Multiple Lipid Bilayers Research Articles

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayersare useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Bacterial membrane vesicles with multiple lipid bilayers: vesicles harboring organelle-like structures.

Bacteria produce outer membrane vesicles (OMVs), which are spherical nanoparticles that are mainly composed of lipopolysaccharides, phospholipids, and outer membrane proteins. OMVs play critical biological roles in stress responses, microbial communication, and bacteria-host interactions. Additionally, they hold great potential for biotechnological applications because of their versatile function in molecular transport while protecting the endogenous substances. While OMVs have been considered lipid monolamellar vesicles for several decades, recent studies have shown that membrane vesicles (MVs) with multiple lipid bilayers, including outer-inner membrane vesicles, multilamellar vesicles, and multivesicular vesicles, are also produced by Gram-negative bacteria. Some internal vesicles contain cytoplasmic components such as DNA and thus function as organelle-like structures within MVs. This review provides recent findings regarding the biogenesis and properties of MVs with complex structures.

Liposomal membrane permeability assessment by fluorescence techniques: Main permeabilizing agents, applications and challenges.

Liposomes are lipid vesicles made of one or multiple lipid bilayers surrounding an internal aqueous core. They are broadly employed as models to study membrane structure and properties. Among these properties, liposome membrane permeability is crucial and widely assessed by fluorescence techniques. The first part of this review is devoted to describe the various techniques used for membrane permeability assessment. Attention is paid to fluorescence techniques based on vesicle leakage of self-quenching probes, dye/quencher pair or cation/ligand pair. Secondly, the membrane-active agents inducing membrane permeabilization is presented and details on their mechanisms of action are given. Emphasis is also laid on the intrinsic and extrinsic factors that can modulate the membrane permeability. Hence, a suitable liposomal membrane should be formulated according to the aim of the study and its application.

Structural Transformation of Amyloid Peptides Interacting with Lipid Membranes

The inherent cytotoxicity of protein aggregates implies a common mechanism for amyloid diseases (Bucciantini et al). However, accumulated evidence suggests that the insoluble fibrils or aggregates are not the culprit. On the other hand, amyloid peptides in soluble form do interact with lipid bilayers, suggesting that the cell plasma membranes are a target of amyloid pathogeneses. In particular, Keyed et al. have demonstrated that amyloid peptides all increase membrane ion conductance without any evidence of discrete channel or pore formation. In this study we try to find the common molecular process of soluble amyloid peptides interacting with lipid membranes that might induce membrane conductivity. It is difficult to study this molecular process for most amyloid peptides because of its propensity to fibrillize at relatively low solution concentration. PrP 106-126 is a random coil in its soluble form. We study its kinetics of structural transformation in the presence of lipid vesicles. The time dependence of the structural changes was analyzed as a function of the lipid concentration. We demonstrate that the soluble peptides transform from random coils to alpha-helices upon binding to lipid bilayers. The helical state is stable, as long as the bound peptide to lipid ratio P/L on the lipid vesicle is below a critical value P/L∗. But as P/L exceeds P/L∗, the peptides transform from the helical state to beta-aggregates. This is consistent with previous studies of penetration and hIAPP in multiple lipid bilayers. Thus we found the common fibrillization process of amyloid peptides interacting with lipid bilayers. Our proposal is that the process of peptides’ transformation from random coils to helices and then to beta-aggregation creates defects in the membranes that allow ion permeation to occur as observed by Keyed et al.

Anesthetic diffusion through lipid membranes depends on the protonation rate.

Hundreds of substances possess anesthetic action. However, despite decades of research and tests, a golden rule is required to reconcile the diverse hypothesis behind anesthesia. What makes an anesthetic to be local or general in the first place? The specific targets on proteins, the solubility in lipids, the diffusivity, potency, action time? Here we show that there could be a new player equally or even more important to disentangle the riddle: the protonation rate. Indeed, such rate modulates the diffusion speed of anesthetics into lipid membranes; low protonation rates enhance the diffusion for local anesthetics while high ones reduce it. We show also that there is a pH and membrane phase dependence on the local anesthetic diffusion across multiple lipid bilayers. Based on our findings we incorporate a new clue that may advance our understanding of the anesthetic phenomenon.

Dangerous Membranes: Viruses That Subvert Autophagosomes

Dangerous Membranes: Viruses That Subvert Autophagosomes

Efficient Photoelectrochemical Energy Conversion using Spinach Photosystem II (PSII) in Lipid Multilayer Films.

The need for clean, renewable energy has fostered research into photovoltaic alternatives to silicon solar cells. Pigment–protein complexes in green plants convert light energy into chemical potential using redox processes that produce molecular oxygen. Here, we report the first use of spinach protein photosystem II (PSII) core complex in lipid films in photoelectrochemical devices. Photocurrents were generated from PSII in a ∼2 μm biomimetic dimyristoylphosphatidylcholine (DMPC) film on a pyrolytic graphite (PG) anode with PSII embedded in multiple lipid bilayers. The photocurrent was ∼20 μA cm−2 under light intensity 40 mW cm−2. The PSII–DMPC anode was used in a photobiofuel cell with a platinum black mesh cathode in perchloric acid solution to give an output voltage of 0.6 V and a maximum output power of 14 μW cm−2. Part of this large output is related to a five-unit anode–cathode pH gradient. With catholytes at higher pH or no perchlorate, or using an MnO2 oxygen-reduction cathode, the power output was smaller. The results described raise the possibility of using PSII–DMPC films in small portable power conversion devices.

