Formation Of Planar Bilayers Research Articles

Mechanical characterization of freestanding lipid bilayers with temperature-controlled phase.

Coexistence of lipid domains in cell membranes is associated with vital biological processes. Here, we investigate two such membranes: a multi-component membrane composed of DOPC and DPPC lipids with gel and fluid separated domains, and a single component membrane composed of PMPC lipids forming ripples. We characterize their mechanical properties below their melting point, where ordered and disordered regions coexist, and above their melting point, where they are in fluid phase. To conduct these inquiries, we create lipid bilayers in a microfluidic chip interfaced with a heating system and optical tweezers. The chip features a bubble trap and enables high-throughput formation of planar bilayers. Optical tweezers experiments reveal interfacial hydrodynamics (fluid-slip) and elastic properties (membrane tension and bending rigidity) at various temperatures. For PMPC bilayers, we demonstrate a higher fluid slip at the interface in the fluid-phase compared to the ripple phase, while for the DOPC:DPPC mixture, similar fluid slip is measured below and above the transition point. Membrane tension for both compositions increases after thermal fluidization. Bending rigidity is also measured using the forces required to extend a lipid nanotube pushed out of the freestanding membranes. This novel temperature-controlled microfluidic platform opens numerous possibilities for thermomechanical studies on freestanding planar membranes.

Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers.

Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single-channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added. © 2018 by John Wiley & Sons, Inc.

Effect of Concentration on the Interfacial and Bulk Structure of Ionic Liquids in Aqueous Solution.

Bio and aqueous applications of ionic liquids (IL) such as catalysis in micelles formed in aqueous IL solutions or extraction of chemicals from biologic materials rely on surface-active and self-assembly properties of ILs. Here, we discuss qualitative relations of the interfacial and bulk structuring of a water-soluble surface-active IL ([C8MIm][Cl]) on chemically controlled surfaces over a wide range of water concentrations using both force probe and X-ray scattering experiments. Our data indicate that IL structuring evolves from surfactant-like surface adsorption at low IL concentrations, to micellar bulk structure adsorption above the critical micelle concentration, to planar bilayer formation in ILs with <1 wt % of water and at high charging of the surface. Interfacial structuring is controlled by mesoscopic bulk structuring at high water concentrations. Surface chemistry and surface charges decisively steer interfacial ordering of ions if the water concentration is low and/or the surface charge is high. We also demonstrate that controlling the interfacial forces by using self-assembled monolayer chemistry allows tuning of interfacial structures. Both the ratio of the head group size to the hydrophobic tail volume as well as the surface charging trigger the bulk structure and offer a tool for predicting interfacial structures. Based on the applied techniques and analyses, a qualitative prediction of molecular layering of ILs in aqueous systems is possible.

Lipid-Dependent Activation and Desensitization Mechanism of MthK

MthK is a prokaryotic Ca2+-activated K+ channel with several high-resolution structures, and serves as a model for investigating gating mechanisms of K+ channels. Previous investigations using proteoliposome-based Tl+ flux assay indicated that MthK underwent desensitization in seconds after Ca2+ activation. In contrast, MthK studied with electrophysiology in a planar lipid bilayer system show no evidence of desensitization. We hypothesized that this is caused by the different bilayer properties between the two preparations. The lipid composition of the bilayers in both preparations is the same (DOPC:POPG, 3:1). However, the differences in curvatures (liposomes vs. planar bilayers) and thicknesses (decane, used for planar bilayer formation, leads to increased bilayer thickness) may affect channel gating. Since MthK does not appear to desensitize in the thicker, decane-containing planar bilayers, we expect that increasing the bilayer thickness in liposomes will cause slower or no desensitization in the flux assays. Conversely, decreasing the bilayer thickness in the planar bilayers will lead to desensitization in the electrophysiology assays. We performed planar bilayer and Tl+ flux experiments using MthK reconstituted in liposomes made with lipids with different lipid acyl chain lengths (PC, from C16:1 to C22:1) while maintaining the POPG mole fraction. Changing the acyl chain length did not affect the voltage-dependent gating behavior of MthK when tested in planar bilayers. In addition, MthK did not undergo desensitization in any lipid compositions tested. On the contrary, varying the acyl chain length affected both MthK activation and inactivation in flux assays. We found that MthK inactivates more slowly in liposomes with longer acyl chain lengths (C20:1 and C22:1), and faster in liposomes with shorter acyl chain lengths (C16:1). Additionally, MthK activation was markedly slower in liposomes with longer acyl-chain lipids. The mechanism for lipid-dependent activation and desensitization of MthK is under investigation.

