In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors
In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors
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DOI：
10.1074/jbc.m301333200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Frommer, WB
中科院分区：
文献类型：
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作者：
Fehr, M;Lalonde, S;Frommer, WB
葡萄糖稳态是葡萄糖供应、跨质膜转运和代谢的功能。为了监测单个细胞中的葡萄糖动力学，通过用两种不同的绿色荧光蛋白变体侧接大肠杆菌周质葡萄糖/半乳糖结合蛋白来开发葡萄糖纳米传感器。在结合底物后，FLIPglu-170 n传感器显示出所连接的发色团之间的荧光共振能量转移的浓度依赖性降低，对葡萄糖的结合亲和力为170 nM。荧光共振能量转移测量与不同的糖表明单糖的广泛的选择性。产生了一个亲和力突变体，其Kd值接近600 μ M，显示出更高的底物特异性，从而允许在生理范围内特异性监测COS-7细胞中可逆的葡萄糖动力学。当外界葡萄糖浓度在0.5 - 10 mM之间时，反映了典型的血液水平，游离胞质葡萄糖浓度保持在接近外界水平的50%。葡萄糖的去除导致细胞中葡萄糖水平降低，证明了可逆性和可视化稳态。即使在存在转运抑制剂细胞松弛素B的情况下，葡萄糖水平也下降，表明代谢迅速。一致地，2-脱氧葡萄糖的添加，其不被传感器识别，影响葡萄糖摄取和代谢速率。在生理范围内，葡萄糖利用，即己糖激酶活性，是不受限制的。此外，结果表明，在COS-7细胞中，胞质葡萄糖浓度可以在至少两个数量级上变化。葡萄糖纳米传感器提供了一种具有众多科学，医学和环境应用的新工具。
Glucose homeostasis is a function of glucose supply, transport across the plasma membrane, and metabolism. To monitor glucose dynamics in individual cells, a glucose nanosensor was developed by flanking the Escherichia coli periplasmic glucose/galactose-binding protein with two different green fluorescent protein variants. Upon binding of substrate the FLIPglu-170n sensor showed a concentration-dependent decrease in fluorescence resonance energy transfer between the attached chromophores with a binding affinity for glucose of 170 nM. Fluorescence resonance energy transfer measurements with different sugars indicated a broad selectivity for monosaccharides. An affinity mutant with a K-d of similar to600 muM was generated, which showed higher substrate specificity, and thus allowed specific monitoring of reversible glucose dynamics in COS-7 cells in the physiological range. At external glucose concentrations between 0.5 and 10 mM, reflecting typical blood levels, free cytosolic glucose concentrations remained at similar to50% of external levels. The removal of glucose lead to reduced glucose levels in the cell, demonstrating reversibility and visualizing homeostasis. Glucose levels dropped even in the presence of the transport inhibitor cytochalasin B, indicating rapid metabolism. Consistently, the addition of 2-deoxyglucose, which is not recognized by the sensor, affects glucose uptake and metabolism rates. Within the physiological range, glucose utilization, i.e. hexokinase activity, was not limiting. Furthermore, the results show that in COS-7 cells, cytosolic glucose concentrations can vary over at least two orders of magnitude. The glucose nanosensor provides a novel tool with numerous scientific, medical, and environmental applications.