Design of In Vivo Indicators for Cyclic GMP

University Of Vermont & State Agricultural College, Burlington VT

Investigators

Abstract

The long-term goal of this project is to understand the spatial and temporal dynamics of the second messenger molecule, cGMP, in live cells. This project focuses on the design of novel in vivo cGMP indicators created by fusing wavelength mutations of green fluorescence protein (GFP) from the jellyfish, Aequorea victoria, to cGMP-binding fragments of a cGMP-dependent protein kinase. Studies will take advantage of changes in fluorescence resonance energy transfer (FRET) between cyan and yellow GFP mutants (CFP and YFP) which are induced by conformational changes upon cGMP binding to the receptor portion of the hybrid-protein. Preliminary results have demonstrated that FRET changes are directly proportional to the concentration of intracellular cGMP and can be used to probe the dynamic behavior of cGMP by non-invasive means. Additional experiments will examine aspects of hybrid protein design that contribute to optimal cGMP-dependent FRET ratio changes. Suitable fluorescent probes will then be tested by employing confocal fluorescence imaging techniques to examine the spatial and temporal changes in intracellular cGMP levels in living cells in response to physiological stimuli. A collaboration will be undertaken to understand the role of cGMP in the regulation of vascular smooth muscle tone by nitric oxide (NO), which decreases tone by direct stimulation of cGMP production. It is thought that cGMP causes relaxation through activation of cGMP-dependent kinase I, which is known to lower cytosolic calcium concentrations in these cells. Studies will correlate changes in intracellular cGMP with various downstream physiological responses, i.e. local and global changes in calcium concentration, and cell relaxation. The in vivo cGMP-indicators which will be developed are the first of their kind and should provide the means to gain essential insights into various other physiological processes involving cGMP-mediated signaling in other cell systems. The project also includes the purchase of accessories for a confocal scanning laser microscope which will be shared by several investigators with NSF funding at the University of Vermont.