开发基于绿色荧光蛋白的 cAMP 指示剂以覆盖广泛的 cAMP 浓度,RSC Advances

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开发基于绿色荧光蛋白的 cAMP 指示剂以覆盖广泛的 cAMP 浓度
RSC Advances ( IF 3.9 ) Pub Date : 2023-05-22 , DOI: 10.1039/d3ra01390a
Sohei Hiasa ₁ , Takeru Fujimori ₁ , Saki Aiki ₂ , Hiroshi Ueda ₃ , Takashi Tsuboi ₂ , Tetsuya Kitaguchi ₃

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细胞内环腺苷 3',5'-单磷酸 (cAMP) 的浓度范围很广，它在影响许多生理过程的通路中作为第二信使介导特定作用。_{在这里，我们开发了具有各种 EC 50}的绿色荧光 cAMP 指示剂，命名为 Green Falcan（基于绿色荧光蛋白的指示剂可视化 cAMP 动力学）值（0.3、1、3、10 μM），用于覆盖广泛的细胞内 cAMP 浓度。Green Falcans 的荧光强度以 cAMP 剂量依赖性方式增加，动态范围超过 3 倍。Green Falcans 对其结构类似物显示出对 cAMP 的高度特异性。当我们在 HeLa 细胞中表达 Green Falcans 时，与之前开发的 cAMP 指标相比，这些指标适用于低浓度范围内 cAMP 动力学的可视化，并且在活细胞中以高时空分辨率可视化 cAMP 在各种途径中的不同动力学。此外，我们证明了 Green Falcans 适用于 R-GECO 的双色成像，R-GECO 是一种红色荧光 Ca ²⁺指示剂，存在于细胞质和细胞核中。这项研究表明，Green Falcans 开辟了一条新途径，通过多色成像了解与各种 cAMP 信号通路中其他分子的分层和协作相互作用。

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Development of green fluorescent protein-based cAMP indicators for covering a wide range of cAMP concentrations

There is a wide range in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate (cAMP), which mediates specific effects as a second messenger in pathways affecting many physiological processes. Here, we developed green fluorescent cAMP indicators, named Green Falcan (Green fluorescent protein-based indicator visualizing cAMP dynamics) with various EC₅₀ values (0.3, 1, 3, 10 μM) for covering the wide range of intracellular cAMP concentrations. The fluorescence intensity of Green Falcans increased in a cAMP dose-dependent manner, with a dynamic range of over 3-fold. Green Falcans showed a high specificity for cAMP over its structural analogues. When we expressed Green Falcans in HeLa cells, these indicators were applicable for visualization of cAMP dynamics in the low concentration range compared to the previously developed cAMP indicators, and visualized distinct kinetics of cAMP in various pathways with high spatiotemporal resolution in living cells. Furthermore, we demonstrated that Green Falcans are applicable to dual-color imaging with R-GECO, a red fluorescent Ca²⁺ indicator, in the cytoplasm and the nucleus. This study shows that Green Falcans open up a new avenue for understanding hierarchal and cooperative interactions with other molecules in various cAMP signaling pathways by multi-color imaging.

更新日期：2023-05-22

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