Bacterial photoactivated adenylyl cyclase (bPAC) has been widely used in signal transduction research. However, due to its low two-photon absorption, bPAC cannot be efficiently activated by two-photon (2P) excitation. Taking advantage of the high two-photon absorption of monomeric teal fluorescent protein 1 (mTFP1), we herein developed 2P-activatable bPAC (2pabPAC), a fusion protein consisting of bPAC and mTFP1. In 2pabPAC, the energy absorbed by mTFP1 excites bPAC by Fürster resonance energy transfer (FRET) at ca. 43% efficiency. The light-induced increase in cAMP was monitored by a red-shifted FRET biosensor for PKA. In 3D MDCK cells and mouse liver, PKA was activated at single-cell resolution under a 2P microscope. We found that PKA activation in a single hepatocyte caused PKA activation in neighboring cells, indicating the propagation of PKA activation. Thus, 2pabPAC will provide a versatile platform for controlling the cAMP signaling pathway and investigating cell-to-cell communication in vivo.

中文翻译：

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FRET辅助bPAC的双光子激活在3D组织中对cAMP信号通路的单细胞激活

细菌光活化腺苷酸环化酶（bPAC）已被广泛用于信号转导研究。但是，由于其低的双光子吸收，不能通过双光子（2P）激发有效地激活bPAC。利用单体蓝绿色荧光蛋白1（mTFP1）的高两个光子吸收，我们在这里开发了2P可激活的bPAC（2pabPAC），这是一种由bPAC和mTFP1组成的融合蛋白。在2pabPAC，能量吸收由mTFP1的激励BPAC通过在Fürster共振能量转移（FRET）CA。效率43％。通过红移FRET生物传感器对PKA监测cAMP的光诱导增加。在3D MDCK细胞和小鼠肝脏中，PKA在2P显微镜下以单细胞分辨率激活。我们发现单个肝细胞中的PKA激活引起邻近细胞中的PKA激活，表明PKA激活的传播。因此，2pabPAC将为控制cAMP信号通路和研究体内细胞间通讯提供一个通用平台。