Solid-Supported Lipid Bilayers...
...mimics of the cell surface....versatile biomaterials
Introduction
The cell membrane is a thin bilayer film that separates the cell contents from the outside world. This thin film is actually a mosaic of lipid molecules and contains many kinds of proteins, glycolipids, receptors, and other species. One of the unique properties of the film is that the species within it are able to diffuse in the two-dimensional plane of the membrane. Because of this property, the cell can carry out various functions like cell-to-cell signaling, creation of ion channels and lipids rafts, and undergo shape deformations, to just name a few.
Here is an artist's rendering of the cell membrane:
Drawing by Dana Burns, in Scientific American, 1985, 253 (4), pp. 86-90, in The molecules of the cell membrane by M.S. Bretscher.
We can create artificial planar analogs of the cell membrane on solid supports that can be used to study various biophysical phenomena, ranging from protein binding to membrane elasticity. These so-called "solid-supported bilayers" can be created on flat glass slides as well as inside microfluidic channels of intricate architecture. The SLBs self-assemble during vesicle fusion, creating large (~ cm) areas of single contiguous bilayer films, as depicted in the diagram below. These SLBs preserve the very important property of membrane fluidity, so that membrane reactions and organizations can proceed like they do in real cells.
We look at SLBs as interesting hybrid biomaterials. Such materials might have many potential applications in bridging inorganic substances with biologically relevant materials. An SLB is a material that houses membrane species in their native environment, preserving molecular structure vital for proper function. Within this environment we can manipulate them, either to separate molecules or selectively aggregate them. Direct management of the molecules within the membrane opens possibilities to study protein aggregation in a controlled manner, as well as creating spatial patterns of species that can be used for applications ranging from combinatorial drug discovery to seed platforms for cell culture. The focus of our group is to explore these possibilities. Currently, we are using SLBs as materials to separate membrane species and study the effect of membrane chemistry on separation efficiency and diffusion.
Separation of membrane components
Separation, purification, and detection of biomembrane species such as lipids and transmembrane proteins are difficult tasks. The processing conditions are often harsh, which can result in alteration of native structures or complete loss of material. Furthermore, it is difficult to detect subtle post-translational changes in these molecules that occur on the cell surface. Typical purification procedures often require one to dissolve the membrane in detergent, sonicate, filter through chromatographic columns, and separation into bands using gel electrophoresis. Procedures that circumvent such drawbacks would represent an attractive alternative and could significantly impact transmembrane proteomics.
Recently, we described a new method to rapidly separate membrane components without exposing the molecules to harsh environments. We employed a solid supported lipid bilayer made of phospholipids and cholesterol as the separation medium to laterally separate membrane-bound species. This procedure is somewhat analogous to gel electrophoresis, except that the SLB replaces the gel. It is well documented that membrane components can be manipulated in SLBs using electrophoresis, including lipids, vesicles tethered to the bilayer using DNA hybridization, and GPI-linked proteins. To conduct separations, however, it is necessary to tune the bilayer chemistry to attenuate the diffusion coefficient of the lipids and, therefore, reduce the diffusive mixing. Cholesterol significantly decreases the lipid diffusion coefficient. As we illustrate below, this analytical-scale separation technique is powerful enough to separate isomers of fluorescently-labeled lipids.
Separation of lipids labeled with two isomers of Texas red subjected to 150V of potential applied parallel to the bilayer. (Movie has been enhanced to speed up the separation) This phospholipid bilayer contains 25 mol% cholesterol which serves to reduce the diffusion and thus diminish band broadening.
Here is an image of a separation of lipids labeled with Texas red isomers and BODIPY fluorophores. The linescan shows that the species are completely separated from each other after about 35 minutes of 100V applied potential.
Our aim in the future is to use solid supported bilayer electrophoresis to separate peripheral and transmembrane proteins. Since the proteins will be separated in their native environment, they should retain their fluidity, orientation, and functionality. Separations will then lead to studies of protein behavior such as crystallization and chemical activity.
Return to the main page
