Triacylglycerol-droplet-induced bilayer spontaneous curvature in giant unilamellar vesicles.

This study investigated the incorporation of triacylglycerol droplets in the bilayers of giant unilamellar vesicles (GUVs) using four triacylglycerols and four phosphatidylcholines by confocal laser scanning microscopy. The triacylglycerol droplets were incorporated between the monolayer leaflets of the GUVs. Among the spherical droplets protruding on only one side of the bilayers, the droplets bound to the outer leaflets outnumbered those bound to the inner leaflets. The more frequent droplet binding to the outer leaflet caused transbilayer asymmetry in the droplet surface density. A vesicle consisting of a single-bilayer spherical segment and a double-bilayer spherical segment was also observed. The yield of these vesicles was comparable with or higher than that of the droplet-incorporating GUVs for many of the phosphatidylcholine-triacylglycerol combinations. In a vesicle consisting of single-bilayer and double-bilayer segments, most of the triacylglycerol droplets were localized on the outermost membrane surface along the segment boundary and in the double-bilayer segment. To rationalize the formation of these vesicle structures, we propose that the transbilayer asymmetry in the droplet surface density induces spontaneous curvature of the bilayer, with the bilayer spontaneously bending away from the droplets. Energy calculations performed assuming the existence of spontaneous curvature of the bilayer corroborated the experimentally determined membrane shapes for the vesicles consisting of unilamellar and bilamellar regions.

Ostwald Ripening of Triacylglycerol Droplets Embedded in Glass-Supported Phospholipid Bilayers.

Lipid droplets are fat storage organelles that consist of a neutral lipid core surrounded by a phospholipid monolayer. Because of their important biological functions, reconstituting model lipid droplets in synthetic phospholipid membranes is of great interest. In the present study, we investigated the incorporation of triacylglycerol droplets into glass-supported phospholipid bilayers by using fluorescence microscopy. We adsorbed triolein emulsions onto a glass surface that was partially covered with planar bilayers. After adsorption, triolein droplets were found to be immobilized in the bilayer membrane. The volume of each bound droplet varied over time. Large droplets grew, whereas small droplets shrank. Additionally, data on fluorescence recovery after photobleaching obtained for a phospholipid probe indicate that phospholipids on and near triolein droplets were fully mobile. Furthermore, photobleaching data obtained for a triacylglycerol probe indicate that triolein molecules diffused between different droplets along the planar bilayer. These results demonstrate Ostwald ripening, where triolein molecules in a small droplet dissolved in the bilayer, diffused laterally, and eventually bound to the interfaces of larger droplets. We investigated the ripening rate by using the average of the cube root of the fluorescence emission obtained for individual droplets. The ripening slowed after the addition of trilinolein to the triolein phase. Finally, we investigated the time dependence of the size distributions of the triolein droplets. The distribution was initially nearly unimodal and subsequently became bimodal.

Hydrocarbon Penetration into Phospholipid Monolayers Formed at Hydrocarbon-Water Interfaces.

Phospholipid monolayers formed at oil-water interfaces are used for various biological applications. However, monolayer structures are not well understood. Herein, we investigated hydrocarbon partitioning in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monolayers formed at hydrocarbon-water interfaces using fluorescence microscopy and pendant drop tensiometry. The monolayers strongly interacted with squalene, n-hexadecane, n-tetradecane, n-dodecane, n-decane, and n-butylcyclohexane. These alkane and alkylcyclohexane molecules remained within the monolayers during area compression. In contrast, the monolayers interacted weakly with n-pentylbenzene and n-butylbenzene. These alkylbenzenes were gradually removed from the monolayers upon area compression and were completely expelled at an area per lipid of ∼70 Å2. Surface pressure analysis indicated that the ability of hydrocarbons to penetrate the monolayers was enhanced in the order of n-butylbenzene < n-pentylbenzene < n-butylcyclohexane < n-hexadecane.

Determining the Dependence of Interfacial Tension on Molecular Area for Phospholipid Monolayers Formed at Silicone Oil-Water and Tricaprylin-Water Interfaces by Vesicle Fusion.

Phospholipid monolayers formed at oil-water interfaces have been used to explore biological interface properties. Thus, monolayer systems need to be quantitatively understood. Previously, we investigated the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers at silicone oil-water interfaces to determine the dependence of interfacial tension, γ, on the area per lipid, a, compared to that of the closely packed monolayers, acp. This study aims to develop a method to obtain the γ-a relationship from the γ-a/acp data by investigating POPC monolayers at the silicone oil-water and tricaprylin-water interfaces. Pendant drop tensiometry was used to obtain the dependence of γ on a/acp. Furthermore, by calculating the surface pressure, Π, from γ and multiplying a/acp with an estimated acp value, the dependence of Π on a was obtained. When a value approximately equal to the a of POPC bilayers was assigned to acp, the resultant Π-a profile partially or approximately completely overlapped with the Π-a isotherms obtained for the monolayers at the air-water interface using a Langmuir trough. The overlap for the silicone oil-water interface occurred at a ≤ 77 Å2, while that for the tricaprylin-water interface occurred in approximately the entire a region. The results indicate that the Π of the condensed monolayers is little affected by bulk oil. Thus, the γ-a relationship for the oil-water interface can be determined by comparing the compression isotherm with the one obtained for the air-water interface.

