Development of Red Fluorescent Protein pH Sensors

Abstract

The regulation of pH plays a crucial role in protein function, metabolic reactions, autophagy, and a wide range of other physiological processes. Cellular activities and diseases are known to perturb the pH in cells. For example, in cancer cells, increased reliance on aerobic glycolysis is thought to contribute to extracellular acidosis in the tumor microenvironment. Therefore, studying changes in pH in different compartments would allow us to better understand the role of pH in signaling and metabolism in both normal and disease states. Live‐cell imaging with fluorescent protein‐based sensors could allow us to monitor pH changes in situ; however, there are currently limited options for these sensors that are able to measure pH dynamics with subcellular resolution in live specimens. Furthermore, most of these genetically‐encoded pH sensors are generated from green fluorescent protein, limiting our ability to perform multiplex imaging to monitor pH simultaneously in different compartments. Therefore, it is the goal of this project to develop a range of genetically‐encoded fluorescent pH sensors. Here, we present the in vitro and live‐cell characterization of a new red fluorescent protein pH sensor. In proof‐of‐concept studies, we measure both intracellular and extracellular pH simultaneously as well as the pH of different subcellular compartments simultaneously. Our results demonstrate our progress towards the measurement of pH in the tumor microenvironment and understanding how pH is affected by aerobic glycolysis.Support or Funding InformationThis work is supported by National Institute of Health R21 NS092010 and R21 EY026425.