Giant Liposomes Research Articles

Barium, Tea and Sodium Sensitive Potassium Channels are Present in the Human Placental Syncytiotrophoblast Apical Membrane

The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno–fetal exchange. There is ample evidence suggesting the presence of potassium (K +) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K + currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K + channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba +2). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba +2 respectively. The more frequent conductance was approximately 73 pS, however several levels of current were detected suggesting the presence of more than one type of K + channel. In addition, sodium (Na +) sensitivity was detected in the patch current thus, over 10 mmol/L Na + reduced the seal current to 38%. These results were corroborated by the total current experiments where the K + current elicited in injected oocytes with apical purified membrane was blocked by Ba +2 and TEA. The total current was also affected by Na +, becoming larger when a Na +-free solution was used. Our results show the existence of at least two types of Ba +2-sensitive K + channels including a TEA sensitive sub-population, and some of them Na + sensitive K + channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K + channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

Permeability Study of Lipid-Coated Capsules with Confocal Laser Scanning Microscope (CLSM)

Abstract Based on our past work on the successfully fabricating giant liposomes, it is necessary to systematically study the capsules’ permeability. In this paper, fluorescence recovery after photo-bleaching (FRAP) experiment was carried out by confocal laser scanning microscope (CLSM) in order to quantify the 6-carboxyfluorescein (6-CF) molecules passing through capsules’ wall. The results showed that the diffusion coefficient of the pure multilayered polyelectrolyte (PE) capsules was similar to the one of partly the lipid-coated PE capsules. The diffusion coefficient for completely coated PE capsules catalyzed selectively by phospholipase A2 is similar to that of partly coated PE capsules.

Cold field emission gun-scanning electron microscopy: A new tool for morphological and ultrastructural analysis of liposomes

Liposomes are lipid vesicles largely investigated in the past 30 years as pharmaceutical carriers. In the development of new liposome-based formulations, the study of liposome surface properties remains a crucial step. For this purpose, microscopy techniques can provide useful information, although each such technique suffers from some limitations. Here, we have used cold field emission gun-scanning electron microscopy (cFEG-SEM) to acquire detailed images of liposome surface. In particular, we observed PEGylated and non-PEGylated liposomes in different size ranges. In the case of nanosized liposomes (mean diameter about 200 nm), a morphological evaluation of the whole preparation was obtained. On the other hand, in the case of giant liposomes (mean diameter about 2 μm), it was possible to observe the different surface ultrastructures of the two formulations. In particular, a regular and only slightly wrinkled surface was observed in the case of non-PEGylated liposomes, while a very irregular surface ultrastructure was visible in the case of PEGylated liposomes. This study shows, for the first time, the potential of cFEG-SEM as a new and powerful tool to obtain information on liposome morphology and, at least in the case of giant liposomes, on ultrastructure of the liposome surface.

Location of Magnetic and Fluorescent Nanoparticles Encapsulated inside Giant Liposomes

In this paper, we demonstrate the production of highly magnetic and fluorescent giant vesicles by encapsulating gamma-Fe2O3-rhodamine B nanoparticles. The liposomes containing the nanoparticles were made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We found that the ionic strength of the initial magnetic fluid is a crucial parameter in controlling the physicochemical properties of the bilayer. At high ionic strength, we obtained very important deformations of liposomes with high magnetic susceptibilities induced by an applied magnetic field. The encapsulation rate was studied using magnetophoresis and photobleaching tests, and the membrane properties were studied using confocal microscopy and elastic measurements.

Aldehyde-encapsulating liposomes impair marine grazer survivorship

In the last decade, there has been an increased awareness that secondary metabolites produced by marine diatoms negatively impact the reproductive success of their principal predators, the copepods. Several oxylipins, products of the enzymatic oxidation of fatty acids, are produced when these unicellular algae are damaged, as occurs during grazing. In the past, the dinoflagellate Prorocentrum minimum, which does not produce the oxylipin 2-trans,4-trans-decadienal (DD), has been used as a live carrier to calculate daily ingestion rates of this molecule by copepod crustaceans. However, since the interaction between oxylipins and live carriers is unknown, the question as to how much and for how long ingestion of these molecules affects copepod reproduction remains a critical point to understanding the functional role of such compounds at sea. In the investigation presented here we used giant liposomes ( approximately 7 mum) as a delivery system for the oxylipin DD, prepared in the same size range as copepod food and containing known amounts of DD. The aim of this work was to relate the ingestion of DD to the reproductive failure of the copepods Temora stylifera and Calanus helgolandicus. Liposomes were very stable over time and after 10 days of feeding, liposomes encapsulating DD reduced egg hatching success and female survival with a concomitant appearance of apoptosis in both copepod embryos and female tissues. Concentrations of DD inducing blockage were one order of magnitude lower that those used in classical feeding experiments demonstrating that liposomes are a useful tool to quantitatively analyze the impact of toxins on copepods.

Lipid Bilayer Elasticity Measurements in Giant Liposomes in Contact with a Solubilizing Surfactant

A new method to probe the modification of the elasticity of phospholipid bilayers is presented. The purpose here concerns the action of a solubilizing surfactant on a vesicle bilayer. This method is based on the measure of the under-field elongation of giant magnetic-fluid-loaded liposomes. The addition of the nonionic surfactant octyl-beta-d-glucopyranoside (OG) to vesicles at sublytic levels increases the elasticity of the membrane, as shown by the value of the bending modulus K(b), which decreases. K(b) measured around 20 kT for a pure 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer indeed reaches a few kT in the case of the mixed OG-DOPC bilayer. The purpose and interest of this study are to allow the determination of the membrane bending modulus before and after the addition of OG on the same magnetic liposome. Moreover, the experimental conditions used in this work allow the control of lipid and surfactant molar fractions in the mixed aggregates. Then, optical microscopy observation can be performed on samples in well-defined regions of the OG-phospholipid state diagram.

