Ex vivo single molecule tools to analyze membrane receptor dynamics

用于分析膜受体动力学的离体单分子工具

• 批准号: 10207891
• 负责人: Christopher I Richards
• 金额: $ 29.91万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10207891
• 关键词: Address; Animal Communication; Animal Disease Models; Animal Model; Animals; Biological; Biological Models; Brain; Brain region; Cardiac; Cell Culture Techniques; Cells; Chemicals; Classification; Communication; Complex; Consumption; Data; Data Analyses; Data Set; Development; Dimerization; Disease; Encapsulated; Environment; Exposure to; Future; Goals; Heart; High Performance Computing; Image; In Vitro; Knock-in; Knock-in Mouse; Lead; Machine Learning; Manuals; Measurement; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Monitor; Mus; Organ; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Physiological Processes; Play; Population; Preparation; Process; Process Measure; Property; Protein Dynamics; Proteins; Regulation; Research; Research Methodology; Research Personnel; Role; RyR2; Ryanodine Receptor Calcium Release Channel; Sampling; Signal Transduction; Structure; System; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Training; Vesicle; alpha-SNAP; animal tissue; base; cell type; cluster computing; convolutional neural network; design; in vivo; insight; large datasets; molecular imaging; nanoscale; nanovesicle; novel; open source; postsynaptic neurons; presynaptic; prototype; receptor; response; sex; single molecule; stoichiometry; technique development; therapeutic target; tissue preparation; tool; trafficking

Membrane receptors play a crucial role in many physiological processes throughout the body by mediating communication between cells. Single molecule imaging has been used extensively to study membrane receptor assembly and stoichiometry in vitro using isolated protein and to a lesser extent cell culture. While studies in isolated cellular systems provide insight into many processes, they lack the context of the complex environment present in an animal where communication between cell types residing in tissue is critical for receptor activity. Single- molecule imaging of native proteins synthesized in vivo would provide a direct approach to measure processes, such as receptor dimerization, that regulate physiological activity. However, current methods are not capable of applying single molecule techniques to study the regulation of protein dynamics taking place within the complex environment of an animal. We have made recent advances in the development of ex vivo single molecule techniques to monitor changes in protein assembly that occur within specific organs. The goal of this proposal is to develop and validate ex vivo single molecule tools that provide new capabilities to monitor the properties of oligomeric membrane receptor assembly, resolve cell type and subcellular region specific single membrane receptors, and automate single molecule data analysis using a machine learning platform. This novel ex vivo technology will enable researchers to take a snap shot in time of the membrane receptor dynamics that took place in the mouse and monitor changes in membrane receptor assembly in response to changes in the animal's physiological environment such as disease and exposure to therapeutics.

膜受体在全身许多生理过程中发挥着至关重要的作用 介导细胞之间的通讯。单分子成像已被广泛用于 使用分离的蛋白质在体外研究膜受体组装和化学计量并 较小程度的细胞培养。虽然对孤立细胞系统的研究提供了对许多 过程中，它们缺乏动物体内存在的复杂环境的背景 组织中细胞类型之间的通讯对于受体活性至关重要。单身的- 体内合成的天然蛋白质的分子成像将提供一种直接的方法 测量调节生理活动的过程，例如受体二聚化。然而， 目前的方法无法应用单分子技术来研究调控 动物复杂环境中发生的蛋白质动力学。我们已经做了 离体单分子技术监测变化的最新进展 特定器官内发生的蛋白质组装。该提案的目标是开发和 验证离体单分子工具，提供监测特性的新功能 寡聚膜受体组装，解析细胞类型和亚细胞区域特异性单 膜受体，并使用机器学习自动进行单分子数据分析 平台。这种新颖的离体技术将使研究人员能够及时拍摄快照 发生在小鼠体内的膜受体动力学并监测膜的变化 受体组装响应动物生理环境的变化，例如 疾病和接受治疗。