Department of Physiology, Hamamatsu University School of Medicine, Handa-yama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka, 431-3192, Japan.
Pflugers Arch. 2009 Nov;459(1):227-34. doi: 10.1007/s00424-009-0705-8. Epub 2009 Aug 13.
Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.
生理现象受受体刺激后多个信号通路调节。在这里，我们结合棱镜全内反射荧光显微镜（PBTIRFM）和明场荧光显微镜（EPI），引入了一种新技术，同时监测多个信号通路。该仪器使我们能够在活细胞中可视化三种信号通路：Ca2+、环腺苷单磷酸（cAMP）/蛋白激酶 A（PKA）和二酰基甘油（DAG）/蛋白激酶 C（PKC）信号。为此目的，使用了三种荧光指示剂：（1）Fura-2 AM 作为钙传感器；（2）Epac1-camp，基于青色荧光蛋白-黄色荧光蛋白荧光共振能量转移的 cAMP 指示剂，作为 cAMP 传感器；（3）C1 标记单体红色荧光蛋白，PKCγ的串联二酰基甘油结合域，作为 DAG 传感器或PKC 激活途径的豆蔻酰化丙氨酸丰富 C 激酶底物标记的 DsRed。DAG 信号通过 PBTIRFM 监测，而 Ca2+和 cAMP 信号通过 EPI 监测。三磷酸腺苷导致三重探针加载的 Cos-7 细胞中产生所有三种第二信使。通过线性解混，评估了这些信号探针之间的光谱重叠。forskolin 也引起 Ca2+、cAMP/PKA 和 DAG/PKC 信号，而乙酰胆碱激活 Ca2+和 DAG/PKC 信号，以及抑制三重探针加载的胰岛素分泌细胞中 cAMP 的产生。因此，结合 PBTIRFM 和 EPI 的光学观察系统为分析活细胞中 G 蛋白偶联受体刺激后这些第二信使等多个信号通路的相互作用提供了很大的进展。
