Measuring dynamic changes in cAMP using fluorescence resonance energy transfer.
Measuring dynamic changes in cAMP using fluorescence resonance energy transfer.
复制标题
使用荧光共振能量转移测量 cAMP 的动态变化。
DOI：
10.1385/1-59259-816-1:259
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发表时间：
2004
影响因子：
--
通讯作者：
M. Zaccolo
中科院分区：
文献类型：
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作者：
Sandrine Evellin;M. Mongillo;Anna Terrin;V. Lissandron;M. Zaccolo
cAMP是一种普遍存在的第二信使，其控制许多细胞事件，包括运动、生长、代谢、收缩和突触可塑性。随着cAMP依赖性信号传导的区室化概念的出现，需要对cAMP的时空细胞内动力学进行详细研究。在这里，我们描述了一种新的方法来监测波动的cAMP在活细胞中，基于使用的遗传编码的生物传感器。与两个合适的绿色荧光蛋白（GFP）突变体融合的主要cAMP效应子蛋白激酶A（PKA）的调节和催化亚基用于测量与细胞内cAMP水平变化相关的荧光共振能量转移（FRET）的变化。该方法允许在时间和空间上以高分辨率研究活细胞中的cAMP波动。
cAMP is a ubiquitous second messenger that controls numerous cellular events including movement, growth, metabolism, contraction, and synaptic plasticity. With the emerging concept of compartmentalization of cAMP-dependent signaling, a detailed study of the spatio-temporal intracellular dynamics of cAMP is required. Here we describe a new methodology for monitoring fluctuations of cAMP in living cells, based on the use of a genetically encoded biosensor. The regulatory and catalytic subunits of the main cAMP effector, the protein kinase A (PKA), fused with two suitable green fluorescent protein (GFP) mutants is used for measuring changes in fluorescence resonance energy transfer (FRET) that correlate with changes in intracellular cAMP levels. This method allows the study of cAMP fluctuations in living cells with high resolution both in time and in space.