PROJECT SUMMARY Ca2+ is a key driving signal for the activation of T lymphocytes, a vital early step in the mounting of an immune response in humans. Impaired Ca2+ signaling results in severe combined immunodeficiency (SCID) and autoimmunity, while excessive Ca2+ signals result in thrombocytopenia and other maladies. Ca2+ signals are generated by Ca2+ influx through plasma membrane (PM) Ca2+ channels (store-operated Ca2+ channels, or SOCCs) summing with Ca2+ extrusion by ATP-driven PM Ca2+ pumps (PMCA4b). SOCCs are known to localize to discrete sites called endoplasmic reticulum (ER)-plasma membrane (PM) junctions (EPJs) where they create Ca2+ microdomains. Local Ca2+ signals in this microdomain drive activation of multiple transcription factors, leading to Ca2+-dependent gene expression and T-cell activation. The duration, amplitude, and dynamics of Ca2+ signals are known to enhance the efficiency and selectivity of transcriptional activation in T cells. However, the features of Ca2+ signals in the EPJ microdomain are completely unknown, due to a lack of suitable tools to measure [Ca2+] selectively at EPJs. Thus, the coupling between specific patterns of local Ca2+ signals and T- cell gene expression and activation remains unclear. In addition, indirect evidence suggests that PMCA4b is also present at EPJs, but its contribution to local signaling in the microdomain is totally unknown. The goal of this research project is to test the hypothesis that PMCA4b activity shapes local Ca2+ signals at EPJ microdomains to regulate gene expression and T-cell activation. To test this hypothesis, I propose two aims. In Aim 1, I will develop, validate, and target a novel genetically-encoded Ca2+ indicator protein to the EPJ to directly measure the ‘signature’ of Ca2+ signals in the microdomain. This will allow us to identify for the first time the types of signals that actually drive T-cell activation and will provide a critical tool for the second Aim. In Aim 2, I will apply CRISPR/Cas9 to fluorescently label endogenous PMCA4b and observe directly its localization to the EPJ, as well as to mutate the endogenous protein to alter its localization and assess the effects of pump localization on local Ca2+ signals, pump activity, and transcriptional activation and gene expression. Completion of the proposed work will uncover the relationship between local Ca2+ signals at EPJ microdomains and Ca2+- dependent gene expression in T cells leading to T-cell activation. In addition, it will reveal new molecular mechanisms that control pump localization and their impact on activation. Finally, this work may also reveal new ways in which Ca2+ signals can be manipulated in T cells, which could suggest new strategies for the treatment of immunodeficiency, autoimmunity, and other maladies in human patients.