Ex Vivo Single-molecule Analysis of Ryanodine Receptor 2 Assembly in Cardiac and Neuronal Tissue.

Understanding receptor assembly is critical for elucidating the mechanisms underlying their function and regulation in physiological processes. While traditional in vitro single-molecule studies rely on isolating proteins from heterologous expression systems, they often fail to capture the in vivo physiological complexity involved in the organization and assembly of cell surface proteins. This protocol employs Total Internal Reflection Fluorescence Microscopy (TIRFM) to study GFP-tagged Ryanodine Receptor 2 (RyR2) molecules encapsulated within nanoscale vesicles. These vesicles, generated from organs rapidly extracted from the animal, effectively provide a snapshot of the receptor's assembly state at the time of extraction, enabling detailed analysis of subunit stoichiometry and receptor organization in response to changes in the animal's physiological environment. This approach utilizes TIRFM and stepwise photobleaching analysis to provide a readout of receptor stoichiometry. Imaging receptors at the single-molecule level facilitates the detection of heterogeneity within the receptor populations assembled in a live animal and enables monitoring of assembly changes associated with disease states. As a result, this method offers a powerful tool for determining the distribution of receptor assemblies and their correlation to changes in their physiological environment.