Résumé
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.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.