Interrogating cyclic AMP signaling using optical approaches.
Interrogating cyclic AMP signaling using optical approaches.
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DOI：
10.1016/j.ceca.2017.02.010
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发表时间：
2017-06
期刊：
影响因子：
4
通讯作者：
Hofer AM
中科院分区：
文献类型：
--
作者：
Jiang JY;Falcone JL;Curci S;Hofer AM
cAMP的光学报告代表了我们研究cAMP信号动力学能力的根本进步。这些荧光传感器可以测量单个细胞或细胞内微区中cAMP的变化，而不是测量cAMP的其他方法所需的整个细胞群。第一个光学cAMP报告基因是由Roger Tsien在20世纪90年代早期引入的基于FRET的传感器，该传感器利用PKA的纯化的调节和催化亚基的解离。这些传感器的实用性通过创建可以通过转染引入细胞的遗传编码版本而得到了极大的改善，其中第一个在2000年发表。随后，使用不同的cAMP结合平台、优化的荧光蛋白和定位于特定微区的靶向基序开发了改进的传感器。当今使用的最常见的传感器是围绕Epac主干设计的基于FRET的传感器。这些依赖于当Epac结合cAMP时其显著的构象变化，改变了Epac侧翼的FRET对之间的信号。已经开发了用于光学询问cAMP的几种其他策略，包括荧光易位报告子、基于二聚化依赖性FP的生物传感器、基于BRET（生物发光共振能量转移）的传感器、非FRET单波长报告子和基于细菌cAMP结合结构域的传感器。其他新描述的哺乳动物cAMP结合蛋白，如Popdc和CRIS，有一天可能会被用于传感器设计。随着工程化荧光蛋白的增殖和自然界中cAMP结合靶点的丰富，cAMP的光学报告物领域在未来几年应该继续看到快速的改进。
Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.