Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.
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DOI：
10.1371/journal.pone.0038591
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Okumoto S
中科院分区：
文献类型：
--
作者：
Gruenwald K;Holland JT;Stromberg V;Ahmad A;Watcharakichkorn D;Okumoto S
谷氨酰胺在氨基酸、蛋白质、神经递质和谷胱甘肽等关键生物分子的新陈代谢中起着核心作用。由于谷氨酰胺代谢是通过多个酶和转运体来调节的，因此细胞内谷氨酰胺浓度可能是时间动态的。此外，同一组织中不同类型细胞(如神经细胞和神经胶质细胞)之间谷氨酰胺代谢的差异对于整个组织的正常功能通常是至关重要的，但通过物理分离来评估这种异种组织中转运蛋白和酶的细胞类型比活性是非常具有挑战性的。因此，在细胞水平上报告谷氨酰胺动态的方法是非常可取的。基因编码的传感器可以针对特定的细胞类型，从而解决这一知识鸿沟。在这里，我们报告了基于改进的青色和黄色荧光蛋白、单体Teal荧光蛋白(MTFP)1和Venus的Föster共振能量转移(FRET)谷氨酰胺传感器的开发。这些传感器对谷氨酰胺是特异的，并且在生理范围内对pH变化稳定。以表达人谷氨酰胺转运蛋白ASCT2的COS7细胞为模型，我们证明了利用这些传感器可以很容易地分析谷氨酰胺转运蛋白的性质。使用不同亲和力的传感器也可以估计给定细胞中谷氨酰胺浓度变化的范围。此外，mTFP1-venus FRET对可以与另一对FRET对mAmetine和tdTomato双链，从而为另一种分子的实时成像打开了可能性。这些新型谷氨酰胺传感器将是在单细胞水平上分析谷氨酰胺代谢特异性的有用工具。
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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影响因子：
4.2
作者：
Conti, F;Melone, M
通讯作者：
Melone, M
影响因子：
25
作者：
Dombeck, Daniel A.;Harvey, Christopher D.;Tian, Lin;Looger, Loren L.;Tank, David W.
通讯作者：
Tank, David W.
影响因子：
3.9
作者：
DENMAN, RB;WEDLER, FC
通讯作者：
WEDLER, FC
DOI：
10.1073/pnas.142089199
发表时间：
2002-07-23
影响因子：
11.1
作者：
Fehr, M;Frommer, WB;Lalonde, S
通讯作者：
Lalonde, S
影响因子：
48
作者：
Ai, Hui-wang;Hazelwood, Kristin L.;Campbell, Robert E.
通讯作者：
Campbell, Robert E.