Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

Abstract

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 Bacillus subtilis 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.

Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity

“…We next sought to establish the extent to which neuronal activity-dependent glucose metabolism might contribute to mitochondrial O-GlcNAcylation. To monitor the metabolic changes that occur following neuronal activity, we measured fructose 1,6-bisphosphate (FBP) levels in neuronal processes using HYlight, a fluorescent sensor that reports glycolytic dynamics 23 (Figure 4A). Because neuronal activity causes transient dips in intracellular glucose levels and also transiently increases neuronal glycolysis 4 , we predicted that FBP levels would decrease as neuronal activity increases.…”

“…The following previously published or commercially DNA constructs were used: pDsRed2-Mito (Clontech Laboratories Inc.), Mito-BFP 60 , HYLight 23 . pAAV.CAG.GCaMP6s.WPRE.SV40 was a gift from Douglas Kim & GENIE Project (Addgene plasmid #100844; http://n2t.net/addgene:100844; RRID: Addgene_100844) 61 .…”

“…Glycolysis dynamics were measured using a genetically encoded fluorescent biosensor for fructose 1,6-bisphosphate (FBP) termed HYlight as described 23 . Neurons were transfected with HYlight and imaged at 11-12 DIV.…”

“…For each imaging session, first pre-stimulus fluorescence baseline (R 0 ) measurements were performed for 5 minutes in the absence of electrical activity as a control, then action potentials were evoked by field stimulation (100mA,10 Hz,600 AP). Time-lapse movies from neurons expressing HYlight were acquired as described previously 23 for 15-20min. Briefly, the neuron images were acquired for each time point by using 488 nm and 405 nm lasers for excitation and emission detection ranges at 492-598 nm and 490-596 nm, respectively.…”

“…The following previously published or commercially DNA constructs were used: pDsRed2-Mito (Clontech Laboratories Inc.), Mito-BFP 60 , HYLight 23 http://n2t.net/addgene:102557; RRID: Addgene_102557) 62 .…”