A novel biosensor to study cAMP dynamics in cilia and flagella
Shatanik Mukherjee,
Vera Jansen,
Jan F Jikeli,
Hussein Hamzeh,
Luis Alvarez,
Marco Dombrowski,
Melanie Balbach,
Timo Strünker,
Reinhard Seifert,
U Benjamin Kaupp,
Dagmar Wachten
Affiliations
Shatanik Mukherjee
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
Vera Jansen
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
Jan F Jikeli
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
Hussein Hamzeh
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
Luis Alvarez
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
Marco Dombrowski
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
Melanie Balbach
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
Timo Strünker
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany; Centrum für Reproduktionsmedizin und Andrologie, Universitätsklinikum Münster, Münster, Germany
Reinhard Seifert
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
U Benjamin Kaupp
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
Dagmar Wachten
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
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 Ca2+, basal SACY activity is suppressed by Ca2+. 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.
