Tracking fructose 1,6-bisphosphate dynamics in liver cancer cells using a fluorescent biosensor
Israel Pérez-Chávez,
John N. Koberstein,
Julia Malo Pueyo,
Eduardo H. Gilglioni,
Didier Vertommen,
Nicolas Baeyens,
Daria Ezeriņa,
Esteban N. Gurzov,
Joris Messens
Affiliations
Israel Pérez-Chávez
VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Signal Transduction and Metabolism Laboratory, Université Libre de Bruxelles (ULB), Brussels, Belgium
John N. Koberstein
HHMI Janelia Research Campus, Ashburn, VA, USA
Julia Malo Pueyo
VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium
Eduardo H. Gilglioni
Signal Transduction and Metabolism Laboratory, Université Libre de Bruxelles (ULB), Brussels, Belgium
Didier Vertommen
de Duve Institute, MASSPROT Platform, UCLouvain, 1200 Brussels, Belgium
Nicolas Baeyens
Laboratoire de Physiologie et de Pharmacologie (LAPP), Université Libre de Bruxelles (ULB), Brussels, Belgium
Daria Ezeriņa
VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Corresponding author
Esteban N. Gurzov
Signal Transduction and Metabolism Laboratory, Université Libre de Bruxelles (ULB), Brussels, Belgium; WELBIO Department, WEL Research Institute, Avenue Pasteur 6, Wavre B-1300, Belgium; Corresponding author
Joris Messens
VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium; Corresponding author
Summary: 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. FBP concentrations 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, making it an invaluable tool for the understanding of cancer metabolism.
