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Guanylate Cyclase and Cyclic GMP pp 89–120Cite as

Visualization of cGMP with cGi Biosensors

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1020))

Abstract

Cyclic guanosine 3′–5′-monophosphate (cGMP) is an important signaling molecule in physiology, pathophysiology, and pharmacological therapy. It has been proposed that the functional outcome of an increase of cGMP in a given cell largely depends on the existence of global versus local cGMP pools. The recent development of genetically encoded fluorescent biosensors for cGMP is a major technical advance in order to monitor the spatiotemporal dynamics and compartmentalization of cGMP signals in living cells. Here we give an overview of the available cGMP sensors and how they can be used to visualize cGMP. The focus is on the fluorescence resonance energy transfer (FRET)-based cGi-type sensors (Russwurm et al., Biochem J 407:69–77, 2007), which are currently among the most useful tools for cGMP imaging in cells, tissues, and living organisms. We present detailed protocols that cover the entire imaging experiment, from the isolation of primary cells from cGi-transgenic mice and adenoviral expression of cGi sensors to the description of the setup required to record FRET changes in single cells and tissues. In-cell calibration of sensors and data evaluation is also described in detail and the limitations and common pitfalls of cGMP imaging are discussed. Specifically, we outline the use of FRET microscopy to visualize cGMP in murine smooth muscle cells (from aorta, bladder, and colon) and cerebellar granule neurons expressing cGi sensors. Most of the protocols can be easily adapted to other cell types and cGMP indicators and can be used as general guidelines for cGMP imaging in living cells, tissues and, eventually, whole organisms.

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Acknowledgments

We thank Barbara Birk, Caroline Vollmers, Erika Mannheim, Ursula Krabbe, and Fred Eichhorst for expert technical assistance, Simone Di Giovanni for advice with neuronal cell culture, Gisela Drews and Peter Krippeit-Drews for providing the superfusion chamber, and Kübra Gülmez, Phillip Messer, Annyesha Mohanty, and Christine Wenz for their contributions to cGMP imaging experiments. Special thanks go to Susanne Feil and Lai Wen for reading the manuscript, to Lai Wen for providing transgenic mice for the calibration experiment, to Thomas Ott for microinjection of transgenes into mouse oocytes, and to Lutz Pott, Anke Gallhoff, and Kirsten Bender for introduction to the adenoviral system. We also thank all past and present members of our laboratories for critical discussions and the Deutsche Forschungsgemeinschaft for financial support.

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Authors and Affiliations

  1. Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany

    Martin Thunemann, Natalie Fomin & Robert Feil

  2. Graduate School of Cellular and Molecular Neuroscience, Universität Tübingen, Tübingen, Germany

    Natalie Fomin

  3. Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, Bochum, Germany

    Christian Krawutschke & Michael Russwurm

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  1. Martin Thunemann
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  2. Natalie Fomin
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  3. Christian Krawutschke
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  4. Michael Russwurm
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  5. Robert Feil
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Editors and Affiliations

  1. Addenbrooke’s Hospital, Department of Medicine, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, United Kingdom

    Thomas Krieg

  2. Toxicology and Clinical Pharmacy, Department of Pharmacology, Universität Tübingen, Auf der Morgenstelle 8, Tübingen, 72076, Germany

    Robert Lukowski

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Thunemann, M., Fomin, N., Krawutschke, C., Russwurm, M., Feil, R. (2013). Visualization of cGMP with cGi Biosensors. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_6

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  • DOI: https://doi.org/10.1007/978-1-62703-459-3_6

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