Artificial Lipid Bilayers Research Articles

Electrochemical triggering of lipid bilayer lift-off oscillation at the electrode interface.

In situ studies of transmembrane channels often require a model bioinspired artificial lipid bilayer (LB) decoupled from its underlaying support. Obtaining free-standing lipid membranes is still a challenge. In this study, we suggest an electrochemical approach for LB separation from its solid support via hydroquinone oxidation. Layer-by-layer deposition of polyethylenimine (PEI) and polystyrene sulfonate (PSS) on the gold electrode was performed to obtain a polymeric nanocushion of [PEI/PSS]3/PEI. The LB was deposited on top of an underlaying polymer support from the dispersion of small unilamellar vesicles due to their electrostatic attraction to the polymer support. Since lipid zwitterions demonstrate pH-dependent charge shifting, the separation distance between the polyelectrolyte support and LB can be adjusted by changing the environmental pH, leading to lipid molecules recharge. The proton generation associated with hydroquinone oxidation was studied using scanning vibrating electrode and scanning ion-selective electrode techniques. Electrochemical impedance spectroscopy is suggested to be a powerful instrument for the in situ observation of processes associated with the LB-solid support interface. Electrochemical spectroscopy highlighted the reversible disappearance of the LB impact on impedance in acidic conditions set by dilute acid addition as well as by electrochemical proton release on the gold electrode due to hydroquinone oxidation.

Regulation of BK Channel Activity by Cholesterol and Its Derivatives.

Cholesterol (CLR) is an essential structural lipid in the plasma membrane of animal cells. In addition, CLR has been widely recognized as a critical modulator of protein function, including ion channels. Voltage- and Ca2+-gated K+ (BK) channels control a wide variety of physiological processes, including cell excitability, smooth muscle contractility, sensory perception, neurotransmitter release, and hormone secretion. Thus, disruption of BK currents has been implicated in the pathophysiology of prevalent human diseases. The current chapter reviews the literature documenting CLR modulation of BK channel function at a variety of levels ranging from organ systems to artificial lipid bilayers. We discuss the use of CLR isomers and structural analogs as a tool to help in discerning the mechanisms underlying CLR-driven modification of BK current. The chapter is finalized with an overview of the phenomenology and potential mechanisms that govern CLR control over the alcohol (ethyl alcohol, ethanol) sensitivity of BK channels. Studies on CLR regulation of BK currents may ultimately pave the way for novel therapeutic approaches to combat prevalent pathophysiological and morbid conditions.

Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes.

Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for imaging of patterns of electrical activity in tissue. Wide application of this technology depends on the availability of sensors with high sensitivity (percent change of fluorescence per 100 mV), high fluorescence quantum yield, and fast response kinetics. A promising approach uses a two-component system consisting of anionic membrane permeable quenchers with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, the quencher must be kept at low concentrations to minimize pharmacological effects, thus limiting sensitivity. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the sensitivity is maximized and is independent of VSD concentration. A series of 13 TBFQ dyes based on the aminonaphthylethenylpyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best of these, TBFQ1, shows a 2.5-fold change in fluorescence per 100 mV change in membrane potential, and the response kinetics is in the 10-20 ms range. This sensitivity is an order of magnitude better than that of commonly used VSDs. However, the fluorescence quantum yield is only 1.6%, which may make this first generation of TBFQ VSDs impractical for in vivo electrical imaging. Nevertheless, the design principles established here can serve as foundation for improved TBFQ VSDs. We believe this approach promises to greatly enhance our ability to monitor electrical activity in cells and tissues.

Disruption of artificial lipid bilayers in the presence of transition metal oxide and rare earth metal oxide nanoparticles

The unique properties of nanoparticles have made them popular materials for a wide variety of applications. Their prevalence has increased research attention to their potential toxicity, and multiple groups have found that certain nanoparticles are biologically hazardous and can induce autophagy, oxidative stress, nucleic acid damage, and cell membrane damage. In particular, multiple studies have investigated interactions between nanoparticles and cell membranes. Here, we use a high throughput artificial lipid bilayer platform capable of high resolution electrical measurements of bilayer conductance to screen for interaction between lipid bilayers and metal oxide and rare earth metal oxide nanoparticles 15–200 nm in diameter composed of: Fe3O4, Eu2O3, Er2O3, Co3O4, ZnO, La2O3, Gd2O3, CeO2, In2O3, CuO, HfO2, and TiO2. We found that all particles, with the exception of Fe3O4, TiO2, and HfO2, exhibited significant interactions with lipid bilayers, causing them to rupture a short time after addition to the solution adjacent to the bilayer. We measured this at several pH values and ionic strengths, finding, in agreement with prior work, that cationic nanoparticle surface charge correlated strongly with nanoparticle-induced disruption of lipid bilayers.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24hr of infection, virulent R.equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R.equi to multiply. This observation is mirrored in the fact that virulent and avirulent R.equi multiply well in extracts of purified lysosomes at pH7.2 but not at pH5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

G-Protein Coupled Receptor Protein Synthesis on a Lipid Bilayer Using a Reconstituted Cell-Free Protein Synthesis System.

