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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Didier Vertommen

LAB HEAD: Joris Messens

PROVIDER: PXD051879 | Pride | 2024-11-14

REPOSITORIES: pride

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			Action	DRS
	EXT522_50kd_1_bis.mzML	Mzml
	EXT522_50kd_1_bis.pdResult	Other
	EXT522_50kd_1_bis.raw	Raw
	EXT522_70kd_bis.mzML	Mzml
	EXT522_70kd_bis.pdResult	Other

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Project description:Metabolic heterogeneity modulates productivity, antibiotic resistance and cancer aggressiveness. Since metabolic fluxes represent the functional output of metabolism, with glycolytic flux correlating with highly-productive phenotypes and cancer, such flux map will be indicative of the cellular metabolic state. Therefore, the quantification of metabolic fluxes is vital to identify the existence of metabolic subpopulations and to understand the process of their emergence at the single-cell level. However, so far inference of metabolic fluxes in individual cells is not possible as no method is available. Here, we developed a biosensor for glycolytic flux measurements in single yeast cells drawing on the robust correlation between fructose-1,6-bisphosphate (FBP) and flux levels in yeast, and using the B. subtilis FBP-binding transcription factor CggR. We followed a systematic engineering approach starting from promoter design, computational protein design and protein engineering, accompanied by strict characterization of the biosensor using different biochemical methods, proteomics, metabolomics and physiological analyses. As proof of principle, we applied the biosensor in vivo in the search for metabolic subpopulations in yeast cultures and, using fluorescence microscopy, we demonstrated that quiescent yeast cells have low glycolytic fluxes in comparison to coexisting dividing cells. We anticipate that our biosensor will contribute with unprecedented resolution for the study of metabolic subpopulations, to understand how and why metabolic subpopulations emerge and, very importantly, give clues on how to counteract the undesirable effects of such.

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