ABSTRACT Synaptic dysfunction is linked to numerous devastating neurological and psychiatric disease. Synaptic transmission is initiated by the calcium dependent release of neurotransmitters. The calcium sensors responsible for triggering vesicle release are the Synaptotagmins (SYT), a 17 protein family. The function of one SYT, SYT7, is less well understood but is thought to be of significant importance due to its broad expression throughout the body and brain, connections to cancer and ALS, and enigmatic function in the central nervous system. Unlike other Synaptotagmins, SYT7 is thought to be found on the plasma membrane and its function remains controversial. The role of SYT7 is still under intense debate due to several outstanding questions regarding its cellular and synaptic localization. Answering these key questions is the focus of my grant proposal and will provide necessary insights into SYT7’s function. This proposal aims to answer these questions by determining SYT7’s cellular and nanoscale localization in cortical neurons using biochemistry and three-color STED super resolution microscopy. Aim 1 will determine whether SYT7 is found on the cell surface of cortical neurons using live-cell surface protein biotinylation and live-cell surface staining in mouse cortical slices and rat primary cortical neurons. Aim 2 will determine how SYT7 is organized at synapses using three-color STED staining for endogenous synaptic proteins. Aim 3 will determine whether neuronal activity regulates the cellular and nanoscale localization of SYT7 using both surface protein biotinylation and live-cell surface staining with three-color STED. This proposal is designed to provide me with intellectual and technical training throughout the fellowship award. I will learn key biochemical assays, ex vivo slice preparations, and will hone my skills in advanced STED super resolution microscopy. With support from this award, the training during this fellowship will provide me with the skills necessary to become a successful physician scientist.