Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity
Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity
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DOI：
10.1016/j.bbrc.2008.02.070
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发表时间：
2008-05-02
影响因子：
3.1
通讯作者：
Hayashi, Yasunori
中科院分区：
文献类型：
--
作者：
Kwok, Showining;Lee, Claudia;Hayashi, Yasunori
Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）在中枢神经系统的兴奋性突触中高度富集，并且与突触可塑性、学习和记忆密切相关。然而，由于缺乏特定的方法，活细胞中CaMKII活性的精确时间和空间调节尚未得到很好的描述。在这里，我们以前的工作的基础上，我们试图产生一个光学探针的荧光寿命成像（FLIM）的CaMKII活性融合的蛋白质与供体和受体荧光蛋白在其氨基和羧基末端。我们首先利用荧光蛋白在荧光光谱分析中向更长波长扩展的优势，优化了荧光蛋白的组合。然后使用数字频域FLIM（DFD-FLIM），我们证明了所得到的蛋白质确实可以检测活细胞中的CaMKII激活。这些FLIM版本的Camui可能有助于阐明CaMKII的体外和体内功能。(C)2008年爱思唯尔公司All rights reserved.
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo. (C) 2008 Elsevier Inc. All rights reserved.