Membrane protein mediated bilayer communication in networks of droplet interface bilayers

James W Hindley,Kalypso Charalambous,Enrique Rodriguez,Oscar Ces,Stuart Haylock,Paula J Booth,Mark S Friddin,Laura M C Barter

doi:10.1038/s42004-020-0322-1

James W Hindley, Kalypso Charalambous + Show 6 more

Open Access

https://doi.org/10.1038/s42004-020-0322-1

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Abstract

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered ‘tissue-like’ networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.

Highlights

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayerencased water droplets in oil
G22C F93W mechanosensitive channel of large conductance (MscL) was expressed and purified from Escherichia coli BL21 (DE3) cells carrying the kanamycinresistant pET-28a vector using a modified protocol reported by Perozo et al.[28]
The scale of the increase in fluorescence is in-line with what has been observed in our lab previously and most likely owes to the small volume of the vesicles[25]. These results demonstrate the successful reconstitution of active MscL into lipid vesicles, which is essential for assembling MscL functionalized DIBs

Summary

Introduction

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayerencased water droplets in oil. The introduction of additional droplets supports the formation of bilayer networks, which can be freely assembled in 2D or 3D3,7–10, while communication between compartments can be facilitated through the addition of chemical and biological motifs, such as the water-soluble pore-forming protein α-hemolysin (αHL), which self-inserts into the membrane These capabilities have enabled the construction of ‘higher-order’ systems that exhibit collective properties, with examples including the engineering of droplet bio-batteries[7] and current rectifiers[11] in addition to a light-sensing network using the light-driven proton pump bacteriorhodopsin[7]. ΑHL-mediated connectivity between droplets has been shown to be triggered in real time using light by exploiting a light-sensitive T7 RNA polymerase coupled to a cell-free expression system[12,13] In view of these ground-breaking advances, it seems clear that the milestone toward realizing the true potential of this platform technology is to demonstrate that water-insoluble membrane proteins can be successfully reconstituted into DIB networks. Reports of the successful reconstitution of water-insoluble membrane proteins into a two droplet (i.e. single bilayer) system have been limited to a handful of different proteins[4,14], including ion channels and transporters expressed using cell-free extracts[15,16]; this has yet to be reported in networks of interconnected DIBs

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