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 dy...

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Autores principales:	Katrin Gruenwald, John Todd Holland, Verlyn Stromberg, Altaf Ahmad, Daisy Watcharakichkorn, Sakiko Okumoto
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c8e189195077477fb59d8d06aa479575
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spelling	oai:doaj.org-article:c8e189195077477fb59d8d06aa4795752021-11-18T07:15:25ZVisualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.1932-620310.1371/journal.pone.0038591https://doaj.org/article/c8e189195077477fb59d8d06aa4795752012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22723868/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Glutamine 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.Katrin GruenwaldJohn Todd HollandVerlyn StrombergAltaf AhmadDaisy WatcharakichkornSakiko OkumotoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 6, p e38591 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Katrin Gruenwald John Todd Holland Verlyn Stromberg Altaf Ahmad Daisy Watcharakichkorn Sakiko Okumoto Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
description	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.
format	article
author	Katrin Gruenwald John Todd Holland Verlyn Stromberg Altaf Ahmad Daisy Watcharakichkorn Sakiko Okumoto
author_facet	Katrin Gruenwald John Todd Holland Verlyn Stromberg Altaf Ahmad Daisy Watcharakichkorn Sakiko Okumoto
author_sort	Katrin Gruenwald
title	Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
title_short	Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
title_full	Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
title_fullStr	Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
title_full_unstemmed	Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
title_sort	visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/c8e189195077477fb59d8d06aa479575
work_keys_str_mv	AT katringruenwald visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors AT johntoddholland visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors AT verlynstromberg visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors AT altafahmad visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors AT daisywatcharakichkorn visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors AT sakikookumoto visualizationofglutaminetransporteractivitiesinlivingcellsusinggeneticallyencodedglutaminesensors
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Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.

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