Domain Sorting in Giant Unilamellar Vesicles Adsorbed on Glass.

Giant unilamellar vesicles (GUVs) adsorb to a solid surface and rupture to form a planar bilayer patch. These bilayer patches are used to investigate the properties and functions of biological membranes. Therefore, it is crucial to understand the mechanisms of GUV adsorption. In this study, we investigate the adsorption of phase-separated GUVs on glass using fluorescence microscopy. GUVs containing liquid-ordered (Lo) and liquid-disordered (Ld) phases underwent domain sorting after adsorption. The Ld domain in the unbound region migrated to the highly curved region near the edge of the adsorbed region. Additionally, the Lo phase grew linearly along the edge of the adsorbed region, creating a thin ring-like domain. After the domain sorting event, the GUV ruptured to form a planar bilayer patch with circular-patterned domains in the initially adsorbed area. We found that domain sorting was promoted by increasing the extent of GUV deformation. These results suggest that both the Ld and Lo domains are reorganized for stabilizing the curved bilayer region in adsorbed GUVs.

Organic and inorganic mixed phase modification of a silver surface for functionalization with biomolecules and stabilization of electromotive force.

A solid-state potentiometric biosensor based on the organic and inorganic mixed phase modification of a silver surface is proposed. Stabilization of the electromotive force and functionalization with biomolecules on the sensing surface were simultaneously achieved using silver chloride chemically deposited with 1,3-diaminopropanetetraacetic acid ferric ammonium salt monohydrate and a self-assembled monolayer with oligonucleotide probes, respectively. The formation of silver chloride and adsorption of alkanethiol on the silver surface were confirmed with X-ray photoelectron spectroscopy. The resulting modified surface reduced the nonspecific binding of interfering biomolecules and achieved a high signal to noise ratio. The electromotive forces of the modified silver thin film electrodes were stable under constant chloride ion concentrations. Hybridization assays were performed to detect microRNA 146. The lower limit of detection was 0.1 pM because of the small standard deviation. The proposed biosensor could be useful as a disposable single-use sensor in medical fields such as liquid biopsies.

Determination of the Coverage of Phosphatidylcholine Monolayers Formed at Silicone Oil-Water Interfaces by Vesicle Fusion.

Phospholipid monolayers at oil-water interfaces are used for various biological applications and often formed by vesicle adsorption. However, the adsorbed structures are not well characterized; therefore, fundamental investigation on vesicle adsorption behavior is necessary for correct understanding of the monolayer systems. Herein, we investigated the adsorption of phosphatidylcholine vesicles onto silicone oil-water interfaces using fluorescence microscopy and pendant drop tensiometry. The interfacial monolayer coverage, S, was determined by assuming S = 1 for tightly packed monolayers. Adsorption for 10 min with a lipid concentration of 0.2 mM resulted in S ≈ 0.4. An increase in lipid concentration (0.5-2 mM) and adsorption time (1 h) moderately increased monolayer coverage (S ≈ 0.6). However, extended adsorption for 24 h only slightly increased coverage (S ≈ 0.7). Monolayers with an S < 0.6 were homogeneous, while those with an S > 0.6 were associated with several vesicular structures. The surface density of these bound vesicles increased with increasing S. We conclude that vesicles readily fused with the interface to form monolayers at S < 0.6 and that their fusogenicity considerably decreased at S > 0.6. These results demonstrate that the ability of vesicles to form monolayers is determined by the interfacial coverage.

Interaction Mechanisms of Giant Unilamellar Vesicles with Hydrophobic Glass Surfaces and Silicone Oil-Water Interfaces: Adsorption, Deformation, Rupture, Dynamic Shape Changes, Internal Vesicle Formation, and Desorption.

