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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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Configuration of a FRET glutamine sensor.(A) Open (cyan) [36] and closed (yellow, glutamine in the binding pocket is indicated in red) [37] conformation of glnH, glutamine binding protein from E.coli. The position of the internal hairpin permissive to an insertion of FP is marked in magenta. (B) Schematic representations of chimeric fusions between mTFP1, glnH and venus sequences.
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pone-0038591-g001: Configuration of a FRET glutamine sensor.(A) Open (cyan) [36] and closed (yellow, glutamine in the binding pocket is indicated in red) [37] conformation of glnH, glutamine binding protein from E.coli. The position of the internal hairpin permissive to an insertion of FP is marked in magenta. (B) Schematic representations of chimeric fusions between mTFP1, glnH and venus sequences.

Mentions: The sites of attachment for the donor and acceptor molecules influence FRET efficiency, because they influence both the distance between fluorophores and dipole-dipole orientation. The crystal structure of glnH in both the open and closed form has been published previously [36], [37]. One of the lobes of glnH contains a large hairpin-like structure close to its N terminus, which allows insertion of the FPs (Fig. 1A, the permissive position is indicated in magenta). It has previously been demonstrated that insertion of ECFP in the corresponding location in the glutamate binding protein ybeJ, which is structurally related to glnH, does not interfere with the binding of glutamate to the chimera protein [38]. Therefore, we systematically tested combinations of these three possible insertion sites for the donor and acceptor proteins (Fig. 1B). For venus, it is known that terminal regions (an N-terminal helix and a C-terminal coil) are not required for the fluorescence [38]. Therefore, in addition to full-length venus, a series of clones that had part of the N- and C- terminal amino acids removed were used in order to find the optimal linker length. Among the constructs examined, we found one functional mTFP1/venus based glutamine sensor, named FLIPQ-TV(mTFP/Venus) 1.0 (Fig. 1B). In this configuration, mTFP1 and venus are located in the same lobe, hence the conformational change in the glnH domain is unlikely to induce a significant change in the distance between the two FPs. However, the binding of glutamine causes a shift of the second lobe, opening up a larger space in the vicinity of C-terminus where the venus molecule is fused (Fig.S1). Such a change is likely to increase the accessible space that the venus protein can occupy due to the decrease in sterical constraint, hence affecting the FRET efficiency between the two FPs. In fact, a number of type II periplasmic binding proteins, in which N- and C- termini are located in the same lobe, can be converted into a functional FRET sensors when the two FPs are fused on the N- and C- termini [38], [39], [40].


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)

Configuration of a FRET glutamine sensor.(A) Open (cyan) [36] and closed (yellow, glutamine in the binding pocket is indicated in red) [37] conformation of glnH, glutamine binding protein from E.coli. The position of the internal hairpin permissive to an insertion of FP is marked in magenta. (B) Schematic representations of chimeric fusions between mTFP1, glnH and venus sequences.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038591-g001: Configuration of a FRET glutamine sensor.(A) Open (cyan) [36] and closed (yellow, glutamine in the binding pocket is indicated in red) [37] conformation of glnH, glutamine binding protein from E.coli. The position of the internal hairpin permissive to an insertion of FP is marked in magenta. (B) Schematic representations of chimeric fusions between mTFP1, glnH and venus sequences.
Mentions: The sites of attachment for the donor and acceptor molecules influence FRET efficiency, because they influence both the distance between fluorophores and dipole-dipole orientation. The crystal structure of glnH in both the open and closed form has been published previously [36], [37]. One of the lobes of glnH contains a large hairpin-like structure close to its N terminus, which allows insertion of the FPs (Fig. 1A, the permissive position is indicated in magenta). It has previously been demonstrated that insertion of ECFP in the corresponding location in the glutamate binding protein ybeJ, which is structurally related to glnH, does not interfere with the binding of glutamate to the chimera protein [38]. Therefore, we systematically tested combinations of these three possible insertion sites for the donor and acceptor proteins (Fig. 1B). For venus, it is known that terminal regions (an N-terminal helix and a C-terminal coil) are not required for the fluorescence [38]. Therefore, in addition to full-length venus, a series of clones that had part of the N- and C- terminal amino acids removed were used in order to find the optimal linker length. Among the constructs examined, we found one functional mTFP1/venus based glutamine sensor, named FLIPQ-TV(mTFP/Venus) 1.0 (Fig. 1B). In this configuration, mTFP1 and venus are located in the same lobe, hence the conformational change in the glnH domain is unlikely to induce a significant change in the distance between the two FPs. However, the binding of glutamine causes a shift of the second lobe, opening up a larger space in the vicinity of C-terminus where the venus molecule is fused (Fig.S1). Such a change is likely to increase the accessible space that the venus protein can occupy due to the decrease in sterical constraint, hence affecting the FRET efficiency between the two FPs. In fact, a number of type II periplasmic binding proteins, in which N- and C- termini are located in the same lobe, can be converted into a functional FRET sensors when the two FPs are fused on the N- and C- termini [38], [39], [40].

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
Related in: MedlinePlus