Mukherjee Shatanik, Jansen Vera, Jikeli Jan F, Hamzeh Hussein, Alvarez Luis, Dombrowski Marco, Balbach Melanie, Strünker Timo, Seifert Reinhard, Kaupp U Benjamin, Wachten Dagmar
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany.
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany.
Elife. 2016 Mar 22;5:e14052. doi: 10.7554/eLife.14052.
The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca(2+), basal SACY activity is suppressed by Ca(2+). Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo.
细胞信使环磷酸腺苷（cAMP）调节多种细胞功能，包括在纤毛和鞭毛中的信号传导。这些亚细胞区室中的cAMP动态变化尚不明确。我们引入了一种基于荧光共振能量转移（FRET）的新型cAMP生物传感器，其具有纳摩尔级的灵敏度，这是其他传感器无法达到的。为了测量精子鞭毛中的cAMP动态变化，我们培育了转基因小鼠，并发现迄今为止测定总cAMP水平的方法并不能反映游离cAMP水平的变化。此外，鞭毛中段和主段中的cAMP动态变化明显不同。鞭毛中唯一的cAMP来源是可溶性腺苷酸环化酶（SACY）。尽管依赖碳酸氢盐的SACY活性需要Ca(2+)，但基础SACY活性受到Ca(2+)的抑制。最后，我们还将该传感器应用于初级纤毛。我们的新型cAMP生物传感器具有独特的特性，能够让我们对cAMP信号传导有新的认识，并揭示体外和体内纤毛功能背后的分子机制。
