lipid molecules between a charged, supported lipid membrane (SLB) and vesicles of opposite charge when the latter adsorb on the SLB. A simple phenomenological bead model has been developed to simulate this process. Beads were defined to be of three types, ‘n’, ‘p’, and ‘0’, representing POPS (negatively charged), POEPC (positively charged), and POPC (neutral but zwitterionic) lipids, respectively. Phenomenological bead–bead interaction potentials and lipid transfer rate constants were used to account for the overall interaction and transfer kinetics. Using different bead mixtures in both the adsorbing vesicle and in the SLB (representing differently composed/charged vesicles and SLBs as in the reported experiments), we clarify under which circumstances a vesicle adsorbs to the SLB, and whether it, after lipid transfer and changed composition of the SLB and vesicle, desorbs back to the bulk again or not. With this model we can reproduce and provide a conceptual picture for the experimental findings.     

A silicate gel for promoting deposition of lipid bilayers

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.     

Theoretical analysis of adhering lipid vesicles with free edges

A theoretical model for describing the adhesion of lipid vesicle with free edges is developed. For adhesion in contact potential or in finite-range potential, the total energy functional is defined as the sum of elastic free energy, the surface energy, the line tension energy and the contact potential or the long-ranged potential. The equilibrium differential equation and boundary conditions for opening-up lipid vesicles are derived through minimizing the total energy functional. Numerical solutions to these equations are obtained under the axial symmetric condition. These numerical solutions can be used to qualitatively explain the influence of the substrate on the open-up lipid vesicles.     

A thermodynamic study of protein-induced lipid lateral phase separation. Effect of lysozyme on mixed lipid vesicles

In this work we study by differential scanning calorimetry (DSC) the lateral phase separation induced by a globular protein (lysozyme) on vesicles built-up by charged (phosphatidic acid) and neutral (phosphatidylcholine) lipids.The adsorption of the positively charged protein onto the negative vesicle surface induces the formation of micro-domains richer in the charged lipid component. This phenomenon is revealed as a splitting of the excess heat capacity peak associated to the melting of the lipid hydrocarbon chains.Also, the peak associated to the protein denaturation is shifted, suggesting the presence of adsorbed proteins onto the vesicle surface. The surface electrostatic potentials, both of proteins and vesicles, have been modulated by pH and ionic strength variations, showing a deep influence of the electric charges in modifying protein adsorption, rate of denaturation (related to unfolding enthalpy variation), and lipid micro-domain formation.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Milk membrane lipid vesicle structures studied with Cryo-TEM

Milk fat globule membrane (MFGM) lipids have been studied in the presence and absence of proteins β-lactoglobulin and β-casein. The aim of this study was to relate the self-assembly structure, e.g. vesicles, formed in aqueous dispersions of MFGM lipids to the lipid composition, electrolyte composition as well as the effect of added milk proteins, i.e. β-lactoglobulin and β-casein. For this purpose, vesicles of phospholipid mixtures, containing dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) at composition corresponding to that of the MFGM, were prepared by extrusion. The morphology of the formed structures of different sample compositions was studied with cryogenic transmission electron microscopy (Cryo-TEM). Mixtures of membrane lipid with a composition (e.g. 80% DOPE, 12% DOPC and 8% SM) that at high lipid content give liquid crystalline phases at the boundary of lamellar to reversed hexagonal phase rather formed microtubular structures than vesicles at high water content. A large proportion of multilamellar vesicles is formed in buffer and divalent salts than in pure water. A small increase in the interlayer spacing of the multilamellar vesicle was observed in the presence of β-casein.     

"Primitive" membrane from polyprenyl phosphates and polyprenyl alcohols

Polyprenyl phosphates, as well as polyprenyl alcohols bearing different isopentenyl C(5) units, have been synthesized. The pH range of spontaneous vesicle formation of polyprenyl phosphates with or without polyprenyl alcohols was defined by fluorescence microscopy. A variety of the acyclic or monocyclic polyprenyl phosphates studied formed stable vesicles in water over a wide range of pHs, and the addition of polyprenyl alcohols allowed the vesicle formation of polyprenyl phosphates at higher pHs. Osmotic swelling of a suspension of unilamellar vesicles using the stopped-flow/light-scattering method enabled us to evaluate the water permeability of polyprenyl phosphate vesicles with or without 10 mol% of free polyprenyl alcohol. The addition of many polyprenyl alcohols to polyprenyl phosphate vesicles decreased the water permeability, and some reduced it even more efficiently than cholesterol.     

Study of Segregation Effects and Fusion Between Lipid Vesicles by Combined Scanning Dilatometric and Calorimetric Measurements

Scanning dilatometric and calorimetric measurements were performed in order to obtain information on correlations between various phenomena involving a lipid vesicle. Scanning dilatometry has been shown to be a fast and reliable tool which gives complementary information to that obtained using differential scanning calorimetry and also, provides a means with which to follow dynamic processes without the introduction of perturbing probes into the lipid matrix. The systems examined were vesicles built up from mixtures of neutral and charged lipids in the presence of mono- and divalent inorganic cations. The studied processes were the gel-liquid crystal transition, lateral phase separation in mixed lipid vesicles and fusion between vesicles.     

