Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells.

BACKGROUND: Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer (FRET) based genetically encoded nanosensors represent a promising tool for measuring the metabolite levels and corresponding rate changes in live cells. Here, we report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of L-lysine is of major interest in industrial biotechnology. RESULTS: The lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor. The LAO was sandwiched between GFP variants i.e. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Affinity, pH stability, specificity and metal ions effects was scrutinized for the in vitro characterization of this nanosensor, named as FLIPK. The FLIPK is specific to lysine and found to be stable with the pH within the physiological range. The calculated affinity (K d ) of FLIPK was 97 µM. For physiological applications, mutants with different binding affinities were also generated and investigated in vitro. The developed nanosensor efficiently monitored the intracellular level of lysine in bacterial as well as yeast cell. CONCLUSION: The developed novel lysine fluorescence resonance energy transfer sensors can be used for in vivo monitoring of lysine levels in prokaryotes as well as eukaryotes. The potential of these sensors is that they can be used as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular lysine during fermentation.

Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor.

Nickel (Ni2+) is an essential mineral nutrient that is required in trace quantities, but exposure to excess amounts can be toxic or carcinogenic. Mechanisms underlying Ni2+ imbalance and toxicity are poorly understood because most analytical methods currently used to probe this metal are invasive or also have toxic effects. To address these problems, a genetically encoded FRET-based probe for nickel metal (FProNiM) was constructed for real-time detection of Ni2+ in live cells. FProNiM was constructed by sandwiching the bacterial periplasmic nickel-binding protein NikA between the fluorescent protein variants ECFP and Venus. Ni2+ binding to FProNiM induced a conformational change that could be detected by a change in fluorescence resonance energy transfer (FRET) ratio (acceptor/donor fluorescence). In vitro studies demonstrated that FProNiM is specific for Ni2+, insensitive to changes in pH from 5.0 to 8.0, and has an affinity (Kd) of 21.6 µM, which allows detection of Ni2+ concentrations from 0.1 to 5000 µM. The sensor variant FProNiM-5 was also expressed in Escherichia coli (E. coli), yeast, and mammalian cells. In each cell type, changes in Ni2+ concentrations were detected with subcellular resolution using confocal microscopy.

Genetically-encoded nanosensor for quantitative monitoring of methionine in bacterial and yeast cells.

Metabolic engineering of microorganisms for production of biological molecules represent a key goal for industrial biotechnology. The metabolic engineering requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. In the present paper, we report the development of genetically-encoded FRET-based nanosensor for methionine as metabolic engineering of microbial strains for the production of l-methionine is of major interest in industrial biotechnology. In this nanosensor, methionine binding protein (MetN) from Escherichia coli (E. coli) K12 was taken and used as the reporter element of the sensor. The MetN was sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Specificity, affinity, pH stability and metal effects was analyzed for the in vitro characterization of this nanosensor, named as FLIPM. The FLIPM is very specific to methionine and found to be stable with the pH within the physiological range. The calculated affinity (Kd) of FLIPM was 203 µM. This nanosensor successfully monitored the intracellular level of methionine in bacterial as well as yeast cell. The data suggest that these nanosensors may be a versatile tool for studying the in vivo dynamics of methionine level non-invasively in living cells.

Genetically encoded FRET-based nanosensor for in vivo measurement of leucine.

Besides fundamental role in protein synthesis, leucine has metabolic roles as energy substrates, precursors for synthesis of other amino acids and as a modulator of muscle protein synthesis via the insulin-signaling pathway. Leucine concentration in cell and tissue is temporally dynamic as the metabolism of leucine is regulated through multiple enzymes and transporters. Assessment of cell-type specific activities of transporters and enzymes by physical fractionation is extremely challenging. Therefore, a method of reporting leucine dynamics at the cellular level is highly desirable. Given this, we developed a series of genetically encoded nanosensors for real-time in vivo measurement of leucine at cellular level. A leucine binding periplasmic binding protein (LivK) of Escherichia coli K12 was flanked with CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) at N-terminus and C-terminus, respectively. The constructed nanosensors allowed in vitro determination of fluorescence resonance energy transfer (FRET) changes in a concentration-dependent manner. These sensors were found to be specific to leucine, and stable to pH-changes within a physiological range. Genetically encoded sensors can be targeted to a specific cell type, and allow dynamic measurement of leucine concentration in bacterial and yeast cells.