ABSTRACT - Project 3 A healthy immune system depends on T cells’ abilities to detect foreign agonist pMHC molecules, at near single molecule levels, among vast numbers of sometimes very similar self-antigens. While the T cell’s fine discrimination capabilities are experimentally well established, understanding how the molecular machinery of the TCR signaling system achieves this is far from transparent. Fundamental issues of noise, variation, and signal fidelity present serious challenges—both for understanding how the T cell works as well as for development of therapeutic strategies utilizing T cells. Recently, a class of phenomena known as protein condensation phase transitions have begun to emerge in biology. Originally identified in the context of nuclear organization and gene expression, a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT. While the role of LAT as a scaffold for the clustering of downstream signaling molecules in T cells has long been recognized, experimental realization that this structure can form through distinct types of phase transition processes is more recent. Protein condensation phase transitions can exhibit a wide range of properties that differ substantially from more linear molecular clustering processes, and thus offer a variety of different ways to regulate the functional output of molecular signaling systems. Project 3 addresses the overarching hypothesis that unique properties of the LAT protein condensation phase transition enable the remarkable sensitivity and selectivity T cells exhibit during antigen recognition. We propose a series of investigations to test aspects of this hypothesis that combine highly quantitative experiments in reconstituted molecular systems with precision single-molecule live cell experiments in primary T cells. We have preliminary data indicating that LAT condensation in live T cells is controlled by an unusual type of kinetic phase transition and that specific molecular features of proximal TCR signaling are tuned to take advantage of this. Confirmation and elucidation of this discovery will form the foundation of our more detailed investigation into the role of the phase transition in TCR signaling. While Projects 1 and 2 focus on proximal signaling feedback mechanisms leading up to LAT phosphorylation and condensate nucleation, and Project 4 examines signaling downstream from the LAT condensate, Project 3 emphasizes experimental and computational characterization of the LAT condensation phase transition itself and measurements of how its properties modulate downstream signal propagation through the following specific aims: 1. Define the factors controlling initiation of LAT condensation in T cells; 2. Engineer non-condensing LAT and LAT-like systems; 3. Define how LAT condensates control downstream signaling to Ras and Ca2+ pathways. Insights originating from this work will likely resolve some conceptual mysteries on mechanistic function of the T cell receptor signaling pathway and highlight alternative angles to engineer and manipulate T cells for therapeutic benefit.