Project Summary The overall goal of this project is to understand how the spatial organization of signaling molecules on the cell membrane regulates signal transduction. Extracellular stimuli are transduced across the plasma membrane, processed, and amplified along the inner leaflet of the lipid bilayer, and then further delivered into the cytoplasm and nucleus. It has been shown that proteins and lipids are organized into membrane domains to mediate signal transduction though the underlying mechanisms are not fully understood. The T cell receptor (TCR) pathway represents an ideal system for studying this phenomenon. Dozens of the components of the TCR pathway are enriched in the T cell microcluster, a membrane-associated micron-sized domain that is essential for TCR signaling. Our recent work suggested that T cell microclusters are phase-separated condensates driven by multivalent protein-protein interactions. What is lacking, however, is an understanding of how the formation of these microclusters is regulated by the local membrane environment, where significant changes occur in both lipid composition and membrane geometry upon TCR activation. Therefore, this proposal aims to determine the mechanism by which lipids and membrane geometry regulate the assembly of T cell microclusters and the associated functional consequences during T cell activation. The following questions will be addressed: How do charged lipids modulate T cell microcluster formation? How does membrane geometry influence microcluster function? How do microclusters affect T cells’ killing activity? Answering these questions will significantly impact the field because it will reveal how the protein machineries and lipid bilayers coordinate to process and amplify the signal from antigen stimuli to cell activation. Moreover, the majority of currently identified phase-separated structures are 3-D droplets located in the nucleus or cytoplasm whereas T cell microclusters are 2-D domains on the membrane. Understanding the functional relationship between T cell microclusters and lipid bilayers is expected to create a new research interface between the field of protein self-assembly and membrane signaling.