title: Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer.
abstract: Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: van der Wal
authors.forename: R
authors.initials: R
authors.lastname: Habets
authors.forename: P
authors.initials: P
authors.lastname: Várnai
authors.forename: T
authors.initials: T
authors.lastname: Balla
authors.forename: K
authors.initials: K
authors.lastname: Jalink
mesh_terms: D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D048429:Cell Size; D017931:DNA Primers; D004789:Enzyme Activation; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D021381:Protein Transport; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D002118:Calcium