Determining the cellular and nanoscale localization of Synaptotagmin VII

确定 Synaptotagmin VII 的细胞和纳米级定位

• 批准号: 10607488
• 负责人: Rachel Cain
• 金额: $ 4.9万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-05 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607488
• 关键词: Agreement; Apoptosis; Applications Grants; Architecture; Award; Biochemical; Biochemistry; Biological Assay; Biotinylation; Bipolar Disorder; Brain; Calcium; Cell Surface Proteins; Cell membrane; Cell surface; Central Nervous System; Color; DLG4 gene; Data; Disease; Docking; Family member; Fellowship; Functional disorder; Head and Neck Cancer; Image; Learning; Link; Malignant Neoplasms; Malignant neoplasm of brain; Membrane Proteins; Mental disorders; Mus; NMDA receptor A1; Neurons; Oncogenes; Physicians; Preparation; Presynaptic Terminals; Prostate; Protein Family; Proteins; Publishing; Rattus; Recycling; Role; Scientist; Site; Slice; Stains; Structural Models; Structure; Surface; Synapses; Synaptic Cleft; Synaptic Transmission; Testing; Tetrodotoxin; Training; Tretinoin; Vertebral column; Vesicle; Work; channel blockers; design; experimental study; inhibitor; insight; nano; nanoscale; nervous system disorder; neurotransmission; neurotransmitter release; overexpression; postsynaptic; presynaptic; protein function; sensor; skills; superresolution microscopy; synaptotagmin; synaptotagmin VII; transmission process; ultra high resolution; vesicular release

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.

摘要 突触功能障碍与许多毁灭性的神经和精神疾病有关。 突触传递由神经递质的钙依赖性释放启动。钙 负责触发囊泡释放的传感器是Synaptotagmins（SYT），一个17蛋白家族。的 一种SYT，SYT7的功能不太清楚，但由于其 在整个身体和大脑中广泛表达，与癌症和ALS的联系，以及在 中枢神经系统与其他突触结合蛋白不同，SYT7被认为存在于血浆中， 膜及其功能仍存在争议。SYT7的作用仍然处于激烈的争论之中， 关于其细胞和突触定位的几个突出问题。回答这些关键问题 是我的拨款申请的重点，并将提供必要的深入了解SYT7的功能。 这项提案旨在通过确定SYT7的细胞和纳米尺度来回答这些问题。 使用生物化学和三色STED超分辨率显微镜在皮质神经元中的定位。目的 1将使用活细胞表面蛋白确定SYT7是否存在于皮质神经元的细胞表面 小鼠皮质切片和大鼠原代皮质神经元中的生物素化和活细胞表面染色。目的2 将使用内源性突触的三色STED染色来确定SYT7在突触中是如何组织的。 proteins.目的3将确定神经元活动是否调节细胞和纳米级定位的 SYT7使用表面蛋白生物素化和活细胞表面用三色STED染色。 这份建议书旨在为我提供整个过程中的知识和技术培训。 奖学金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。在此奖项的支持下， 奖学金将为我提供必要的技能，成为一个成功的医生科学家。