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Elife. 2016 Mar 22;5. pii: e14052. doi: 10.7554/eLife.14052.

A novel biosensor to study cAMP dynamics in cilia and flagella.

Author information

1
Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany.
2
Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany.
3
Centrum für Reproduktionsmedizin und Andrologie, Universitätsklinikum Münster, Münster, Germany.

Abstract

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.

KEYWORDS:

FRET; cAMP; cell biology; ciliary; flagella; mouse; sperm

PMID:
27003291
PMCID:
PMC4811770
DOI:
10.7554/eLife.14052
[Indexed for MEDLINE]
Free PMC Article

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