PROJECT SUMMARY CAR-T therapy relies on the creation of synthetic T-cell receptors (TCR) to recognize cancer cells antigens as well as activate the full immune response, something endogenous TCRs cannot do on their own. However, this reliance on synthetic receptor molecules limits both the range and effectiveness of cancer immunotherapy. Developing new modes of cancer immunotherapy depends on finding new targets that activate the full T cell signaling response. One such target may be the signaling complex that forms immediately proximal to the TCR between the scaffold protein LAT and its kinase ZAP70. This signaling complex appears to organize all the downstream pathways activated following antigen binding. Moreover, it was recently found in vitro that LAT and ZAP70 form liquid droplets in a process termed “protein phase separation” and that these protein droplets could nucleate actin filaments. However, it remains unknown how LAT-ZAP70 phase separation affects signal transduction in living cells. Here, I propose to determine whether synthetic phase separation of LAT-ZAP70 is sufficient to activate the signaling pathways associated with antigen binding in both non-Tcells as well as Jurkat cells. To do so, this project will utilize recently developed optogenetic tools that cause proteins to phase separate when stimulated with light. Preliminary results from my studies suggest that synthetically inducing LAT-ZAP70 protein phase separation with these optogenetic tools does indeed activate downstream signaling pathways in non-T cells, but that causing LAT and ZAP70 to interact in other ways, such as protein heterodimerization, does not activate these same pathways. To understand how protein phase separation activates these signaling pathways, I will develop a two-color optogenetic system that causes LAT-ZAP70 to heterodimerize and/or phase separate in a single cell. Thus, I can compare how the same number of molecules interacting as heterodimers or liquid droplets activates signaling pathways. I will also compare how well heterodimers or droplets activate signaling pathways in the presence of T-cell specific factors, including the membrane phosphatase CD45, a proposed negative regulator of LAT-ZAP70 complex activity. Finally, I plan to determine whether light-induced phase- separation of LAT-ZAP70 in Jurkat T cells results in the secretion of cytokines and other key outcomes of T cell activation. Overall, this project will determine whether and how protein phase separation of LAT- ZAP70 is sufficient to activate the full T cell signaling response.