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， 介导细胞间的交流单分子成像已被广泛用于 研究膜受体组装和化学计量在体外使用分离的蛋白质， 细胞培养程度较低。虽然在孤立的细胞系统中的研究提供了对许多 过程，它们缺乏动物中存在的复杂环境的背景， 存在于组织中的细胞类型之间的通讯对于受体活性是至关重要的。单身- 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，如受体二聚化。然而，在这方面， 目前的方法不能应用单分子技术来研究调节 蛋白质动力学在动物体内复杂环境中的作用。我们取得了 在开发离体单分子技术以监测 在特定器官内发生的蛋白质组装。该提案的目标是发展和 验证离体单分子工具，提供新的能力，以监测的性质 寡聚体膜受体组装，分辨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化单分子数据分析 平台这种新颖的离体技术将使研究人员能够及时拍摄 膜受体动态，发生在小鼠和监测膜的变化 受体组装，以响应动物生理环境的变化， 疾病和暴露于治疗剂。

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.