Fluorescence ratio imaging of cyclic AMP in single cells

Abstract

FLUORESCENCEimaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells¹. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca²⁺, H⁺, Na⁺, K⁺, Mg²⁺and Cl^-. We now report a fluorescent indicator for the adenosine 3′ 5′cyclic monophos-phate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase² in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R₂C₂. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.