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 Gα_q/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.