Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of β-cell glycolytic heterogeneity and dynamics.
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Early online date||26 Jul 2022|
|Publication status||Published (in print/issue) - 2 Aug 2022|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS. We thank Markus Grompe and Sunghee Chai for the β-cell–specific adeno-associated virus vector; Xander Viray and James Frank for help with calcium measurements; Matthew Schleisman for assistance with FACS; Yibing Jia for help with DNA sequencing; Stephanie Kaech for her expertise in microscopy; Erkan Karakas and Carsten Shultz for helpful discussions; and Paul Klevit and Melissa Kirigiti from the Oregon National Primate Research Center for technical assistance in preparing islets. Portions of this work were carried out in the Oregon Health and Science University Imaging Core, Flow Cytometry Core, Molecular Technologies Core, and by R01 AG055431 (to R.H.G.).
Copyright © 2022 the Author(s).
- Biosensing Techniques
- Repressor Proteins - metabolism
- fructose 1,6-bisphosphate