Development of a red fluorescent protein-based cGMP indicator applicable for live-cell imaging

Area of Science:

• Molecular Biology
• Cellular Physiology
• Biotechnology

Background:

• Cyclic guanosine 3", 5 omino-monophosphate (cGMP) acts as a crucial second messenger in diverse physiological pathways.
• Existing indicators for cGMP may have limitations in sensitivity, spectral properties, or compatibility with advanced imaging techniques.

Purpose of the Study:

• To develop and characterize a novel red fluorescent protein-based indicator for sensitive and specific detection of intracellular cGMP.
• To evaluate the utility of the new indicator in live-cell imaging, including dual-color imaging and optogenetics applications.
• To investigate the role of cGMP in enteroendocrine cells and its interplay with calcium and L-arginine stimulation.

Main Methods:

• Development of a red fluorescent protein-based indicator, termed Red cGull, engineered for cGMP binding.
• Characterization of Red cGull's fluorescence properties, including dynamic range, EC50, and excitation/emission spectra.
• Live-cell imaging experiments using Red cGull in various cell types, including enteroendocrine cell lines, to monitor cGMP dynamics.
• Assessment of Red cGull's compatibility with optogenetics tools and its performance in dual-color imaging setups.

Main Results:

• Red cGull exhibits a significant sixfold increase in fluorescence intensity in response to cGMP, with an EC50 of 0.33 μM.
• The indicator has excitation and emission peaks at 567 nm and 591 nm, respectively, suitable for red fluorescence imaging.
• Live-cell imaging demonstrated Red cGull's utility for dual-color imaging and co-application with optogenetics.
• Red cGull successfully detected increased cGMP levels in enteroendocrine cells upon L-arginine stimulation, revealing inhibition by Ca2+.

Conclusions:

• Red cGull is a highly sensitive and photostable red fluorescent indicator for monitoring cGMP dynamics in live cells.
• Its spectral properties and compatibility with advanced imaging techniques make it a valuable tool for cell signaling research.
• The indicator facilitates the study of cGMP's role in physiological processes and its crosstalk with other signaling pathways, such as calcium signaling.