Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry.

Robert V Law,Paula J Booth,Mark S Friddin,Jordan Gregg,Oscar Ces,James W Hindley,Robert Strutt,John D Harling

doi:10.1039/d0sc03889j

Abstract

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

Highlights

Introduction(BLMs) or aperture suspended bilayers15), including increased stability,[14] compartmentalisation of droplet content and the ability to support droplet volumes spanning three orders of magnitude from ml to pl.[16,17]
The eld of bottom-up synthetic biology aims to reconstitute the form, function and behaviour of biological organisms from self-assembled chemical systems.[1,2,3,4,5] To this end, different pathways have been explored to create compartmentalised biomimetic microstructures capable of supporting functions such as chemical synthesis,[6,7,8] environment sensing,[9,10] information transduction[11] and motility.[12]
By supplying different lipids to each droplet compartment,[21] droplet interface bilayers (DIBs) offer a route for controlling membrane asymmetry, a feature that is ubiquitous in native biological membranes[22] and is essential in facilitating core biological functions such as apoptosis and phagocytosis.[23]

Summary

Introduction

(BLMs) or aperture suspended bilayers15), including increased stability,[14] compartmentalisation of droplet content and the ability to support droplet volumes spanning three orders of magnitude from ml to pl.[16,17]. Both GOF mutants have been reconstituted and activated in DIBs41–45 with greater ease than their wild-type counterpart, they still require individual droplets to be prepared or manipulated by the user, meaning that ux pathways cannot be de ned in real-time This becomes a problem when constructing droplet networks, especially given that network architecture and composition can be used to de ne the ow of molecular information throughout the tissue.[46] To this end, recent work has focused on using external stimuli such as light[19] or temperature[47] to dynamically control network activation, this has only been achieved for the water-soluble alpha-toxin alpha haemolysin and not for waterinsoluble membrane proteins, highlighting the need to develop methods that offer new, orthogonal ways to de ne information ow across a bilayer network. Our method to control the full gating of the G22C F93W loss-offunction (LOF) channel in a DIB system using membrane patterning could be applied to other mutants and serve as a new strategy to control molecular ux in droplet networks, helping to design and build minimal tissues capable of increasingly complex information processing

Results

Discussion

Conclusions