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Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.

Gruenwald K, Holland JT, Stromberg V, Ahmad A, Watcharakichkorn D, Okumoto S - PLoS ONE (2012)

Bottom Line: These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range.Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule.These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, Virginia, USA.

ABSTRACT
Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

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Elimination of cellular glutamine through the ASCT2 transporter in the presence of external amino acids, visualized using FLIPQ-TV3.0_8 m sensor.(A) Cytosolic glutamine is exported by the addition of extracellular Ala, Ser, Cys, Thr, and D-ser. Timepoints when extracellular glutamine (red boxes) or other amino acids (blue boxes) were added to the perfusion media are indicated as boxes above the graph. (B) Addition of Pro, Lys, His (filled boxes) does not alter cytosolic glutamine concentration, whereas the addition of Ala (blue boxes) promotes the export of glutamine. Solid and dashed lines represent two individual cells measured in the same experiment. All amino acids were added at 5 mM external concentrations.
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pone-0038591-g007: Elimination of cellular glutamine through the ASCT2 transporter in the presence of external amino acids, visualized using FLIPQ-TV3.0_8 m sensor.(A) Cytosolic glutamine is exported by the addition of extracellular Ala, Ser, Cys, Thr, and D-ser. Timepoints when extracellular glutamine (red boxes) or other amino acids (blue boxes) were added to the perfusion media are indicated as boxes above the graph. (B) Addition of Pro, Lys, His (filled boxes) does not alter cytosolic glutamine concentration, whereas the addition of Ala (blue boxes) promotes the export of glutamine. Solid and dashed lines represent two individual cells measured in the same experiment. All amino acids were added at 5 mM external concentrations.

Mentions: The cellular glutamine concentration stayed high after the removal of extracellular glutamine, indicating that the cellular metabolism is not sufficient to lower the level of glutamine to the detection range of these sensors. However, the addition of small, neutral amino acids such as Ala quickly reversed the cellular glutamine concentration (Fig. 6 and Fig. S7A). ASCT2 is an obligatory exchanger, and is able to mediate glutamine efflux in the presence of other extracellular amino acids [62]. Therefore we tested an array of amino acids for the ability to induce glutamine efflux. Ala, Thr, Cys, Ser, and D-ser, which are recognized by ASCT2, were able to induce glutamine efflux, whereas His, Pro, and Lys did not promote glutamine efflux (Fig. 7). The transport of glutamine was also dependent on extracellular sodium, consistent with previous observations [48] (Fig. 8). Since all of these results corroborate with the characteristics of ASCT2, we concluded that FLIPQ-TV sensors can be utilized to monitor cytosolic concentrations of glutamine, and that they can be used to investigate physiological parameters of the transporter, such as substrate specificity and dependence on extracellular sodium. Since genetically encoded sensors can be targeted to a single cell type using appropriate promoters, it would be possible to utilize these sensors to examine glutamine transport and metabolism in a specific cell type in vivo. Such an experimental setup will be particularly useful for tissues that consist of heterologous cell types with distinct glutamine metabolism (i.e. glial cells and neurons). For example, the molecular identity of the transporter that is responsible for providing glutamine to neuronal cells is still controversial in some cases [62], [63]. Presumably genetically encoded sensors could be utilized in combination with pharmacological techniques to analyze the transport system responsible for glutamine uptake in the context of live tissue, or even in an intact animal [64], [65], [66].


Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.

Gruenwald K, Holland JT, Stromberg V, Ahmad A, Watcharakichkorn D, Okumoto S - PLoS ONE (2012)

Elimination of cellular glutamine through the ASCT2 transporter in the presence of external amino acids, visualized using FLIPQ-TV3.0_8 m sensor.(A) Cytosolic glutamine is exported by the addition of extracellular Ala, Ser, Cys, Thr, and D-ser. Timepoints when extracellular glutamine (red boxes) or other amino acids (blue boxes) were added to the perfusion media are indicated as boxes above the graph. (B) Addition of Pro, Lys, His (filled boxes) does not alter cytosolic glutamine concentration, whereas the addition of Ala (blue boxes) promotes the export of glutamine. Solid and dashed lines represent two individual cells measured in the same experiment. All amino acids were added at 5 mM external concentrations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375291&req=5

pone-0038591-g007: Elimination of cellular glutamine through the ASCT2 transporter in the presence of external amino acids, visualized using FLIPQ-TV3.0_8 m sensor.(A) Cytosolic glutamine is exported by the addition of extracellular Ala, Ser, Cys, Thr, and D-ser. Timepoints when extracellular glutamine (red boxes) or other amino acids (blue boxes) were added to the perfusion media are indicated as boxes above the graph. (B) Addition of Pro, Lys, His (filled boxes) does not alter cytosolic glutamine concentration, whereas the addition of Ala (blue boxes) promotes the export of glutamine. Solid and dashed lines represent two individual cells measured in the same experiment. All amino acids were added at 5 mM external concentrations.
Mentions: The cellular glutamine concentration stayed high after the removal of extracellular glutamine, indicating that the cellular metabolism is not sufficient to lower the level of glutamine to the detection range of these sensors. However, the addition of small, neutral amino acids such as Ala quickly reversed the cellular glutamine concentration (Fig. 6 and Fig. S7A). ASCT2 is an obligatory exchanger, and is able to mediate glutamine efflux in the presence of other extracellular amino acids [62]. Therefore we tested an array of amino acids for the ability to induce glutamine efflux. Ala, Thr, Cys, Ser, and D-ser, which are recognized by ASCT2, were able to induce glutamine efflux, whereas His, Pro, and Lys did not promote glutamine efflux (Fig. 7). The transport of glutamine was also dependent on extracellular sodium, consistent with previous observations [48] (Fig. 8). Since all of these results corroborate with the characteristics of ASCT2, we concluded that FLIPQ-TV sensors can be utilized to monitor cytosolic concentrations of glutamine, and that they can be used to investigate physiological parameters of the transporter, such as substrate specificity and dependence on extracellular sodium. Since genetically encoded sensors can be targeted to a single cell type using appropriate promoters, it would be possible to utilize these sensors to examine glutamine transport and metabolism in a specific cell type in vivo. Such an experimental setup will be particularly useful for tissues that consist of heterologous cell types with distinct glutamine metabolism (i.e. glial cells and neurons). For example, the molecular identity of the transporter that is responsible for providing glutamine to neuronal cells is still controversial in some cases [62], [63]. Presumably genetically encoded sensors could be utilized in combination with pharmacological techniques to analyze the transport system responsible for glutamine uptake in the context of live tissue, or even in an intact animal [64], [65], [66].

Bottom Line: These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range.Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule.These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, Virginia, USA.

ABSTRACT
Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

Show MeSH