Permeation through Lipid Bilayers by Adhesion of Giant Vesicles on Decorated Surfaces

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.

Download Full-text

Electroformation from patterned single-layered supported lipid bilayers for formation of giant vesicles with narrow size distribution

Applied Physics Express ◽

10.7567/apex.7.117001 ◽

2014 ◽

Vol 7 (11) ◽

pp. 117001

Author(s):

Nobuo Misawa ◽

Toshinori Motegi ◽

Ryugo Tero

Keyword(s):

Size Distribution ◽

Lipid Bilayers ◽

Supported Lipid Bilayers ◽

Narrow Size Distribution ◽

Giant Vesicles

Download Full-text

Giant Vesicles and Their Use in Assays for Assessing Membrane Phase State, Curvature, Mechanics, and Electrical Properties

Annual Review of Biophysics ◽

10.1146/annurev-biophys-052118-115342 ◽

2019 ◽

Vol 48 (1) ◽

pp. 93-119 ◽

Cited By ~ 32

Author(s):

Rumiana Dimova

Keyword(s):

Electrical Properties ◽

Lipid Bilayers ◽

Material Properties ◽

Phase State ◽

External Factors ◽

Membrane Composition ◽

Membrane Phase ◽

Membrane Systems ◽

Giant Vesicles ◽

Vesicle Preparation

Giant unilamellar vesicles represent a promising and extremely useful model biomembrane system for systematic measurements of mechanical, thermodynamic, electrical, and rheological properties of lipid bilayers as a function of membrane composition, surrounding media, and temperature. The most important advantage of giant vesicles over other model membrane systems is that the membrane responses to external factors such as ions, (macro)molecules, hydrodynamic flows, or electromagnetic fields can be directly observed under the microscope. Here, we briefly review approaches for giant vesicle preparation and describe several assays used for deducing the membrane phase state and measuring a number of material properties, with further emphasis on membrane reshaping and curvature.

Download Full-text

Rate of Permeabilization of Giant Vesicles by Amphiphilic Polyacrylates Compared to the Adsorption of These Polymers onto Large Vesicles and Tethered Lipid Bilayers

Langmuir ◽

10.1021/la900261s ◽

2009 ◽

Vol 25 (13) ◽

pp. 7506-7513 ◽

Cited By ~ 18

Author(s):

F. Vial ◽

F. Cousin ◽

L. Bouteiller ◽

C. Tribet

Keyword(s):

Lipid Bilayers ◽

Giant Vesicles

Download Full-text

Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076640 ◽

1983 ◽

Vol 41 ◽

pp. 608-609

Author(s):

Neng-Bo He ◽

S.W. Hui

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Lipid Membranes ◽

Surface Film ◽

Biological Membranes ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Black Lipid Membranes ◽

Fine Mesh ◽

Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

Download Full-text

Effect of Integral Proteins on Frozen Membrane Fracture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003287 ◽

1980 ◽

Vol 38 ◽

pp. 756-759 ◽

Cited By ~ 1

Author(s):

S. Kirchanski ◽

D. Branton

Keyword(s):

Lipid Bilayers ◽

Freeze Fracture ◽

Covalent Bond ◽

Erythrocyte Membranes ◽

Glycophorin A ◽

Experimental Protocol ◽

Transmembrane Glycoprotein ◽

Bond Breakage ◽

Human Erythrocyte Membranes ◽

Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

Download Full-text

Multiresponsive Behavior of Biomembranes and Giant Vesicles

10.1016/s2451-9634(19)x0003-5 ◽

2019 ◽

Keyword(s):

Giant Vesicles

Download Full-text

Late Stages of the “Pearling" Instability in Lipid Bilayers

Journal de Physique II ◽

10.1051/jp2:1997180 ◽

1997 ◽

Vol 7 (9) ◽

pp. 1185-1204 ◽

Cited By ~ 6

Author(s):

J. L. Coveas ◽

S. T. Milner ◽

W. B. Russel

Keyword(s):

Lipid Bilayers ◽

Late Stages

Download Full-text

Improved micro-impedance spectroscopy to determine cell barrier properties

The EuroBiotech Journal ◽

10.2478/ebtj-2020-0017 ◽

2020 ◽

Vol 4 (3) ◽

pp. 150-155 ◽

Cited By ~ 1

Author(s):

Md. Mehadi Hasan Sohag ◽

Olivier Nicoud ◽

Racha Amine ◽

Abir Khalil-Mgharbel ◽

Jean-Pierre Alcaraz ◽

...

Keyword(s):

Impedance Spectroscopy ◽

Epithelial Cells ◽

Lipid Bilayers ◽

Cultured Cells ◽

Cell Model ◽

Measurement Techniques ◽

Cell Monolayer ◽

Microfluidic Channel ◽

Small Surface ◽

Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

Download Full-text