Constructing supported lipid bilayers from native cell membranes

Abstract

The aim of this MSc-project was to investigate whether it is possible to incorporate native cell membrane components from Jurkat T cells into supported lipid bilayers, SLBs, to produce a model membrane system better resembling the native cell membranes in T cells. The procedure of forming native-like SLBs (nSLB) was adapted from an existing protocol and is done through the deposition of hybrid vesicles onto a cleaned glass slide. The hybrid vesicles are composed of synthetic lipids (PEGylated lipids and POPC) and native cell membrane components from the Jurkat T cells. Upon contact with the substrate the hybrid vesicles rupture to form the nSLB. By using this experimental protocol naturally occurring cell membrane components, such as membrane proteins, can be transferred to the bilayer. Formation of the hybrid vesicles is done through bath sonication to fuse synthetic vesicles and native membrane vesicles (NMVs). First, the most optimal sonication parameters to ensure a good mixing of the two types of vesicles was evaluated. It was found that sonicating at 35°C 45 min gave a good mixing of NMVs and synthetic vesicles. These hybrid vesicles were next used to form the nSLB, which was characterized in detail. The diffusivity of lipids in the nSLB was measured using fluorescence recovery after photobleaching and found to be 1.00 µm2/s. This was 16 % lower compared to a similar SLB without native material and similar to the diffusivity measured by others for nSLBs, indicating that an nSLB has formed. The immobile fraction of lipids was 21% for the nSLB which was significantly higher than the 5% measures for an SLB without native material. This could be due to unruptured vesicles, which could also be observed in the fluorescence microscopy images. Antibodies targeted at different T-cell proteins were finally used to investigate how well these proteins had been incorporated in the nSLB and whether they were mobile. Both the proteins CD45 and TCR could be detected at a surface concentration < 100 molecules/μm2 in this way. However, essentially all of the antibodies were immobile on the nSLB, which could be due to the proteins interacting with the underlying support or being confined to vesicles. This needs to be taken into consideration if using this as a model membrane system for T cells, but for experiments where the mobility is not of primary concern then the developed nSLB could make it possible to study the interaction with T-cell membrane proteins under more controlled conditions.

Popular Abstract

The cell membrane plays an important part in many of the cell’s functions. It forms a boundary between the internal and external environment of the cell and governs the exchange of essential molecules, for the cell’s survival, with its surroundings. The behaviour of the membrane and the proteins present in it, greatly affects our immune system and health. In fact, a majority of drugs in the market target some protein integrated into the cell membrane. With the presence of certain proteins embedded into the membrane, the cells in the immune system are able to communicate with each other to fight back any pathogens posing a threat against the body. The T-cell is one such cell in the immune system which is central in forming an immune response against bacteria or viruses.
Studying the cell membrane and the molecules therein can therefore seem to be an obvious endeavour but is quite difficult since it is so complex. For example, there are more than 600 lipid types in most cell membranes and the contribution from each cell membrane component can be hard to quantify. A popular alternative within research has been to instead use supported lipid bilayers (SLBs), a mimic of a real cell membrane formed on a solid support. Contrary to cell membranes, the SLB is comprised of only one or two lipid types and normally only have one type of membrane protein integrated into it. This makes it easier to study the function of individual proteins. Although this system has been of great use to study how membranes behave, it is in some instances too simple when compared to a live cell membrane. The main aim of this work has been to try and form SLBs which contain a larger fraction of lipids and membrane proteins that are naturally occurring in native cell membranes. Specifically, I tried to produce SLBs that resemble the cell membrane of the immune cells called T cells.
This was done by forming structures called vesicles. These structures can be imagined as planar bilayer, a membrane, which closes in on itself to form a sphere. One of the main tasks was to try and incorporate components from a real T cell membrane into these vesicle structures. This was done by optimizing a technique called sonication. When the vesicles are deposited onto a solid support, they deform and rupture to form a patch of SLB. In a real cell membrane, the lipids and proteins are able to move around in the bilayer. It is therefore interesting to investigate how fast the components are moving in the more cell-like SLB compared to the traditional SLBs which doesn’t contain any components from real cell membranes. The results show that the lipids move a bit slower in the more cell-like SLB. Similar to how it is more difficult to move around in a more crowded room, the lipids move slower when facing more obstacles from the extra added components into bilayer from a real cell membrane.
I further investigated how much the SLB resembled the T-cell membrane by using antibodies. Antibodies are proteins which only bind specifically to a certain protein. By using antibodies I could detect if the membrane proteins one would expect to find on the membrane of a T cell, are present on the newly formed SLB. A few proteins could be detected on the surface of the SLB. However, the proteins seem to be quite stationary and does not move around very well. This problem is multi-facetted can be because the proteins are very large and more massive objects require more energy to move around. An additional reason may be that the proteins are still part of vesicles which haven’t ruptured, in which case they are not able to move in the bilayer that has formed.
In summation, this has been an investigation into whether it is possible to create a platform to better model the cell membrane of a T cell. An experimental protocol to produce a SLB, with cell membrane components, was formed. Although the membrane proteins seem to be largely stationary, the resulting SLB could still be used to study the binding mechanism of the proteins in the SLB which does not require proteins to move. This work forms a basis which could be further expanded upon to improve the produced cell-like SLB. The tool could then be used to further study how T cells interact with other cells in the immune system, giving more insights into how the body fights off diseases. Knowing more about this process could also optimize and improve some of the strategies, used by the pharmaceutical industry, developing drugs against a certain illness.

• author: Dam, Tommy
• supervisor: Peter Jönsson
• organization: Biophysical Chemistry
• course: KFKM05 20181
• year: 2018
• type: H3 - Professional qualifications (4 Years - )
• subject: Chemistry
• keywords: T cells, Model membrane systems, Supported lipid bilayers, SLB, biophysical chemistry, biofysikalisk kemi
• language: English
• id: 8952458
• date added to LUP: 2018-06-26 10:02:23
• date last changed: 2018-06-26 10:02:23