J. Matthew Rhett, PhD Instructor, Department of Surgery, Medical University of South Carolina
Gap junctions (GJs) are large aggregates of intercellular channels that facilitate the diffusion of small molecules and ions between two interacting cells. GJ intercellular channels are formed through the interaction of two half-channels, called hemichannels, composed of oligomerized connexin protein subunits. Both GJs and hemichannels have numerous important physiological and pathological roles in tissue functions including propagation of the action potential in the heart, tumor growth and metastasis, the inflammatory response and adaptive immunity, wound healing, and electrical synaptic transmission in the central nervous system. The most widely expressed connexin isoform is Cx43, and its regulation in the abovementioned processes has been a major focus of GJ research. Over the past two decades protein-protein interaction with the cytoplasmic carboxyl terminus of Cx43 has come to the fore as an endogenous mechanism for controlling the GJ life cycle, channel gating, and channel-independent functions. We have used the Duolink proximity ligation assay (PLA) as a technique to study protein interactions with Cx43 in cultured cells. Two unique aspects of the technology – specifically, subcellular localization and the binary nature of the labeling – in combination with standard immunofluorescent confocal imaging techniques have yielded unexpected insights into GJ ultrastructure, action potential conduction, and the mechanistic regulation of Cx43 trafficking and hemichannel accretion to GJ plaques. In this context, the practical application of, appropriate controls for, and interpretation of Duolink PLAs will be explicated.