Supported double bilayers: an experimental model to probe biomembrane properties

The structure and stability of biological membranes is vital for living cells. Each cell is surrounded by a cell membrane and a large variety of membranes is found inside cells as well: the nucleus, mitochondria, chloroplasts and many other cell organelles hold membranes and have a membrane-like envelop.  The most fundamental role of the cell membrane is to act as a physical barrier. Apart from their barrier function, membranes serve as a matrix for molecules (in particular proteins) that carry out specific tasks. See Figure 1.
Figure 1. Left: Cartoon of a biological membrane. Right: a lipid vesicle (liposome).
Figure 1. Left: Cartoon of a biological membrane. Right: a lipid vesicle (liposome).

The matrix of each biomembrane is a lipid bilayer. Topologically complex and dynamic processes, such as endocytosis and exocytosis, require that biomembranes are on the edge of stability and this potentially clashes with their barrier function. Small variations in the lipid composition can have large effects on the membrane properties.

At PCC we use lipid bilayers as models for biomembranes and intend to study their properties in a new experimental system: a supported double lipid membrane configuration in a flow cell. In Figure 2 the construction of this supported double bilayer is schematically presented. Through vesicle adsorption the first bilayer covers the substrate. The second (outer) one will be connected to the first one by flexible biotin-streptavidin tethered spacers.

Ultimately, we aim to study the properties of the outer lipid membrane using simultaneously a combination of total internal reflection fluorescence (TIRF) microscopy and force measurements with an atomic force microscope. However, since it is probably not easy to construct the double bilayer in an AFM/TIRF flow cell, we first want to have a proof-of-principle that we can construct the double bilayer in a simpler set-up: a reflectometer. This is the subject of this BSc/MSc thesis project.

Figure 2. Schematic illustration of the formation of supported double bilayers: a and b) formation of the proximal layer by adsorption of vesicles containing biotinylated lipids (biotin on a flexible spacer). The formation of the second bilayer: c) streptavidin, a 52.8 kDa protein, has four binding sites for biotin and a sufficient number is added so that approximately half the binding sites remain free. d) Subsequently, new biotinylated vesicles are added, which will bind to the streptavidin layer. e) After rupture of the vesicles the double bilayer configuration is formed.
Figure 2. Schematic illustration of the formation of supported double bilayers: a and b) formation of the proximal layer by adsorption of vesicles containing biotinylated lipids (biotin on a flexible spacer). The formation of the second bilayer: c) streptavidin, a 52.8 kDa protein, has four binding sites for biotin and a sufficient number is added so that approximately half the binding sites remain free. d) Subsequently, new biotinylated vesicles are added, which will bind to the streptavidin layer. e) After rupture of the vesicles the double bilayer configuration is formed.

In a reflectometer it is possible to adsorb vesicles on a silica surface in a controlled way and simultaneously measure very accurately the adsorbed amount of lipids. So, using this technique we can monitor step-by-step the formation of the supported double bilayer in a quantitative way. Once it is proven that the method works, many relevant experiments can be conducted. For example, we can determine the stability of the (outer) membrane as a function of salt concentration and do this for different lipid compositions. Another example is to look into the adsorption of nanoparticles on the outer membrane and study their effect on the bilayer stability.