Phospholipid monolayers at oil-water interfaces are often obtained via vesicle adsorption. However, the interaction mechanisms of vesicles with these oil-water interfaces remain unclear. Herein, we studied the adsorption of giant unilamellar vesicles (GUVs) of approximately 2-5 µm diameter onto silicone oil-water interfaces and glass surfaces modified with hexamethyldisilazane (HMDS) and octadecyltrimethoxysilane (ODTMS) using fluorescence microscopy. The GUVs exhibited various modes of interaction, adsorbing on the silanized glass surfaces without sizable deformation, whereas GUVs bound to the silicone oil-water interface exhibited large deformation. After adsorption, GUV rupture occurred within 350, 110, and 3 ms on HMDS-modified glass, ODTMS-modified glass, and silicone oil-water interface, respectively. On glass surfaces, GUV rupture was often initiated and proceeded with pore formation near the surface. The monolayer patches formed by GUV rupture on HMDS-modified glass remained for at least 1 h over an area approximately twice of that estimated from the original GUV. On the ODTMS-modified glass and silicone oil surfaces, the monolayer patch structures disappeared in milliseconds owing to lipid diffusion across the interface. When adsorbed on the oil-water interface, the GUVs spontaneously underwent dynamic shape changes, internal vesicle formation, and desorption without rupture. Thus, it can be concluded that these different pathways arose from different lipid-surface affinities.

Binding of Lipopolysaccharide and Cholesterol-Modified Gelatin on Supported Lipid Bilayers: Effect of Bilayer Area Confinement and Bilayer Edge Tension.

Binding of amphiphilic molecules to supported lipid bilayers (SLBs) often results in lipid fibril extension from the SLBs. Previous studies proposed that amphiphiles with large and flexible hydrophilic regions trigger lipid fibril formation in SLBs by inducing membrane curvature via their hydrophilic regions. However, no experimental studies have verified this mechanism of fibril formation. In this work, we investigated the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted bilayer areas were employed to identify the mechanism of fibril generation. We show that the main cause of lipid fibril formation is an approximately 20% expansion in the bilayer area rather than increased membrane curvature. The data indicate that bilayer area confinement plays a critical role in morphological changes of SLBs even when bound amphiphilic molecules have a large hydrophilic domain. We also show that bilayer area change after LPS insertion is dependent on the patch shape of the SLB. When an SLB patch consists of a broad bilayer segment connected to a long thin streak, bilayer area expansion mainly occurs within the bilayer streak. The results indicate that LPS insertion causes net lipid flow from the broad bilayer region to the streak area. The differential increase in area is explained by the instability of planar bilayer streaks that originate from the large energetic contribution of line tension arising along the bilayer edge.

Induced rupture of vesicles adsorbed on glass by pore formation at the surface-bilayer interface.

Supported lipid bilayers (SLBs) are often formed by spontaneous vesicle rupture and fusion on a solid surface. A well-characterized rupture mechanism for isolated vesicles is pore nucleation and expansion in the solution-exposed nonadsorbed area. In contrast, pore formation in the adsorbed bilayer region has not been investigated to date. In this work, we studied the detailed mechanisms of asymmetric rupture of giant unilamellar vesicles (GUVs) adsorbed on glass using fluorescence microscopy. Asymmetric rupture is the pathway where a rupture pore forms in a GUV near the edge of the glass-bilayer interface with high curvature and then expansion of the pore yields a planar bilayer patch. We show that asymmetric rupture occasionally resulted in SLB patches bearing a defect pore. The defect formation probability depended on lipid composition, salt concentration, and pH. Approximately 40% of negatively charged GUVs under physiological conditions formed pore-containing SLB patches, while negatively charged GUVs at low salt concentration or pH 4.0 and positively charged GUVs exhibited a low probability of defect inclusion. The edge of the defect pore was either in contact with (on-edge) or away from (off-edge) the edge of the planar bilayer. On-edge pores were predominantly formed over off-edge defects. Pores initially formed in the glass-adsorbed region before rupture, most frequently in close contact with the edge of the adsorbed region. When a pore formed near the edge of the adsorbed area or when the edge of a pore reached that of the adsorbed area by pore expansion, asymmetric rupture was induced from the defect site. These induced rupture mechanisms yielded SLB patches with an on-edge pore. In contrast, off-edge pores were produced when defect pore generation and subsequent vesicle rupture were uncoupled. The current results demonstrate that pore formation in the surface-adsorbed region of GUVs is not a negligible event.

Induction of intermembrane adhesion by incorporation of synthetic adhesive molecules into cell membranes.