Membrane proteins are important drug targets which play a pivotal role in various cellular activities. However, unlike cytosolic proteins, most of them are difficult-to-express proteins. In this study, to synthesize and produce sufficient quantities of membrane proteins for functional and structural analysis, we used a bottom-up approach in a reconstituted cell-free synthesis system, the PURE system, supplemented with artificial lipid mimetics or micelles. Membrane proteins were synthesized by the cell-free system and integrated into lipid bilayers co-translationally. Membrane proteins such as the G-protein coupled receptors were expressed in the PURE system and a productivity ranging from 0.04 to 0.1 mg per mL of reaction was achieved with a correct secondary structure as predicted by circular dichroism spectrum. In addition, a ligand binding constant of 27.8 nM in lipid nanodisc and 39.4 nM in micelle was obtained by surface plasmon resonance and the membrane protein localization was confirmed by confocal microscopy in giant unilamellar vesicles. We found that our method is a promising approach to study the different classes of membrane proteins in their native-like artificial lipid bilayer environment for functional and structural studies.

Exploring photosensitization as an efficient antifungal method

Lipid bilayers containing ergosterol show signs of destruction when they are treated with singlet oxygen, due to the conversion of ergosterol into its peroxy derivative. Applying this previous knowledge, an antifungal method was explored using Candida tropicalis as model, and membrane permeation under photosensitization conditions became evident. These data were complemented through AFM images of artificial lipid bilayers, using cholesterol or ergosterol as structural sterols, showing their corresponding morphologies at the nanoscale. Based on these results, an antifungal method was developed, which shows evidence of the extent of membrane permeation during photosensitization. Such photosensitization offers an effective alternative treatment, especially in membranes with a high ergosterol content, suggesting that this procedure constitutes an easy and efficient antifungal method.

Clostridium perfringens Enterotoxin: The Toxin Forms Highly Cation-Selective Channels in Lipid Bilayers

One of the numerous toxins produced by Clostridium perfringens is Clostridium perfringens enterotoxin (CPE), a polypeptide with a molecular mass of 35.5 kDa exhibiting three different domains. Domain one is responsible for receptor binding, domain two is involved in hexamer formation and domain three has to do with channel formation in membranes. CPE is the major virulence factor of this bacterium and acts on the claudin-receptor containing tight junctions between epithelial cells resulting in various gastrointestinal diseases. The activity of CPE on Vero cells was demonstrated by the entry of propidium iodide (PI) in the cells. The entry of propidium iodide caused by CPE was well correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. CPE formed ion-permeable channels in artificial lipid bilayer membranes with a single-channel conductance of 620 pS in 1 M KCl. The single-channel conductance was not a linear function of the bulk aqueous salt concentration indicating that point-negative charges at the CPE channel controlled ion transport. This resulted in the high cation selectivity of the CPE channels, which suggested that anions are presumably not permeable through the CPE channels. The possible role of cation transport by CPE channels in disease caused by C. perfringens is discussed.

Interaction between the barley allelochemical compounds gramine and hordenine and artificial lipid bilayers mimicking the plant plasma membrane

Some plants affect the development of neighbouring plants by releasing secondary metabolites into their environment. This phenomenon is known as allelopathy and is a potential tool for weed management within the framework of sustainable agriculture. While many studies have investigated the mode of action of various allelochemicals (molecules emitted by allelopathic plants), little attention has been paid to their initial contact with the plant plasma membrane (PPM). In this paper, this key step is explored for two alkaloids, gramine and hordenine, that are allelochemicals from barley. Using in vitro bioassays, we first showed that gramine has a greater toxicity than hordenine towards a weed commonly found in northern countries (Matricaria recutita L.). Then, isothermal titration calorimetry was used to show that these alkaloids spontaneously interact with lipid bilayers that mimic the PPM. The greater impact of gramine on the thermotropic behaviour of lipids compared to hordenine was established by means of infrared spectroscopy. Finally, the molecular mechanisms of these interactions were explored with molecular dynamics simulations. The good correlation between phytotoxicity and the ability to disturb lipid bilayers is discussed. In this study, biophysical tools were used for the first time to investigate the interactions of allelochemicals with artificial PPM.

