Live Imaging of glucose homeostasis in nuclei of COS-7 cells
Live Imaging of glucose homeostasis in nuclei of COS-7 cells
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DOI：
10.1023/b:jofl.0000039347.94943.99
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发表时间：
2004-09-01
影响因子：
2.7
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
测量组织深处的亚细胞葡萄糖水平可以为不同细胞之间葡萄糖代谢的区室化和专业化提供新的见解。如前所示，由两种GFP变体和大肠杆菌周质葡萄糖/半乳糖结合蛋白组成的基于FRET的葡萄糖传感器在COS 7细胞的胞质溶胶中成功表达，并用于测定胞质葡萄糖水平。由于其他细胞层对激发和发射光的影响导致信号强度损失，记录位于组织深层的细胞中的胞质荧光强度通常是困难的。这些干扰效应可以通过限制荧光团仅占据测定的组织体积的一部分来减少。这可以通过将荧光团限制在组织中每个细胞的子隔室（诸如细胞核）来实现。葡萄糖传感器靶向于COS 7-细胞的细胞核。为了确定核葡萄糖水平是否可用于跟踪胞质变化，当细胞用0.5至10 mM范围内的外部葡萄糖攻击时，定量核葡萄糖浓度，并与胞质水平进行比较。两个隔室中的内部葡萄糖浓度相似，相当于外部浓度的50%。两者合计，这些结果表明，核葡萄糖水平可用于间接确定胞质水平，允许更可靠的定量荧光强度，并提供了一个工具，不仅在细胞培养物中，而且在组织中的测量。
Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to similar to50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.