The goal of this project is fluorescently visualize ATP release and extracellular accumulation at the surface of stimulated microglia. The development of this innovative technology has the potential to enable spatiotemporal imaging of microglial extracellular signaling. For this project, I am exploiting the presence of the cell's glycocalyx to attach ATP-sensitive biosensors at the sites of ATP accumulation. There are two aims to this project: 1) to synthesize a novel, polyhistidine binding moiety that covalently modifies the glycocalyces of living cells and binds recombinant biosensors to measure ion and metabolite efflux and accumulation; 2) to visualize and measure ATP release from pannexin channels in C5a stimulated microglia. The completion of these aims will yield a transformative set of chemical-biological tools and methodologies to investigate the physiology and pathophysiology of pannexin-dependent activity in glia, and potentially in living animals.
The stimulated release of pannexin-dependent ATP from microglia is essential for neuroinflammatory responses in the brain. Accordingly, dysregulated release of ATP and over-inflammation has been linked to neuronal loss, brain damage, some forms of neuropathic pain. By visualizing the spatiotemporal release and accumulation of ATP and other ions and metabolites in cells, tissues and potentially model mammalian organisms, I aim to understand how these channel-mediated signals regulate brain function in healthy and diseased individuals.