Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors

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

Configuration of a FRET glutamine sensor.

<p>(A) Open (cyan) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038591#pone.0038591-Hsiao1" target="_blank">[36]</a> and closed (yellow, glutamine in the binding pocket is indicated in red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038591#pone.0038591-Sun1" target="_blank">[37]</a> conformation of glnH, glutamine binding protein from <i>E.coli</i>. 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.</p

Affinities of FLIPQ-TV3.0 point mutants.

<p>Affinities of FLIPQ-TV3.0 point mutants.</p

Affinities and substrate specificities of FLIPQ-TV3.0 sensors.

<p>(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.</p

Responses of cos7 cells co-expressing the FLIPQ-TV3.0_8 m sensor and ASCT2-mCherry to external glutamine in the absence (A) or in the presence of 13.4 mM (∼10% of normal Hank’s buffer) of extracellular sodium.

<p>Timepoints when 5 mM extracellular glutamine (red box) or 5 mM Ala (blue box) are indicated as boxes above the graph. Solid and dashed lines represent two individual cells measured in the same experiment.</p

<i>In vivo</i> glutamine measurements using FLIPQ-TV3.0_8 m and 100 μ sensors.

<p>(A) The venus/mTFP1 ratio of cos7 cells co-expressing FLIPQ-TV3.0_8 m sensor and ASCT2-mCherry. The cells were perfused with HEPES-buffered Hank’s buffer. Timepoints when extracellular glutamine (red) and 5 mM Ala (blue) was added to the perfusion media are indicated as boxes above the graph. Solid and dashed lines represent two individual cells measured in the same experiment. (B) The intensities of mTFP1 and venus channels in the experiment shown in (A). The values were corrected for photobleaching and normalized to the baseline. (C) and (D) A similar experiment as in (A) and (B), performed with cos7 cells expressing the FLIPQ-TV3.0_100 μ sensor.</p

Elimination of cellular glutamine through the ASCT2 transporter in the presence of external amino acids, visualized using FLIPQ-TV3.0_8 m sensor.

<p>(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.</p

Improvement of the FLIPQ-TV sensor through semi-high throughput screening.

<p>(A) Schematic representation of FLIPQ-TV 3.0_R75K sensor, in which linker sequences (indicated as Ln1–3) were sequentially altered through random mutagenesis. The linker sequence of the resulting clone (FLIPQ-TV 3.0_R75K) is indicated. (b) Emission spectra of the FLIPQ-TV3.0_R75K sensor in the absence (black squares) or in the presence of 1 mM glutamine (open squares).</p