Fabrication of a Planar Zwitterionic Lipid Bilayer on Titanium Oxide

There is great demand to fabricate planar phospholipid bilayers on biocompatible materials. The preferred method of forming bilayers on these substrates is the spontaneous adsorption and rupture of phospholipid vesicles. However, in the case of titanium oxide, model vesicles composed solely of zwitterionic phospholipids do not follow this self-assembly pathway under physiological conditions, prompting the use of complex bilayer materials and less-facile methods. Herein, we report a novel pH-based strategy for fabricating zwitterionic bilayers on titanium oxide in a simple and robust manner. Depending on the pH conditions under which lipid vesicles adsorb onto titanium oxide, quartz crystal microbalance-dissipation (QCM-D) monitoring demonstrated that the self-assembly pathway can in fact result in planar bilayer formation. The pH of the solution could then be adjusted to physiological levels with no effect on the mass and viscoelastic properties of the bilayer. Moreover, fluorescence recovery after photobleaching (FRAP) measurements indicated a high degree of lateral lipid diffusivity within the bilayer at physiological pH, commensurate with its role as a cell membrane mimic. Compared to existing protocols, this strategy permits the fabrication of a more diverse array of planar bilayers on titanium oxide by tuning the self-assembly pathway of lipid vesicle adsorption onto solid substrates.

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 ∼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.

Induced structural changes of a supported planar bilayer after exposure to halothane — A real-time atomic force microscopy study

Atomic force microscopy (AFM) was used to study the effect of the general anesthetic halothane on a supported DOPC phospholipid bilayer, under conditions of high anaesthetic loading. The presence of the anaesthetic in the lipid-induced restructuring of the supported bilayer, erosion of the bilayer from the mica surface, and the formation of lipid aggregates. Addition of halothane to lipid vesicles prior to adsorption on the mica surface inhibited the lysis of adsorbed vesicles and thus no planar bilayer formation was observed.Key words: lipid bilayer restructuring, atomic force microscopy, halothane, anesthesis.

AFM Visualization of Mobile Influenza A M2 Molecules in Planar Bilayers

We report the observation of influenza A M2 (M2) incorporated in a dipalmitoylphosphatidylcholine (DPPC) supported planar bilayer on mica, formed by use of a modified vesicle fusion method from proteoliposomes and visualized with contact mode atomic force microscopy. Incubation of proteoliposomes in a hyperosmotic solution and increased DPPC/M2 weight ratios improved supported planar bilayer formation by M2/DPPC proteoliposomes. M2's extra-bilayer domains were observed as particles estimated to protrude 1–1.5 nm above the bilayer surface and <4 nm in diameter. Particle density was 5–18% of the nominal tetramer density. Movement of observable M2 particles was independent of the probe tip. The mean lateral diffusion coefficient ( D) of M2 was 4.4 ± 1.0 × 10 −14 cm 2/s. Eighty-two percent of observable particles were mobile on the observable timescale ( D > 6 × 10 −15 cm 2/s). Protein-protein interactions were also observed directly.

Adhesion and rupture of liposomes mediated by electrostatic interaction monitored by thickness shear mode resonators.

