Tracking the dynamics of the glycolytic metabolite fructose 1,6-bisphosphate in liver cancer cells with a fluorescent biosensor

HYlight is a genetically encoded fluorescent biosensor that ratiometrically monitors fructose 1,6-bisphosphate (FBP), a key glycolytic metabolite. Given the role of glucose in liver cancer metabolism, we expressed HYlight in human liver cancer cells and primary mouse hepatocytes. Through in vitro, in silico, and in cellulo experiments, we showed HYlight's ability to monitor FBP changes linked to glycolysis, not gluconeogenesis. HYlight's affinity for FBP was ~1 µM and stable within physiological pH range. HYlight demonstrated weak binding to dihydroxyacetone phosphate, and its ratiometric response was influenced by both ionic strength and phosphate. Therefore, simulating cytosolic conditions in vitro was necessary to establish a reliable correlation between HYlight’s cellular responses and FBP concentrations. They were found to be in the lower micromolar range, far lower than previous millimolar estimates. Altogether, this biosensor approach offers real-time monitoring of FBP concentrations at single-cell resolution, potentially revolutionizing our understanding of cancer metabolism.