Ex vivo single molecule tools to analyze membrane receptor dynamics

膜受体在整个身体的许多生理过程中起着至关重要的作用 调节细胞间的通讯。单分子成像已被广泛应用于 用分离蛋白研究膜受体的体外组装和化学计量比 较小程度的细胞培养。虽然对隔离细胞系统的研究提供了对许多 过程，它们缺乏动物体内存在的复杂环境的背景 驻留在组织中的细胞类型之间的通信对受体的活性至关重要。单人- 对体内合成的天然蛋白质进行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，如受体二聚化。然而， 目前的方法不能应用单分子技术来研究这种调节 在动物复杂的环境中发生的蛋白质动力学。我们已经做出了 体外单分子技术监测血管紧张素转换酶变化研究进展 发生在特定器官内的蛋白质组装。这项提议的目标是发展和 验证体外单分子工具，提供新的功能来监测 寡聚膜受体组装，分辨细胞类型和亚细胞区域特异性单一 膜受体，并使用机器学习自动化单分子数据分析 站台。这项新颖的体外技术将使研究人员能够及时拍摄 在小鼠体内发生的膜受体动力学和监测膜的变化 对动物生理环境的变化作出反应的受体组装 疾病和接受治疗的机会。

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.