New Technologies for detecting extracellular fluxes

Abstract: The goal of this Focused Technology and Development grant is to repurpose intracellular fluorescent ion and metabolite sensors to detect extracellular fluxes. The three orthogonal approaches directly target the cell’s glycocalyx: the nanometer thick, sugar‐coating where ion and metabolite concentrations vary greatly during ion channel and membrane transporter activity. Aim 1 describes an approach to attach any fluorescent protein sensor to cells metabolically-labeled with an unnatural azidosugar. Aim 2 is a non-genetic approach that labels all mammalian cells—including human cells—with both small molecule and protein fluorescent sensors. Aim 3 is a chemical genetic approach to achieve cell-type-specific labeling with small molecule and protein fluorescent sensors. Completion of the aims will convert these working prototypes that utilize three major classes of fluorescent sensors (small molecule, protein, and FRET) into technologies that enable the visualization of ions and metabolites at the surface of a primary cells, tissues, and potentially in living animals.