Modulation of cell adhesion by synthetic materials is useful for a wide range of biomedical applications. Here, we characterized cell adhesion mediated by a semisynthetic molecule, cholesteryl-modified gelatin (chol-gelatin). We found that this hybrid molecule facilitated cell adhesion by connecting two apposed membranes via multiple cholesterol moieties on the gelatin molecules, whereas unmodified gelatin did not bind to cell membranes. Analyses revealed that the rate of the formation of cell adhesions was increased by displaying more cholesterol moieties on the cell membrane. In contrast, the area of the cell adhesion site was unchanged by increasing the number of cholesterol molecules, suggesting that chol-gelatin may suppress cell spreading. Such restriction was not observed in cell adhesion mediated by the mutant of physiological adhesion protein CD2, which lacked its cytoplasmic domain and was unable to connect to cytoplasmic actin filaments, but had a similar affinity for its ligand compared with the chol-gelatin-cell membrane interaction. Further analysis suggested the restriction of cell spreading by chol-gelatin was largely independent of the modulation of the surface force, and thus we hypothesize that the restriction could be in part due to the modulation of cell membrane mechanics by membrane-incorporated chol-gelatin. Our study dissected the two roles of the hybrid molecule in cell adhesion, namely the formation of a molecular connection and the restriction of spreading, and may be useful for designing other novel synthetic agents to modulate various types of cell adhesions.

Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.

T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.

Packing density changes of supported lipid bilayers observed by fluorescence microscopy and quartz crystal microbalance-dissipation.

Various properties of supported lipid bilayers such as diffusion and lipid partitioning are well characterized. However, little attention has been paid to their molecular packing density. In this work, the adsorption of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) vesicles on glass and silicon dioxide was investigated using fluorescence microscopy, quartz crystal microbalance-dissipation (QCM-D), and atomic force microscopy. Fluorescence recovery after photobleaching data showed that the adsorption of large unilamellar vesicles (LUVs) on glass yielded supported bilayers with full mobility under alkaline (pH 8.3) and acidic (pH 3-4) conditions. These fluid bilayers exhibited quite different diffusion constants; those at alkaline pH were ~10 times larger than those at acidic pH. The reason for this pH dependence was clarified by investigation of the rupture of giant unilamellar vesicles (GUVs) on glass. Fluorescence data revealed that the area of planar bilayer patches increased at alkaline pH. Thus, we conclude that the rapid diffusion in alkaline solution arises from the decreased molecular density. QCM-D data showed that dissipation increased in a stepwise manner during vesicle fusion on silicon dioxide at alkaline pH. We attribute this behavior to the decrease in packing density of planar bilayers.

Detergent-mediated formation of polymer-supported phospholipid bilayers.

Supported phospholipid bilayers can be formed by established methods such as vesicle fusion and the Langmuir-Blodgett (LB) technique. However, challenges remain in regards to creating supported bilayers from various lipid compositions, using various support surfaces, and incorporating membrane proteins. Here we report a detergent removal method as an alternative means of supported bilayer formation. The process consists of three steps: (1) incubation of phospholipid-poly(ethylene glycol) (PEG)-grafted glass with lipid-detergent micelles; (2) detergent removal by washing the surface with vesicles; and (3) incubation with the vesicles to complete lipid adsorption. These procedures yielded fluid planar bilayers of zwitterionic lipids. Because fluid structures were not obtained by vesicle fusion, the detergent seemed necessary to produce the polymer-supported bilayers. While anionic phospholipids inhibited the attachment of fluid bilayers in the absence of calcium ions, supported bilayers with almost full mobility were obtained from lipid mixtures containing 10-20 mol % anionic lipids in the presence of calcium ions. The incorporation of the anionic lipids in the bulk-facing leaflet was demonstrated by the binding of dye-labeled annexin V.

Field-effect detection using phospholipid membranes.

The application of field-effect devices to biosensors has become an area of intense research interest. An attractive feature of field-effect sensing is that the binding or reaction of biomolecules can be directly detected from a change in electrical signals. The integration of such field-effect devices into cell membrane mimics may lead to the development of biosensors useful in clinical and biotechnological applications. This review summarizes recent studies on the fabrication and characterization of field-effect devices incorporating model membranes. The incorporation of black lipid membranes and supported lipid monolayers and bilayers into semiconductor devices is described.

Mechanisms of supported bilayer detection using field-effect devices.

The adsorption of positively charged supported lipid bilayers (SLBs) on field-effect devices was studied using various salt solutions to make it possible to understand the signal generation mechanisms of the devices. The flat-band voltage change that occurred with SLB formation was dependent on the type of monovalent cations contained in the solution. Zeta potential data showed that the intrinsic charge of the bilayers was almost constant in the presence of any of the examined alkali ions. The results suggest that the lipid charge does not solely determine the flat-band voltage. The binding affinity of alkali ions to silicon nitride showed a similar trend to the dependence of the flat-band voltage change on alkali ions. The results indicate that the specific interaction of small inorganic ions with the device surface has a significant influence on the magnitude of the signal response that occurs with SLB formation. It appears that salt ions specifically bound to the device surface and the charge of the bilayers both contribute to the signal generation mechanisms.

Detection of supported lipid bilayers using their electric charge.

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.