Abstract
In order to observe the spatial and temporal pattern of intracellular signalling in intact cells, we have developed a variety of sensors that are based on fluorescence resonance energy transfer (FRET) between CFP and YFP, or similar fluorophores, fused to various signaling proteins [1]. For the detection of the second messengers we have developed similar sensors based on cAMP or cGMP binding domains derived from various proteins that were likewise fused to CFP and YFP or analogous fluorescent proteins. These sensors respond to changes in intracellular second messenger concentrations with changes in FRET. For cGMP, various sensors were developed based on fragments of cGMP-dependent protein kinase or regulatory GAF-domains, which differed in amplitude, speed of reaction and in sensitivity to cGMP [2]. When expressed in primary cells or cell culture lines, these sensors allowed spatially and temporally resolved imaging of cGMP concentrations in intact cells. More rapid variants of these sensors also permit the monitoring of oscillating changes in cGMP levels, generated, for example, by pulsatile stimulation of cGMP synthesis by sodium nitroprusside.
Highlights
3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Meeting abstracts – A single PDF containing all abstracts in this Supplement is available here.
In order to observe the spatial and temporal pattern of intracellular signalling in intact cells, we have developed a variety of sensors that are based on fluorescence resonance energy transfer (FRET) between CFP and YFP, or similar fluorophores, fused to various signaling proteins [1]
For the detection of the second messengers we have developed similar sensors based on cAMP or cGMP binding domains derived from various proteins that were likewise fused to CFP and YFP or analogous fluorescent proteins
Summary
3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Meeting abstracts – A single PDF containing all abstracts in this Supplement is available here. . Rapid monitoring of intracellular cGMP Viacheslav O Nikolaev and Martin J Lohse* Address: Institute of Pharmacology and Toxicology, University of Würzburg, Germany Email: Martin J Lohse* - lohse@toxi.uni-wuerzburg.de * Corresponding author from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany.
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