Odaka Haruki, Arai Satoshi, Inoue Takafumi, Kitaguchi Tetsuya
Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore; Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore.
PLoS One. 2014 Jun 24;9(6):e100252. doi: 10.1371/journal.pone.0100252. eCollection 2014.
Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.
环磷酸腺苷（cAMP）是一种普遍存在的第二信使，它介导许多主要由细胞表面受体激活引发的细胞反应。人们已经创建了各种基于荧光共振能量转移的比率型cAMP指示剂，用于在单细胞水平监测cAMP的时空动态。然而，基于单一荧光蛋白的cAMP指示剂发展较差，其动态范围和亮度需要改进。基于我们之前基于黄色荧光蛋白的cAMP指示剂Flamindo，我们开发了一种改进的黄色荧光cAMP指示剂，命名为Flamindo2。通过优化发色团附近的连接肽，Flamindo2与Flamindo相比，动态范围扩大了2倍，亮度增加了8倍。我们发现，响应cAMP时，Flamindo2的荧光强度降低至25%。分别使用Flamindo2和核定位信号Flamindo2（nlsFlamindo2）对COS7细胞的细胞质和细胞核进行活细胞cAMP成像，结果表明，福斯可林以不同的动力学升高每个区室中的cAMP水平。此外，使用Flamindo2和红色荧光Ca2+指示剂R-GECO对cAMP和Ca2+进行双色成像，结果表明，去甲肾上腺素在单个HeLa细胞中诱导cAMP和Ca2+升高。我们的研究表明，Flamindo2可用于与其他细胞内信号分子进行多色成像，是一种有用的工具，也是活细胞成像细胞内cAMP动态的替代工具。