Polystyrene-b-poly(acrylic acid) vesicle size control using solution properties and hydrophilic block length

Polymeric vesicles have attracted considerable attention in recent years, since they are a model for biological membranes and have versatile structures with several practical applications. In this study, we prepare vesicles from polystyrene-b-poly(acrylic acid) block copolymer in dioxane/water and dioxane/THF/water mixtures. We then examine the ability of additives (such as NaCl, HCl, or NaOH), solvent composition, and hydrophilic block length to control vesicle size. Using turbidity measurements and transmission electron microscopy (TEM) we show that larger vesicles can be prepared from a given copolymer by adding NaCl or HCl, while adding NaOH yields smaller vesicles. The solvent composition (ratio of dioxane to THF, as well as the water content) can also determine the vesicle size. From a given copolymer, smaller vesicles can be prepared by increasing the THF content in the THF/dioxane solvent mixture. In a given solvent mixture, vesicle size increases with water content, but such an increase is most pronounced when dioxane is used as the solvent. In THF-rich solutions, on the other hand, vesicle size changes only slightly with the water concentration. As to the effect of the acrylic acid block length, the results show that block copolymers with shorter hydrophilic blocks assemble into larger vesicles. The effect of additives and solvent composition on vesicle size is related to their influence on chain repulsion and aggregation number, whereas the effect of acrylic acid block length occurs because of the relationship among the block length, the width of the molecular weight distribution, and the stabilization of the vesicle curvature.     

Effects of newcastle disease virus glycoproteins on the structural and thermal behaviour of 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine lipid membranes under osmotic stress conditions

The interaction of hem agglutininneuraminidase (HN) and fusion (F) glycoproteins with swollen vesicles of 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC) was investigated under transition from gel to fluid phase. X-ray studies of the structure of lipid/HN-F mixtures in normal and swollen vesicles have shown that the lamellar bilayer structure predominate in the gel and liquid crystalline phases. A swollen lipid phase, in which the mean repeat distance of lipid bilayers is larger than in the other phases was found. The nature of this phase is similar to the anomalous bilayer swelling reported in literature. The presence of HN and F in the vesicles led to the coexistence of structures with low and high lamellar order, showing larger repeat distance in comparison with the pure lipid. This finding was attributed to the increase in the lipid bilayer thickness due to the HN-F included in the free water layer. The thermal behaviour of the system was not affected by the vesicle swelling. The data showed the existence of gel and liquid crystalline lamellar phases and changes in lipid/HN-F specific heats, mainly due to the concentration effect of the HN-F and its location in the free water layer.     

Electrophoretic transport and dynamic deformation of bio‐vesicles

Study of the deformation dynamics of cells and other sub‐micron vesicles, such as virus and neurotransmitter vesicles are necessary to understand their functional properties. This mechanical characterization can be done by submerging the vesicle in a fluid medium and deforming it with a controlled electric field, which is known as electrodeformation. Electrodeformation of biological and artificial lipid vesicles is directly influenced by the vesicle and surrounding media properties and geometric factors. The problem is compounded when the vesicle is naturally charged, which creates electrophoretic forcing on the vesicle membrane. We studied the electrodeformation and transport of charged vesicles immersed in a fluid media under the influence of a DC electric field. The electric field and fluid‐solid interactions are modeled using a hybrid immersed interface‐immersed boundary technique. Model results are verified with experimental observations for electric field driven translocation of a virus through a nanopore sensor. Our modeling results show interesting changes in deformation behavior with changing electrical properties of the vesicle and the surrounding media. Vesicle movement due to electrophoresis can also be characterized by the change in local conductivity, which can serve as a potential sensing mechanism for electrodeformation experiments in solid‐state nanopore setups.     

A detailed look at vesicle fusion

Many different hypotheses on the molecular mechanisms of vesicle fusion exist. Because these mechanisms cannot be readily asserted experimentally, we address the problem by a coarse-grained molecular dynamics simulations study and compare the results with the results of other techniques. The simulations performed include the fusion of small and large vesicles and exocytosis, i.e., the fusion of small vesicles with flat bilayers. We demonstrate that the stalk, the initial contact between two fusing vesicles, is initiated by lipid tails that extend spontaneously. The stalk is revealed to be composed of the contacting monolayers only, yet without hydrophobic voids. Anisotropic and radial expansion of the stalk have been theorized; we show that stalk evolution can proceed via both pathways starting from similar setups and that water triggers the transition from elongated stalk to hemifusion diaphragm.     

Migration of poly-L-lysine through a lipid bilayer

When a giant vesicle, composed of neutral and anionic lipid (90:10 mol %), comes into contact with various poly-l-lysines (MW 500-29 300), ropelike structures form within the vesicle interior. By using fluorescence lipids and epi-fluorescence microscopy, we have shown that both neutral and anionic lipids are constituents of the ropes. Evidence that the ropes are also comprised of poly-l-lysine comes from two experiments: (a) direct microinjection of poly(acrylic acid) into rope-containing vesicles causes the ropes to contract into small particles, an observation consistent with a polycation/polyanion interaction; and (b) direct microinjection of fluorescein isothiocyanate (a compound that covalently labels poly-l-lysine with a fluorescent moiety) into rope-containing vesicles leads to fluorescent ropes. The results may be explained by a model in which poly-l-lysine binds to the vesicle exterior, forms a domain, and enters the vesicle through defects or at the domain boundary. The model helps explain the ability of poly-l-lysine to mediate the permeation of a cancer drug, doxorubicine, into the vesicle interior.     

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.