Ex Vivo Single Molecule Tools to Analyze Membrane Receptor Dynamics

Membrane receptors play a crucial role in many physiological processes in the central nervous system (CNS) including mediating communication between neurons and glial 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 in 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. This is particularly true in the central nervous system (CNS) where neuronal and glial interactions shape processes ranging from protein expression to cellular function and communication. 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 the CNS. The goal of this proposal is to develop and validate ex vivo single molecule tools that provide new capabilities to monitor brain region specific properties of oligomeric membrane receptor assembly, resolve presynaptic and postsynaptic 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 CNS and monitor changes in membrane receptor assembly in response to changes in the animal’s physiological environment such as disease and exposure to therapeutics.