A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments.

Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM) in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni(2+)-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on Ni(2+)-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab')2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.