Computational Modeling and Validation of Thermally Induced Electrical Capacitance Changes for Lipid Bilayer Membranes Irradiated by Pulsed Lasers.

Neural stimulation has widespread applications in investigating brain functions, restoring impaired neural functions, and treating numerous neurological/psychiatric diseases. Use of infrared pulses to stimulate neurons (infrared neural stimulation) offers a direct and non-invasive technique. Recent research has demonstrated that transient heating associated with the absorption of infrared light by the local aqueous medium around the cell membrane can stimulate nerves. One mechanism for this stimulation is due to a thermally induced increase in the membrane electrical capacitance, which causes cell depolarization as well as action potential production under certain physiological conditions. A theoretical and computational model helps better understand the mechanism of thermally induced electrical capacitance changes and optimize the stimulus parameters. In this article, we develop the existing theoretical models for membrane electrical capacitance and its thermally induced changes. We improve the formulation of Gouy-Chapman-Stern theory by Genet et al. and Shapiro et al. with the addition of a diffuse layer to the electrical double layer and by modifying the relation of Stern layer capacitance, to calculate the membrane capacitive charge and capacitive current. We also present a new method to calculate the membrane electrical capacitance and the rate of its thermally induced changes. In our calculations, two new factors are considered including the temperature dependence of the surface charge density and the hydrophobic core dielectric constant of the lipid bilayer. Our developed model predicts rates of 0.3 and 0.26%/°C for the thermally induced capacitance changes of the artificial lipid bilayer under two different sets of conditions previously reported by Shapiro et al. and Carvalho-de-Souza et al., respectively. Our model is in very good agreement with the corresponding experimental values given by these groups. The presented model is also able to calculate the membrane capacitive currents and investigate the voltage dependence of this current.

Evidence from ITIR-FCS Diffusion Studies that the Amyloid-Beta (Aβ) Peptide Does Not Perturb Plasma Membrane Fluidity in Neuronal Cells

The amyloid-beta (Aβ) peptide, commonly found in elevated levels in the brains of patients with Alzheimer's disease (AD) and in the cerebrospinal fluid of individuals presenting mild cognitive impairment, is thought to be one of the major factors resulting in the onset of AD. Although observed and studied at the molecular level for several decades, the exact disease pathology of AD is still not totally clear. One way in which Aβ is thought to affect neurons is by influencing cell membrane fluidity, which could result in abnormal synaptic or signaling function. The effects of Aβ on the fluidity of biological membranes have been studied using numerous membrane models such as artificial lipid bilayers and vesicles, living cells and membranes extracted from animal models of AD, yet there is still no consensus as to what effects Aβ has, if any, on membrane fluidity. As one of the most precise and accurate means of assaying membrane dynamics, we have thus chosen fluorescence correlation spectroscopy to investigate the issue, using fluorescent membrane-targeted probes on living cells treated with Aβ(1–42) oligomers and observing possible changes in membrane diffusion. Effects of Aβ on viability in different cell types varied from no detectable effect to extensive cell death by 72 h post-exposure. However, there was no change in the fluidity of either ordered membrane domains or the bulk membrane in any of these cells within this period. Our conclusion from these results is that perturbation of membrane fluidity is not likely to be a factor in acute Aβ-induced cytotoxicity.

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane.

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.

Patterning artificial lipid bilayer on nanostructured surfaces.

Artificial lipid bilayer on solid substrate plays an important role as an interface between nanotechnology and biology. In this study, grid structures were patterned on Au-Nb-glass substrate and artificial bilayer was prepared on these structures. The fluidity was checked using fluorescence recovery after photobleaching (FRAP), and neuronal adhesion was monitored on such structure using EphrinA5-tethered lipid bilayer. EphrinA5 is a ligand that binds to the Eph receptors of rat cortical neurons and influences cellular adhesion. Our result elucidated that influence of these nanopatterned protein-tethered lipid bilayer on cellular guidance and signaling can address many underlying mechanisms of cellular functioning and help us to understand and differentiate the signaling procedure in cancer and neurodegenerative diseases.

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Cholesterol induced asymmetry in DOPC bilayers probed by AFM force spectroscopy

Cholesterol induced mechanical effects on artificial lipid bilayers are well known and have been thoroughly investigated by AFM force spectroscopy. However, dynamics of cholesterol impingement into bilayers at various cholesterol concentrations and their effects have not been clearly understood. In this paper we present, the effect of cholesterol as a function of its concentration in a simple single component dioleoylphosphatidylcholine (DOPC) bilayer. The nature of measured breakthrough forces on a bilayer with the addition of cholesterol, suggested that it is not just responsible to increase the mechanical stability but also introduces irregularities across the leaflets of the bilayer. This cholesterol induced asymmetry across the (in the inner and outer leaflets) bilayer is related to the phenomena of interleaflet coupling and is a function of cholesterol concentration probed by AFM can provide an unprecedented direction on mechanical properties of lipid membrane as it can be directly correlated to biophysical properties of a cell membrane.