Formation of lipid bilayers inside microfluidic channel array for monitoring membrane-embedded nanopores of phi29 DNA packaging nanomotor

An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing.

Automation Highlights from the Literature

Automation Highlights from the Literature

TIRF Imaging of Lipid Bilayer Arrays

A TIRF imaging platform for the simultaneous interrogation of multiple lipid bilayers in an arrayed format has been developed. Here, a proof of principle demonstration is made with the quantification of Calcium flux through the trans membrane pore, alpha-hemolysin. The effect of pore blocking by cyclodextrins is also demonstrated. Utilising a droplet on hydrogel method of lipid bilayer formation, an array of multiple lipid bilayers are illuminated simultaneously by the evanescent field of laser light undergoing total internal reflection at the surface of the glass hydrogel support layer. The droplet based method of bilayer production is ideally suited to an array platform, with the segregated contents of each droplet affording the opportunity to incorporate various membrane proteins, or simultaneously screen a variety of molecular candidates for their interaction with incorporated membrane components. The system offers the opportunity of asymmetric bilayer generation and the option of rapidly screening environmental dependencies for protein function. The scalable and automatable nature of the approach, together with the ability to tailor droplet contents, creates many exciting opportunities for characterisation of membrane protein behaviour and ligand interactions, the focus of future studies with the platform.

Development of an Efficient Transdermal Delivery System of Small Interfering RNA Using Functional Peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI, not the MLB coat or lack thereof. In conclusion, the measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2000 MLBs/mm³, about half that reported for early stage nuclear cataracts from the US, the forward scattering could be more than 30% of the incident light. Therefore, the extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients.

Development of an automation technique for the establishment of functional lipid bilayer arrays

In the present work, a technique for establishing multiple black lipid membranes (BLMs) in arrays of micro structured ethylene tetrafluoroethylene (ETFE) films, and supported by a micro porous material was developed. Rectangular 8 × 8 arrays with apertures having diameters of 301 ± 5 µm were fabricated in ETFE Teflon film by laser ablation using a carbon dioxide laser. Multiple lipid membranes could be formed across the micro structured 8 × 8 array ETFE partitions. Success rates for the establishment of cellulose-supported BLMs across the multiple aperture arrays were above 95%. However, the time course of the membrane thinning process was found to vary considerably between multiple aperture bilayer experiments. An airbrush partition pretreatment technique was developed to increase the reproducibility of the multiple lipid bilayers formation during the time course from the establishment of the lipid membranes to the formation of bilayers. The results showed that multiple lipid bilayers could be reproducible formed across the airbrush-pretreated 8 × 8 rectangular arrays. The ionophoric peptide valinomycin was incorporated into established membrane arrays, resulting in ionic currents that could be effectively blocked by tetraethylammonium. This shows that functional bimolecular lipid membranes were established, and furthermore outlines that the established lipid membrane arrays could host functional membrane-spanning molecules.

Formation of Bilayer Lipid Membranes with Microfluidic Devices

Membrane proteins play very important roles in cells. They are also useful in various industrial fields, including drug discovery, and highly sensitive ion-channel-based biosensors. Here, I describe our two approaches toward membrane protein chips: an array of membrane proteins on (1) planar lipid bilayers and (2) giant vesicles. First, a highly reproducible method was developed for planar lipid bilayer reconstitution. Planar lipid bilayers are formed at apertures by flowing lipid organic solution and buffer alternately into an integrated microfluidic channel. Using this technique, multiple lipid bilayers are formed simultaneously, and channel currents through peptide ion channels were recorded. Second, I describe a method for the preparation of lipid vesicles inspired by forming soap bubbles from a soap film. In our “blowing” method, lipid vesicles are blown out of the pre-formed bio-functional planar lipid membrane, directly encapsulating ejected materials. We believe that these devices are useful for an efficient and rapid analysis of singlespecies-specific membrane proteins.

The epidermal permeability barrier.

The permeability barrier of the skin which prevents transcutaneous water loss and penetration of harmful drugs from the environment is localized in the horny layer of the epidermis. Multiple lipid bilayers obstructing the intercellular space of the stratum corneum fulfill this function. In contrast to cellular membranes consisting predominantly of phospholipids, these lamellae contain mostly ceramides, cholesterol and free fatty acids. The lamellae are derived from the contents of lamellar granules (LGs) which are synthesized in the viable epidermal layers by the keratinocytes. LGs display stacks of small disks each of which represents a flattened vesicle or liposome. Prior to terminal differentiation, the disks are exocytosed into the intercellular space and fused to form uninterrupted sheetlike lamellae. The singular lipid composition of LG-disks and of stratum corneum-lamellae reflects the multistage process of barrier formation. It also renders these structures well suited to provide for a barrier function.