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.
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ABSTRACT: We report the development of a method to analyze receptor and β-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged β2-adrenergic receptor (β2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged β-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, β-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to β-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting β-arrestin interaction found with β2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.Microscopy and Microanalysis 01/2013; · 2.50 Impact Factor