Engineering plant membranes using droplet interface bilayers.

A J Flemming,O Ces,R Macey,M S Friddin,N E Barlow,N J Brooks,I R Gould,L M C Barter,E Smpokou,C Turnbull

doi:10.1063/1.4979045

Abstract

Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.

Highlights

Droplets are manipulated into contact, which can be achieved in a number of ways including manual or robotic droplet anchors,[3,4] electric fields,[5] optical traps,[6] compressible substrates,[7] via magnetic beads,[8] or using droplet microfluidic systems.[9,10]
We address this issue by reporting the assembly of asymmetric Droplet interface bilayers (DIBs) designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time
We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs

Summary

Introduction

Droplets are manipulated into contact, which can be achieved in a number of ways including manual or robotic droplet anchors,[3,4] electric fields,[5] optical traps,[6] compressible substrates,[7] via magnetic beads,[8] or using droplet microfluidic systems.[9,10]. A key drawback of the DIB platform is that the compositions of the bilayers assembled are typically oversimplified and are not representative of biological membranes, where the relationship between lipid composition and membrane function is understood to play a key role This limitation presents a bottleneck to the applicability of DIBs to biological systems, such as plant membranes, where representative lipid compositions are vital for studying the tightly regulated function of intracellular nanochannels such as plasmodesmata,[20,21] and for uncovering the biological engineering rules that regulate and control the efficient translocation of endogenous proteins, small molecules, or agrochemicals[22] across plant membranes. Our findings suggest that our approach could be scaled to assemble higher-order networks of model plant DIBs to study the translocation of endogenous proteins, small molecules, and agrochemicals in plants

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Results

Conclusion