Project Summary T cells are central effectors of adaptive immunity, mediating critical defenses against infections and cancer. Aberrant T cell activation is associated with autoimmune diseases and immunodeficiency disorders. To elicit proper immune responses upon antigenic stimulation, T cell activation must be tightly regulated to integrate and transduce signals detected at the plasma membrane. Despite decades of research, the mechanisms regulating this transduction are not fully understood. Recent reports have demonstrated that key T cell signal transduction proteins form biomolecular condensates at the T cell immunological synapse via liquid-liquid phase separation. Specifically, the transmembrane adaptor protein Linker for Activation of T cells (LAT), which is essential for T cell activation, is phosphorylated upon T cell receptor ligation. Phosphorylated LAT produces multiple interactions with multivalent effector proteins, including Grb2 and Sos1, which lead to assembly of condensates with fluid properties in vitro and in living cells. How LAT condensate assembly and disassembly are regulated and how LAT condensates regulate T cell signaling in cells is unknown. Notably, LAT is recruited to ordered, cholesterol-rich membrane domains known as lipid rafts, and such recruitment has been implicated in T cell activation. Since LAT is involved in both membrane rafts and condensate assembly, I hypothesize that the interplay between membrane rafts and LAT condensates tunes T cell signaling. My preliminary data supports this hypothesis both in vitro and in Jurkat T cells. The goal of this proposal is to understand how LAT condensates couple to membrane rafts to cooperatively regulate T cell signaling. Our overall hypothesis is that the LAT mediates biophysical and functional coupling between membrane rafts and condensates to regulate T cell activation. To evaluate how LAT condensate assembly mediates membrane lipid organization, experiments proposed in Aim 1 will characterize the biophysical properties of membranes coupled to LAT condensates both in vitro and in live cells. Aim 2 will evaluate the effects of membrane lipid remodeling on LAT condensate properties and downstream T cell signaling upon antigenic stimulation. To this end, in vitro reconstituted membrane models are used to precisely manipulate lipid composition to study the effect on LAT condensate formation. These studies will be complemented by dietary lipid supplementation or pharmacological manipulations in live cells to study the effect of membrane lipid remodeling on LAT condensate assembly, and how it regulates signaling pathways downstream of TCR engagement. Successful completion of this work will provide evidence and mechanistic insights on how membrane rafts and LAT condensates mutually regulate to tune T cell signaling. These findings will advance our understanding of spatiotemporal regulation of T cell activation, thereby can potentially shed light on novel therapeutic strateg...