Article

Simultaneous real-time imaging of signal oscillations using multiple fluorescence-based reporters.

The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, Department of Physiology & PharmacologyThe University of Western Ontario, London, ON, Canada.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 756:273-81. DOI: 10.1007/978-1-61779-160-4_15
Source: PubMed

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.

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