Ex vivo single molecule tools to analyze membrane receptor dynamics

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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.