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

Methods Mol Biol. 2011:756:273-81. doi: 10.1007/978-1-61779-160-4_15.

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.

MeSH terms

  • Calcium / analysis*
  • Calcium / metabolism*
  • Calcium Signaling*
  • Carboxylic Acids / analysis
  • Cell Line
  • Humans
  • Luminescent Proteins / analysis
  • Luminescent Proteins / genetics
  • Microscopy, Confocal / methods*
  • Protein Kinase C / analysis*
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Kinase C beta
  • Protein Transport
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, Metabotropic Glutamate / metabolism
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • Carboxylic Acids
  • Luminescent Proteins
  • Oregon Green 488 carboxylic acid
  • Receptors, G-Protein-Coupled
  • Receptors, Metabotropic Glutamate
  • Recombinant Fusion Proteins
  • fluorescent protein 583
  • metabotropic glutamate receptor type 1
  • Protein Kinase C
  • Protein Kinase C beta
  • Calcium