Studying Dynamic Plasma Membrane Binding of TCR-CD3 Chains During Immunological Synapse Formation Using Donor-Quenching FRET and FLIM-FRET

Over the last decade, advancements in the time and space resolution of microscopy technologies have enabled dissection of the molecular events involved in T cell Immunological Synapse (IS) formation. Using a combination of Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imagining Microscopy (FLIM), we have demonstrated dynamic plasma membrane binding by cytoplasmic domains of T cell receptor (TCR)-associated CD3 chains and other T cell transmembrane receptors. We have developed methods for imaging such membrane binding both at steady state and during receptor triggering at the IS. Plasma membrane binding by cytoplasmic domains may represent a novel mechanism for regulating the signaling function of important receptors in the immune system.