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.
Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, Virginia, USA.
Katrin
K
Gruenwald
John Todd
JT
Holland
Verlyn
V
Stromberg
Altaf
A
Ahmad
Daisy
D
Watcharakichkorn
Sakiko
S
Okumoto
D000818:Animals; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism; D057054:Molecular Imaging; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D002352:Carrier Proteins; D008164:Luminescent Proteins; C037853:glutamine transport proteins; D005973:Glutamine