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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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Affinities and substrate specificities of FLIPQ-TV3.0 sensors.(A) Saturation curves of FLIPQ-TV3.0 sensors with altered affinities. (B) Substrate specificities of FLIPQ-TV3.0_1.5 μ (black), 50 μ (hatched), 100 μ (white), 2 m (horizontal stripes), and 8 m (gray) sensors to Gln, Glu, Asn and Asp.
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pone-0038591-g004: Affinities and substrate specificities of FLIPQ-TV3.0 sensors.(A) Saturation curves of FLIPQ-TV3.0 sensors with altered affinities. (B) Substrate specificities of FLIPQ-TV3.0_1.5 μ (black), 50 μ (hatched), 100 μ (white), 2 m (horizontal stripes), and 8 m (gray) sensors to Gln, Glu, Asn and Asp.

Mentions: The FLIPQ-TV1.0 had very low FRET efficiency change upon binding of glutamine (Fig. 2B, ΔR/R0 = 0.033). To further improve the FRET efficiency change of FLIPQ-TV sensors, linker sequences between the binding protein and fluorophores were modified using a semi-high throughput approach. Linker sequences at the N- and C- termini of mTFP1, and N- terminus of venus were altered sequentially through random mutagenesis (Fig. 3A and Fig.S2) to select for clones with an improved FRET efficiency change. In order to avoid the potential saturation of the sensors due to contamination by glutamine from bacterial lysate, a version of the sensor that had ∼20 times lower affinity compared to the sensor based on wild-type glnH (FLIPQ-TV_R75K, Table 1 and Fig. 4A) was used as the starting clone for the optimization.


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)

Affinities and substrate specificities of FLIPQ-TV3.0 sensors.(A) Saturation curves of FLIPQ-TV3.0 sensors with altered affinities. (B) Substrate specificities of FLIPQ-TV3.0_1.5 μ (black), 50 μ (hatched), 100 μ (white), 2 m (horizontal stripes), and 8 m (gray) sensors to Gln, Glu, Asn and Asp.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038591-g004: Affinities and substrate specificities of FLIPQ-TV3.0 sensors.(A) Saturation curves of FLIPQ-TV3.0 sensors with altered affinities. (B) Substrate specificities of FLIPQ-TV3.0_1.5 μ (black), 50 μ (hatched), 100 μ (white), 2 m (horizontal stripes), and 8 m (gray) sensors to Gln, Glu, Asn and Asp.
Mentions: The FLIPQ-TV1.0 had very low FRET efficiency change upon binding of glutamine (Fig. 2B, ΔR/R0 = 0.033). To further improve the FRET efficiency change of FLIPQ-TV sensors, linker sequences between the binding protein and fluorophores were modified using a semi-high throughput approach. Linker sequences at the N- and C- termini of mTFP1, and N- terminus of venus were altered sequentially through random mutagenesis (Fig. 3A and Fig.S2) to select for clones with an improved FRET efficiency change. In order to avoid the potential saturation of the sensors due to contamination by glutamine from bacterial lysate, a version of the sensor that had ∼20 times lower affinity compared to the sensor based on wild-type glnH (FLIPQ-TV_R75K, Table 1 and Fig. 4A) was used as the starting clone for the optimization.

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