Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Summary

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Abstract

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added. Then, bilayers are charged with Ni and his-tagged recombinant proteins are added. Finally, lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting, and sites of receptor degradation.

Protocol

1. Preparing the lipids

1. In order to link proteins to the upper leaflet of a bilayer, we use Ni²⁺ chelating lipids, which anchor recombinant proteins with poly-histidine tags. Certain cell types may adhere non-specifically to Ni²⁺-coated bilayers, but T-cells generally do not, making this method optimal for studying T cell activation.
2. The lipid bilayer solution is made by preparing a solution of 20% Ni²⁺ chelating lipid and 80% phosphatidylcholine in the appropriate amounts in a glass tube with 1-2 ml of chloroform-methanol.
   1. Evaporate the solvent under a stream of nitrogen gas in a warm (30-37°C)....