National Research Center "Kurchatov Institute", Moscow 123182, Russia.
P.K. Anokhin Institute of Normal Physiology, Moscow 125315, Russia.
Int J Mol Sci. 2019 Jul 16;20(14):3488. doi: 10.3390/ijms20143488.
A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (K of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.
目前有多种遗传编码的钙指示剂可用于观察培养细胞和体内的钙动力学。其中只有一种称为 NIR-GECO1，它在光谱的近红外区域显示荧光。NIR-GECO1 是基于源自细菌光感受器的近红外荧光蛋白 mIFP 设计的。然而，NIR-GECO1 对钙离子的反应相反，其激发光谱不适用于常用的 640nm 激光器。我们使用小的近红外细菌光感受器 GAF-FP 和钙调蛋白/M13-肽对，开发了一种称为 GAF-CaMP2 的近红外钙指示剂。在体外，GAF-CaMP2 对钙离子呈 78%的正响应和高亲和力（K 为 466nM）。它的激发和发射最大值分别为 642nm 和 674nm。与体外的 GAF-FP 相比，GAF-CaMP2 的亮度低 2 倍，成熟速度快 5.5 倍，pH 稳定性低。GAF-CaMP2 的光稳定性比 smURFP 蛋白高 2.9 倍。在培养的哺乳动物细胞的细胞质中表达时，GAF-CaMP2 与 sfGFP 的融合显示出 2.9 倍的比率响应，动态范围为 169%。最后，我们成功地应用了比率版本的 GAF-CaMP2，用于使用四色荧光显微镜同时可视化哺乳动物细胞中三个细胞器的钙瞬变。
