Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.     

Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.     

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.     

Micrometer-sized supported lipid bilayer arrays for bacterial toxin binding studies through total internal reflection fluorescence microscopy

In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.     

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.     

Bilayer Edges Catalyze Supported Lipid Bilayer Formation

Supported lipid bilayers (SLB) are important for the study of membrane-based phenomena and as coatings for biosensors. Nevertheless, there is a fundamental lack of understanding of the process by which they form from vesicles in solution. We report insights into the mechanism of SLB formation by vesicle adsorption using temperature-controlled time-resolved fluorescence microscopy at low vesicle concentrations. First, lipid accumulates on the surface at a constant rate up to ∼0.8 of SLB coverage. Then, as patches of SLB nucleate and spread, the rate of accumulation increases. At a coverage of ∼1.5 × SLB, excess vesicles desorb as SLB patches rapidly coalesce into a continuous SLB. Variable surface fluorescence immediately before SLB patch formation argues against the existence of a critical vesicle density necessary for rupture. The accelerating rate of accumulation and the widespread, abrupt loss of vesicles coincide with the emergence and disappearance of patch edges. We conclude that SLB edges enhance vesicle adhesion to the surface and induce vesicle rupture, thus playing a key role in the formation of continuous SLB.     

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO₂ beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.     

Reaction characteristics and mechanisms of lipid bilayer vesicle fusion

Small unilamellar lipid bilayer vesicles were prepared from brain phosphatidylserine, egg phosphatidylcholine, and synthetic dipalmitoylphosphatidylcholine, and were fused into larger structures by freezing and thawing, addition of calcium chloride, and passage through the lipid phase transition temperature. Fusion reactions were studied by electron microscopy, light scattering, and use of fluorescent probes. Fusion was accompanied by leakage of lipid vesicle constituents and of water-soluble solutes in the inner vesicle compartments, and by uptake of these types of components from the external solution. Such leakage was greater during fusion by freezing than by Ca²⁺. Passage through the transition temperature produced a moderate degree of fusion, without loss of membrane components. It is concluded that each fusion method gives rise to a characteristic size or narrow range of sizes of fusion products. The fraction of small vesicles fused into larger structure depends on the method of vesicle preparation, composition of the lipid bilayer, and composition of the external solution. Fusion is induced by creation of a discontinuity in the bilayer or by removal of water associated with the bilayer. The amount of water removed controls the extent of fusion. This is maximized in bilayers when in the liquid-crystal phase, as against the gel phase, in vesicles made by ethanol injection, as against sonication, and in charged bilayers, as against neutral ones.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle–vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis.     

Lipid Mixing and Content Release in Single-Vesicle, SNARE-Driven Fusion Assay with 1-5 ms Resolution

A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5-10 ms. At 60% phosphatidylethanolamine lipid composition, productive and dead-end hemifusion account for 65% of all fusion events. However, quantitative analysis shows that calcein is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.     

Early steps of supported bilayer formation probed by single vesicle fluorescence assays

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.     

Preparation of giant liposomes

A method is described for the preparation of giant unilamellar lipid vesicles that are stable in electrolyte solution. In general, it involves dialysis of lipid and indifferent solute in a water-miscible organic solvent against an aqueous buffer. During dialysis the concentration of organic solvent decreases so that vesicles form under conditions where their internal contents are continuously hyperosmotic. Interlamellar attractive forces are neutralized, even between bilayer membranes with no net charge, and giant vesicles are generated in large numbers. The population is heterogeneous but most large vesicles have diameters between 10 and 100 μm. The method is simple. One procedure involves dialysis for a day or more of a methanol solution of phosphatidylcholine, supersaturated with methylglucoside, against an aqueous phase containing up to 1 M univalent electrolyte. The procedure is effective over a wide range of temperature and pH.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Productive hemifusion intermediates in fast vesicle fusion driven by neuronal SNAREs

An in vitro fusion assay uses fluorescence microscopy of labeled lipids to monitor single v-SNARE vesicle docking and fusion events on a planar lipid bilayer containing t-SNAREs. For vesicles and bilayer comprising phosphatidylcholine (POPC, 84-85% by mol) and phosphatidylserine (DOPS, 15% by mol), previous work demonstrated prompt, full fusion (τ_fus = 25 ms). Substitution of 20-60% phosphatidylethanolamine (DOPE) for phosphatidylcholine in the v-SNARE vesicle with either 0 or 20% DOPE included in the t-SNARE bilayer gives rise to hemifusion events. Labeled lipids diffuse into the planar bilayer as two temporally distinct waves, presumably hemifusion of the outer leaflet followed by inner leaflet (core) fusion. The fusion kinetics with DOPE is markedly heterogeneous. Some vesicle/docking site pairs exhibit prompt, full fusion while others exhibit hemifusion. Hemifusion events are roughly half productive (leading to subsequent core fusion within 20 s) and half dead-end. In qualitative accord with expectations from studies of protein-free vesicle-vesicle fusion, the hemifusion rate k_hemi is 15-20 times faster than the core fusion rate k_core, and the fraction of hemifusion events increases with increasing percentage of DOPE. This suggests similar underlying molecular pathways for protein-free and neuronal SNARE-driven fusion. Removal of phosphatidylserine from the v-SNARE vesicle has no effect on docking or fusion.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.