Patch clamp characterization of a non‐selective cation channel of ER membranes purified from Beta vulgaris taproots

The ER fraction from red beet taproot was purified on sucrose gradient and giant liposomes, suitable for patch clamping, were formed by dehydration-rehydration of the lipid film. Single-channel recordings on excised and attached patches revealed a large conductance (165 pS) cation (P(Cl-)/P(K+) < 0.03) channel with equal conductance and relative permeability for Na+ and K+. This non-selective cation channel was also highly permeable for Ca2+. We failed to detect any single-channel currents activated by a direct application of d-myo-inositol 1,4,5 trisphosphate, despite the fact that the ER membranes were native.

2P-231 微小操作技術によるジャイアントリポソームからの膜チューブ形成実験の理論解析(生体膜/人工膜・ダイナミクス,第46回日本生物物理学会年会)

2P-231 微小操作技術によるジャイアントリポソームからの膜チューブ形成実験の理論解析(生体膜/人工膜・ダイナミクス,第46回日本生物物理学会年会)

2S8-2 巨大リポソームを用いて観察された様々な条件下で起きる膜穿孔(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-2 巨大リポソームを用いて観察された様々な条件下で起きる膜穿孔(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-7 Making giant liposomes of controlled size and composition : toward de novo cells(2S8 Giant Liposome Research Front Line,The 46th Annual Meeting of the Biophysical Society of Japan)

2S8-7 Making giant liposomes of controlled size and composition : toward de novo cells(2S8 Giant Liposome Research Front Line,The 46th Annual Meeting of the Biophysical Society of Japan)

2S8-3 単一GUV法を用いたペプチド/蛋白質と脂質膜の相互作用の研究(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-3 単一GUV法を用いたペプチド/蛋白質と脂質膜の相互作用の研究(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-1 MEMSによるベシクル形成(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-1 MEMSによるベシクル形成(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-5 新しい人工細胞モデル : 分子コンピュータ内蔵リポソーム(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-5 新しい人工細胞モデル : 分子コンピュータ内蔵リポソーム(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-4 モデル生体膜における相分離と浸透圧差による変形ダイナミクス(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-4 モデル生体膜における相分離と浸透圧差による変形ダイナミクス(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-6 蛍光セルソータを用いたリポソーム構造および内部反応のダイナミクス計測(2S8 リポソーム戦線,第46回日本生物物理学会年会)

2S8-6 蛍光セルソータを用いたリポソーム構造および内部反応のダイナミクス計測(2S8 リポソーム戦線,第46回日本生物物理学会年会)

Spontaneous Generation of Giant Liposomes from an Oil/Water Interface

Spontaneous Generation of Giant Liposomes from an Oil/Water Interface

Secondary structure and distribution of fusogenic LV-peptides in lipid membranes

LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains. These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the beta-branched residue content of SNARE TMDs (approximately 50%) enhances fusogenicity while maintaining a largely alpha-helical structure in liposomal membranes. A further increase in valine content or introduction of a glycine/proline pair favors beta-sheet formation. In planar bilayers, the alpha-helices adopt oblique angles relative to the bilayer normal and the ratio of alpha-helix to beta-sheet responds more sensitively to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning alpha-helix. Beta-sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility of the core.

Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.

Gramicidin Ion Channel-Based Biosensors: Construction, Stochastic Dynamical Models, and Statistical Detection Algorithms

This paper deals with the experimental construction, stochastic modeling, and statistical signal processing of a novel, artificially constructed biosensor comprised of biological ion channels. Such nanoscale biosensors have been built by incorporating dimeric gramicidin A (bis-gA) ion channels into bilayer membranes of giant unilamellar liposomes, and then excising small patches of the membrane loaded with ion channels. We present a stochastic model for the response of the biosensor and present statistical model validation tests to verify the adequacy of the model. We show that in the presence of specific target molecules, the statistics of the gating mechanisms of the gA channels are altered. By capturing the change in real time, we devise a maximum-likelihood detector to detect the presence of target molecules. To test the sensitivity of this model, we conducted patch-clamp experiments with two compounds known to inhibit conduction of the gA channels. We found experimentally that the real-time detection algorithm was able to accurately identify the addition of the compounds even when the alterations in the patch-clamp recordings were very small. This algorithm provides the sensitive detection system for ongoing development of lipid-based nanosensors.

Dynamic Processes in Endocytic Transformation of a Raft-Exhibiting Giant Liposome

The dynamic response of a raft-exhibiting giant liposome to external stimuli, such as the addition of Triton X-100 or osmotic stress, was studied. We observed that daughter vesicles are generated inside of the liposome through endocytic budding. It was found that the budding to generate daughter vesicles is classified into two different routes, simple budding through the invagination of a whole raft and budding from the boundary of a raft accompanied by waving motion. Smaller rafts show a preference for simple budding, whereas large rafts mainly adopt the other process. We discuss the mechanism of this difference in terms of the kinetic pathway of internalization by considering the line energy and bending energy of the membrane.