TY - JOUR
T1 - Tracking fructose 1,6-bisphosphate dynamics in liver cancer cells using a fluorescent biosensor
AU - Pérez-Chávez, Israel
AU - Koberstein, John N.
AU - Malo Pueyo, Julia
AU - Gilglioni, Eduardo H.
AU - Vertommen, Didier
AU - Baeyens, Nicolas
AU - Ezeriņa, Daria
AU - Gurzov, Esteban N.
AU - Messens, Joris
N1 - Funding Information:
We thank Edwige Martin-Valiente and Chlo\u00E9 Goemans for confocal microscopy assistance; Steven Janvier for mass spectrometry experimental advice; Mariana Nunes, and Anne Van Praet for the isolation of primary mouse hepatocytes; Erick Arroba Nu\u00F1ez, Francisco Ribeiro Coelho De Melo Costa for their technical assistance; Khadija Wahni, Antonio Ruiz Albor, Irene Clares Pedrero, and Andrea J\u00E1\u00F1ez Pedrayes for helpful discussions. We thank BioRender software for the creation of several figures. We thank the VIB Metabolomics Core facility for determining the FBP concentrations.FundingI.P.C. was supported by FNRS-FRIA Scholarship ( FC47359 ). J.M. was supported by a VIB grant. J.M.P. was supported by an FWO fellowship ( 1193524N ). E.N.G. was supported by a European Research Council Consolidator grant METAPTPs (grant agreement no. GA817940 ), FNRS-WELBIO grant ( 35112672 ), FNRS-PDR grant ( 40007740 ), FNRS-TELEVIE grant ( 40007402 ), and ULB Foundation. N.B. was supported by the ULB foundation .
Publisher Copyright:
© 2024 The Author(s)
PY - 2024/12/20
Y1 - 2024/12/20
N2 - 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.
AB - 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.
UR - https://doi.org/10.1016/j.isci.2024.111336
UR - http://www.scopus.com/inward/record.url?scp=85209576097&partnerID=8YFLogxK
U2 - 10.1016/j.isci.2024.111336
DO - 10.1016/j.isci.2024.111336
M3 - Article
C2 - 39640569
VL - 27
JO - iScience
JF - iScience
SN - 2589-0042
IS - 12
M1 - 111336
ER -