BPS2026 - Biosensors for organization and dynamics of Lck kinase activity in immune signaling

Immune receptor triggering is a complex process mediated through dramatic reorganization of receptors and signaling molecules at the plasma membrane. After ligand binding to the T cell receptor (TCR), the Src-family kinase (SFK) Lck performs the first receptor phosphorylation step, initiating the signal transduction cascade. While receptors organize in distinct signaling assemblies upon activation, questions remain about how active SFKs are recruited to receptors and how kinase activity is regulated proximal to receptor assemblies. SFKs are typically activated through trans-autophosphorylation, therefore local density and dynamic organization of SFKs on the membrane is a potential key factor in regulation of kinase activity. Localization and concentration of Lck in membrane domains could play the dual regulatory role of bringing Lck in proximity to immune receptors and locally amplifying Lck activity. Testing this hypothesis will require tools that can measure both Lck activation state and Lck organization on sub-diffraction length scales characteristic of receptor signaling assemblies and membrane domains. SPECTr (small protein exposure for conformational tracking) is a single-molecule biosensor designed to exploit conformational changes of SFKs, localizing individual active SFK molecules. The innovation of SPECTr comes from the ability to measure both activity and nanoscale organization of proteins in live cells. We have adapted SPECTr to measure Lck conformational dynamics to produce “SPECTr-Lck”. We have verified the biosensor’s functional activity and optimized fluorophore labeling for super-resolution imaging. We will use SPECTr-Lck in multicolor super-resolution imaging experiments to simultaneously observe rearrangement of receptors and active kinases. By perturbing the stability of plasma membrane domains, we can address the working hypothesis of the role of Lck organization in TCR signaling. Tracking the distribution of active Lck across the membrane in live cells will provide insight into the dynamic regulation of TCR triggering.