Blue fluorescent cGMP sensor for multiparameter fluorescence imaging.
Blue fluorescent cGMP sensor for multiparameter fluorescence imaging.
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DOI：
10.1371/journal.pone.0009164
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发表时间：
2010-02-11
期刊：
影响因子：
3.7
通讯作者：
Oka K
中科院分区：
文献类型：
--
作者：
Niino Y;Hotta K;Oka K
环 GMP (cGMP) 通过与环 AMP (cAMP) 和 Ca2+ 等其他信号分子合作调节许多生理过程。 cGMP 基因编码传感器是基于荧光蛋白之间的荧光共振能量转移 (FRET) 开发的。然而，为了分析这些第二信使之间的动态关系，由于显着的光谱重叠，在单个细胞中组合使用现有传感器是不够的。单波长指示剂是避免此问题的有效替代方案，但基于单一荧光蛋白的生物传感器的颜色变化是有限的。在本研究中，为了构建新型彩色荧光传感器，我们使用暗 FRET 受体将基于 FRET 的传感器转换为单波长指示器。我们开发了一种蓝色荧光 cGMP 生物传感器，它与使用青色和黄色荧光蛋白 (CFP/YFP) 的基于 FRET 的 cAMP 传感器在光谱上兼容。我们将它们共转染，并将 Ca2+ 的红色荧光探针加载到细胞中，并完成了这些环核苷酸和 Ca2+ 的三参数荧光成像，证实了这种组合在单个细胞中单独监测它们动态的适用性。这种蓝色荧光传感器和使用该 FRET 对的方法将有助于多参数荧光成像，以了解复杂的信号转导网络。
Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.
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