The Molecular Structure of Human Red Blood Cell Membranes From Highly Oriented, Solid Supported Multi-Lamellar Membranes

The preparation of Red Blood Cell (RBC) Ghosts is a well-known protocol in biological and medical research and describes the extraction of the membrane from RBCs. Another well-known protocol is the preparation of highly ordered stacks of artificial lipid bilayers on silicon wafers. There are various attempts to adapt this protocol to a native cell membrane. For the first time we were able to combine both described techniques and to prepare highly ordered stacks of RBC membranes on silicon wafers [1]. These systems can now be used as inexpensive and safe platforms for testing the effect of drugs and bacteria on RBC membranes in-vitro using biophysical techniques, such as X-ray and neutron diffraction, optical spectroscopy and atomic force microscopy. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. [1] Himbert S. et al., The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes, Scientific Reports 7, Article number: 39661 (2017).

The Study of Platelet Receptors Using Artificial Lipid Bilayers.

Artificial lipid bilayers are powerful tools that can be used to model the interactions between platelets and membrane-bound ligands. To mimic the interaction of platelets with membrane-bound ligands, biotinylated lipids can be used to couple monobiotinylated recombinant ligands to the upper leaflet of an artificial lipid bilayer using streptavidin to bridge the two. Artificial lipid bilayers are generated by preparing liposomes, treating glass coverslips to make them hydrophilic and by assembling the bilayer in a specialized flow chamber. Finally platelets can be added to the flow chamber and the localization of fluorescently labeled molecules followed using microscopy.

RyR2 Inhibition by Dantrolene Requires both Calmodulin and FKBP12.6

Dantrolene protects cardiac muscle from arrhythmia and heart failure by inhibiting Ca2+ release from the sarcoplasmic reticulum (SR). Single-channel recordings were obtained from SR calcium release channels (RyR2) isolated from sheep and human heart and incorporated into artificial lipid bilayers. Dantrolene inhibition was measured by exposing RyR2 both in the absence and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2+] (100 nM), dantrolene (50 μM) reduced the mean open probability of RyR2 to 45 ± 6% in the presence of CaM (n = 7, p<0.05, Student's paired t-test) but had no significant effect when CaM was absent (95 ± 9%, n = 20, p = 0.24). Dantrolene exhibited a hyperbolic dose response in the presence of CaM with an IC50 of 0.16 ± 0.03 μM and with a saturating relative Po (Emax) of 52 ± 4%. Emax increased to 1 as cytoplasmic Ca2+ was increased to levels >1 μM. In saponin-permeablised mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene reduced the frequency and amplitude of Ca2+ waves, with an IC50 of 0.3 μM. However, when cells were depleted of CaM, dantrolene had no effect. Thus, CaM is essential for inhibitory action of dantrolene. Incubation of RyR2 to rapamycin prior to single-channel recording, known to dissociate FKBP12.6 from RyR2, removed the inhibitory action of dantrolene. This could be restored by adding FKBP12.6 to the bathing media. We conclude that the dantrolene inhibition required the presence of both CaM and FKBP12.6 in the RyR2 complex.

Imaging the Interactions Between B Cells and Antigen-Presenting Cells.

In vivo, B cells are often activated by antigens that are displayed on the surface of antigen-presenting cells (APCs). Binding of membrane-associated antigens to the B cell receptor (BCR) causes rapid cytoskeleton-dependent changes in the spatial organization of the BCR and other B cell membrane proteins, leading to the formation of an immune synapse. This process has been modeled using antigens attached to artificial planar lipid bilayers or to plasma membrane sheets. As a more physiological system for studying B cell-APC interactions, we have expressed model antigens in easily transfected adherent cell lines such as Cos-7 cells. The model antigens that we have used are a transmembrane form of a single-chain anti-Igκ antibody and a transmembrane form of hen egg lysozyme that is fused to a fluorescent protein. This has allowed us to study multiple aspects of B cell immune synapse formation including cytoskeletal reorganization, BCR microcluster coalescence, BCR-mediated antigen gathering, and BCR signaling. Here, we provide protocols for expressing these model antigens on the surface of Cos-7 cells, transfecting B cells with siRNAs or withplasmids encoding fluorescent proteins, using fixedcell and live cell fluorescence microscopy to image B cell-APC interactions, and quantifying APC-induced changes in BCR spatial organization and signaling.