Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

Abstract

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup

“…Because of these limitations, free‐standing membranes have been gaining popularity as they satisfy some of the fundamental criteria: they are fluid and both sides are in contact with the aqueous medium. Historically, vesicles and black lipid membranes were among the first suspended membranes to be successfully used. Over the years, many setups have been developed that provide multiple ways to form suspended membranes, e.g., on a hole between two separate compartments, at the tip of pipettes, between two emulsion droplets, on holey substrates, on nanodisks, and in microfabricated systems .…”

“…One common difficulty is to recreate asymmetric bilayer. Their preparation often leads to the inclusion of an organic solvent layer that affects the biophysical properties of the membrane and they are extremely limited in terms of lipid composition . Also, the stability of the bilayers is often an issue which is resolved in the vast majority of planar bilayer setups by using a non‐physiological lipid, DPhPC (1,2‐diphytanoyl‐sn‐glycero‐3‐phosphocholine) .…”

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

“…Examples of hybrid asymmetric vesicles are scarce and most reported systems consist of a bilayer formed from two different lipid types42, 43, 44, 45 or two different polymers 51, 52. To our knowledge there is only one reported example of such a system, but no clear evidence was provided to fully support the membrane asymmetry because of the impossibility to perform a fluorescence quenching assay 45.…”

“…This is energetically unfavorable and happens at rather slow rates and with the help of enzymes. Flip‐flop rates have been evaluated for different types of lipids and lipid bilayers in several synthetic systems such as supported bilayers or large unilamellar vesicles 44, 47, 64, 65, 66, 67. However, owing to the much larger sizes of biological cells, membrane properties such as curvature (and thus lipid diffusion rates) would be more accurately represented by giant vesicle systems.…”

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

“…C) Fabrication of cell‐sized asymmetric lipid vesicles by a jet flow microfluidic device. Reproduced with permission . Copyright 2016, Springer Nature.…”