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.
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