Native supported cell-membrane mimics for membrane-protein separation

The lipid-bilayer membrane that encloses all cells and their internal organelles has due to its important role in biology and medicine been subject to intense investigations. Many functions of the cell membrane are controlled by proteins that transduce signals across the lipid bilayer, either via molecular-transport reactions or structural rearrangements. An evidence of their important role is the fact that they are the targets of more than half of today’s drugs, and significant effort has therefore been devoted to the development of bioanalytical instruments capable of measuring membrane-protein controlled reactions. 

However, the enormous complexity of cell membranes, being built up by numerous different lipids and proteins, combined with the fact that membrane proteins must reside in this environment to function, makes functional characterization of this class of protein extremely demanding. In particular, isolation procedures needed for in-depth studies of an individual type of membrane protein is complicated by the high tendency of membrane proteins to lose function upon detergent-based solubilization procedures.

This demanding challenge has spurred intense activities directed towards membrane-protein separation and enrichment protocols applicable directly on natural cell-membranes, with the aim to bypass the destructive solubilization step. Supported lipid bilayers, which are continuous two dimensional cell-membrane mimics (originally developed for biosensor applications) have due to preserved lateral mobility emerged as a promising system for this type of membrane-protein separation. Up to now, however, the creation of planar supported cell-membrane mimics from compositionally complex cell-membrane derived vesicles has remained relatively elusive. 

Our group has contributed to resolve this challenge by first learning how to control the motion of a supported lipid bilayer in a microfluidic channel [1], and then use this principle to induce fusion of adsorbed cell-membrane derived vesicles with the front edge of a hydrodynamically driven lipid bilayer (Fig. 1a) [2]. Moreover, hydrodynamic forces generated in the microfluidic channels were successfully utilized for both separation and accumulation of membrane-associated proteins (Fig. 1b) [2-4].


Figure 1. (a) Schematic illustration of a continuous lipid bilayer derived from native cell membrane, (b) micrograph of the separation of two types of fluorescently labeled membrane-associated proteins, (c) microfluidic setup, (d) polymer-cushioned planar cell membrane, and (e) electrophoretic separation of transmembrane proteins.


In running projects, the group is further developing these microfluidic-based (Fig. 1c) concepts with the aim to improve the spatial mobility also of membrane proteins that span across the entire lipid bilayer, which due to their close contact with the underlying support typically become immobile. One approach is to utilize polymer cushions (Fig. 1d) in order to separate the lipid membrane from the underlying support and thereby maintain the mobility of transmembrane proteins. Another strategy is to introduce a conductive polymer to the underlying substrate (Fig. 1e). The aim is in this case to tune the interaction between protein and support and thereby also influence the spatial mobility of the lipid membrane and its components.

Once local enrichment of transmembrane proteins is successfully accomplished, it is our aim to employ surface analytical tools, such as SPR, TIRF microscopy, electrical impedance spectroscopy and QCM-D, for functional studies of membrane proteins in a near-native cell-membrane environment.


Selected references

1.         Jonsson, P., et al., Shear-Driven Motion of Supported Lipid Bilayers in Microfluidic Channels. Journal of the American Chemical Society, 2009. 131(14): p. 5294-5297.

2.         Simonsson, L., et al., Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation. Journal of the American Chemical Society, 2011. 133(35): p. 14027-14032.

3.         Jonsson, P., A. Gunnarsson, and F. Hook, Accumulation and Separation of Membrane-Bound Proteins Using Hydrodynamic Forces. Analytical Chemistry, 2011. 83(2): p. 604-611.

4.         Johansson, B., et al., Hydrodynamic separation of proteins in supported lipid bilayers confined by gold barriers. Soft Matter, 2013. 9(39): p. 9414-9419.

 

Project members

Fredrik Höök (project leader)
Lisa Simonsson
Hudson Pace
Hanna Gustafsson
Sabina Burazerovic
Björn Johansson


Funding

Swedish Research Council
Swedish Foundation for Strategic Research
Göran Gustafsson Foundation

Published: Mon 20 Oct 2014. Modified: Tue 20 Sep 2016