Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Erwin London,Qingqing Lin,David Holowka

doi:10.1371/journal.pone.0087903

Abstract

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

Highlights

A common feature of eukaryotic cell membranes is the nonrandom distribution of lipid species in the inner and outer leaflets of the lipid bilayer, which is called lipid asymmetry
Our first aim was to define methods to prepare asymmetric vesicles with various controlled amounts of cholesterol, and with a composition that would roughly approximate that of mammalian plasma membranes
Cholesterol-containing asymmetric large unilamellar vesicles (LUV) with POPC, SM or a SM/POPC mixture in the outer leaflet and POPE/POPS in the inner leaflet were prepared by HPaCD-induced lipid exchange, and the lipid composition of the resulting vesicles assayed by HPTLC

Summary

Introduction

A common feature of eukaryotic cell membranes is the nonrandom distribution of lipid species in the inner and outer leaflets of the lipid bilayer, which is called lipid asymmetry. In mammalian plasma membranes aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) are predominantly exposed on the cytosolic leaflet, whereas phosphatidylcholine (PC) and sphingolipids, including sphingomyelin (SM), are predominantly located on the outer leaflet [1,2]. This transmembrane (TM) lipid asymmetry provides the two sides of plasma membrane with different biophysical properties and influences numerous cellular functions. Lipid asymmetry imparts asymmetry of lipid charge across the bilayer, with a higher anionic charge at the cytofacial surface of membranes This may influence membrane protein topology and the establishment of TM protein orientation during biosynthesis [5]

Methods

Results

Conclusion