Adhesion and spreading of negatively charged unilamellar vesicles composed of POPG/POPC and DPPG/DPPC on positively charged self-assembly monolayers of 11-amino-1-undecanethiol were monitored by means of thickness shear mode (TSM) resonators with a fundamental frequency of 5 MHz. Changes of frequency and motional resistance upon vesicle adsorption were recorded as a function of surface charge density and lyotropic phase state of the lipids. From the readout of the TSM resonator, changes of the shape of the vesicles as well as the formation of supported lipid bilayers can be inferred in a quantitative manner. Increasing surface charge densities on the vesicles, which are tunable by the POPG content, led to decreasing frequency and resistance changes. At very high PG content, a lower limit of 3-12 Hz was found, indicative of the formation of planar bilayers due to vesicle rupture induced by the strong electrostatic interaction forces. Vesicles composed of DPPG/DPPC were less susceptible to deformation and rupture, a fact that can be attributed to the higher bending rigidity of DPPG/DPPC liposomes. More than 70 mol% of DPPG were needed to induce adhesion-controlled rupture of surface-attached vesicles, while only 30-50% of POPG were sufficient to form planar lipid bilayers on the quartz.

Supported planar bilayer formation by vesicle fusion: the interaction of phospholipid vesicles with surfaces and the effect of gramicidin on bilayer properties using atomic force microscopy

We have used magnetic alternating current mode atomic force microscopy (MAC–AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.

Liposome and phospholipid adsorption on a platinum surface studied in a flow cell designed for simultaneous quartz crystal microbalance and ellipsometry measurements

A flow cell was designed for simultaneous mass measurements with a quartz crystal microbalance and ellipsometry and was used for studying the adsorption processes of different liposomes, prepared from phospholipids and cholesterol, on both bare and lipid monolayer-covered platinum surfaces. Ellipsometric and gravimetric data were collected during recirculation of liposomes in 0.15 M NaCl. Experiments with ATP-containing liposomes with subsequent lysis were also performed. The results are discussed in terms of both intact liposome adhesion and intact liposome adhesion in combination with planar bilayer formation.

Lipid vesicle adsorption versus formation of planar bilayers on solid surfaces

The absorption and spreading behavior of lipid vesicles composed of either palmitoyloleoylphosphatidylcholine (POPC) or Escherichia coli lipid upon contact with a glass surface was examined by fluorescence measurements. Fluorescently labeled lipids were used to determine 1) the amount of lipid adsorbed at the surface, 2) the extent of fusion of the vesicles upon contact with the surface, 3) the ability of the adsorbed lipids to undergo lateral diffusion, and 4) the accessibility of the adsorbed lipids by external water soluble molecules. The results of these measurements indicate that POPC vesicles spread on the surface and form a supported planar bilayer, whereas E. coli lipid vesicles adsorb to the surface and form a supported vesicle layer. Supported planar bilayers were found to be permeable for small molecules, whereas supported vesicles were impermeable and thus represented immobilized, topologically separate compartments.

Formation of planar bilayers from artificial or native membrane vesicles

FEBS LettersVolume 122, Issue 1 p. 77-79 Full-length articleFree Access Formation of planar bilayers from artificial or native membrane vesicles Hansgeorg Schindler, Hansgeorg Schindler Biozentrum, University of Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this author Hansgeorg Schindler, Hansgeorg Schindler Biozentrum, University of Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this author First published: December 15, 1980 https://doi.org/10.1016/0014-5793(80)80405-4Citations: 135AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 H. Schindler, U. Quast, Proc. Natl. Acad. Sci. USA, 77, (1980), 3052– 3056. 2 N. Nelson, R. Anholt, J. Lindstrom, M. Montal, Proc. Natl. Acad. Sci. USA, 77, (1980), 3057– 3061. 3 H. Schindler, J.P. Rosenbusch, Proc. Natl. Acad Sci. USA, 75, (1978), 3751– 3755. 4 H. Schindler, Biochim. Biophys. Acta, 555, (1979), 316– 336. 5 C. Huang, Biochemistry, 4, (1969), 344– 352. 6 H. Schindler, G. Feher, Biophys. J., 16, (1976), 1109– 1113. Citing Literature Volume122, Issue1December 15, 1980Pages 77-79 ReferencesRelatedInformation