Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer

Agonist-induced intracellular Ca2+ signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, a nd oscillatory behavior. These Ca2+ changes have been well characterized, b ut detailed kinetic analyses of PLC activation in single living cells is la cking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been emp loyed to monitor PLC activation in single cells, based on (confocal) imagin g of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate, Here we descr ibe fluorescence resonance energy transfer between pleckstrin homology doma ins of PLC delta1 tagged with cyan and yellow fluorescent proteins as a sen sitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy tra nsfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal expe riments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites, C haracterization of responses to various agonists by this method reveals tha t stimuli that elicit very similar Ca2+ mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca2+ transie nt.