Determining the cellular and nanoscale localization of Synaptotagmin VII

摘要 突触功能障碍与许多毁灭性的神经和精神疾病有关。 突触传递是由钙依赖的神经递质释放启动的。钙 负责触发囊泡释放的传感器是突触素(SYT)，这是一个17蛋白家族。这个 一个SYT，SYT7的功能不太清楚，但被认为是非常重要的，因为它 在身体和大脑中广泛表达，与癌症和ALS有关，以及神秘的功能 中枢神经系统。与其他突触素不同，SYT7被认为是在血浆中发现的 膜及其功能仍存在争议。SYT7的作用仍在激烈的争论中，因为 关于其细胞和突触定位的几个悬而未决的问题。回答这些关键问题 是我拨款提案的重点，并将为了解SYT7的S功能提供必要的见解。 这项提议旨在通过确定SYT7的S细胞和纳米尺度来回答这些问题 用生化和三色STED超分辨显微镜定位皮质神经元。目标 1将使用活细胞表面蛋白确定是否在皮质神经元的细胞表面发现SYT7 小鼠皮质切片和大鼠原代皮质神经元的生物素化和活细胞表面染色。目标2 将使用内源性突触的三色STED染色来确定SYT7在突触中的组织方式 蛋白质。目标3将确定神经元活动是否调节细胞和纳米级的定位 SYT7采用表面蛋白生物素化和活细胞表面三色STED染色。 这项建议旨在为我提供知识和技术培训 奖学金获得者。我将学习关键的生化分析，体外切片制备，并将磨练我的技能 先进的STED超分辨率显微镜。在该奖项的支持下，在此期间的培训 奖学金将为我提供成为一名成功的内科科学家所必需的技能。

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.