title: Simultaneous real-time imaging of signal oscillations using multiple fluorescence-based reporters.
abstract: It is now well understood that G protein-coupled receptor (GPCR)-mediated cell signalling is subject to extensive spatial-temporal control, and that a meaningful understanding of this complexity requires techniques to study signalling at the molecular and sub-cellular level. This complexity in cell signal pattern begins with ligand binding to the receptor and its coupling to a variety of different effector systems. These signal transduction cascades within a cell involve a very complex series of molecular events requiring the generation of multiple second messenger responses and the activation a multiple effector proteins. In the present chapter, we will describe methodology for the simultaneous assessment of the spatial-temporal measurement of increases in intracellular Ca2+ concentrations and the activation of protein kinase C (PKC) in response to the agonist activation of a Gαq/11-coupled GPCR. Specifically, we will describe a confocal imaging approach to simultaneously measure oscillilations in intracellular Ca2+ levels and PKC translocation to the plasma membrane in response to mGluR1 stimulation in transiently transfected human embryonic kidney (HEK293) cells. The changes in intracellular Ca2+ were imaged using the fluorescent indicator Oregon Green 488 BAPTA and a recombinant PKCβII-DsRed fusion protein was used to image the sub-cellular distribution of the PKCβII isoform.
authors.affiliation: The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, Department of Physiology & PharmacologyThe University of Western Ontario, London, ON, Canada.
authors.forename: Lianne B
authors.initials: LB
authors.lastname: Dale
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
mesh_terms: D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D020013:Calcium Signaling; D002264:Carboxylic Acids / Q000032:analysis; D002460:Cell Line; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods*; D011493:Protein Kinase C / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D002264:Carboxylic Acids; D008164:Luminescent Proteins; C436864:Oregon Green 488 carboxylic acid; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; C104077:metabotropic glutamate receptor type 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002118:Calcium