MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor
MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor
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DOI：
10.1371/journal.pone.0082009
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发表时间：
2013-12-02
期刊：
影响因子：
3.7
通讯作者：
Merkx, Maarten
中科院分区：
文献类型：
--
作者：
Lindenburg, Laurens H.;Vinkenborg, Jan L.;Merkx, Maarten
镁在细胞中具有重要的结构、催化和信号作用，但很少有工具可以实时和亚细胞分辨率地成像这种金属离子。在这里，我们报告了第一个遗传编码的Mg2+传感器，MagFRET-1。该传感器基于人类中心蛋白3 （HsCen3）的高亲和力Mg2+结合结构域，该结构域经历了从熔融球状载子形式到紧密折叠的Mg2+结合状态的转变。将Cerulean和Citrine荧光结构域融合到HsCen3的末端，产生了MagFRET-1，该结构结合了生理上相关的Mg2+亲和力（K-d = 148 mu M），并且由于Cerulean和Citrine之间FRET效率的变化，Mg2+结合后发射比增加了50%。金属结合位点的突变产生的MagFRET变体，其Mg2+亲和力相对于MagFRET-1减弱了2- 100倍，从而覆盖了广泛的Mg2+浓度范围。在HEK293细胞的原位实验表明，MagFRET-1可以靶向细胞质和细胞核。表达magfret -1的HEK293细胞被洋地黄苷渗透后，对细胞外Mg2+浓度的变化有明显的反应，而完整的细胞则没有类似的变化。虽然MagFRET-1也对Ca2+敏感，但这种亲和力被充分减弱（K-d为10 μ M），使该传感器对HEK293细胞中已知的Ca2+刺激不敏感。虽然MagFRET传感器用于细胞内Mg2+成像的潜力和局限性需要进一步确定，但我们希望这些基因编码和比例荧光Mg2+传感器可以证明在理解细胞内Mg2+稳态和信号传导方面非常有用。
Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (K-d = 148 mu M) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (K-d of 10 mu M) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling.