Single Molecule Observation of TCR Signaling Complexes in Living T Cells

Abstract

Dense protein complexes such as those found in the immunological synapses of antigen-activated lymphocytes are sites of critical biological activity, but their study is complicated by the large number of protein species present. Array tomography is a proteomic imaging method capable of addressing this highdimensional problem through iterative immunostaining, but would be difficult to apply to spatially-restricted regions of interest such as complexes within the immunological synapse that form in the area of contact between lymphocytes and antigen-presenting surfaces. We have developed a novel variant of this technique which embeds a thin (~500 nm) slice of material in LR White, making proteomic multiplexing possible. We are using this method with physically unroofed resting and activated B cells to study signaling in antigen-receptor microclusters in B cell plasma membranes. Here we present this method, and our current work in its application to the B cell membrane.