Advertisement

Simultaneous Real-Time Imaging of Signal Oscillations Using Multiple Fluorescence-Based Reporters

  • Lianne B. Dale
  • Stephen S. G. FergusonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

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.

Key words

Intracellular Ca2+ Protein kinase C Oscillation Fluorescence Laser scanning confocal microscopy 

References

  1. 1.
    Niswender, C.M. and Conn, P.J. (2010) Metabotropic Glutamate Receptors: Physiology, Pharmacology, and Disease. Annu Rev Pharmacol Toxicol 50, 295–322.PubMedCrossRefGoogle Scholar
  2. 2.
    Woehler, A. and Ponimaskin, E.G. (2009) G Protein-mediated Signaling: Same Receptor, Multiple Effectors. Curr Mol Pharmacol 2, 23748.PubMedCrossRefGoogle Scholar
  3. 3.
    Steinberg, S.F. (2008) Structural Basis of Protein Kinase C Isoform Function. Physiol Rev 88, 1341–1378.PubMedCrossRefGoogle Scholar
  4. 4.
    Newton, A.C. (2010) Protein Kinase C: poised to signal. Am J Physiol Endocrinol Metab 298, E395–E402.PubMedCrossRefGoogle Scholar
  5. 5.
    Policha, A., Daneshtalab, N., Chen, L., Dale, L.B., Altier, C., Khosravani, H., Thomas, W.G., Zamponi, G.W. and Ferguson, S.S. (2006) Role of angiotensin II type 1A receptor phosphorylation, phospholypase D, and extracellular calcium. J Biol Chem 281, 2634026349.PubMedCrossRefGoogle Scholar
  6. 6.
    Dale L.B., Babwah A.V., Bhattacharya M., Kelvin D.J. and Ferguson S.S. (2001) Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required. J Biol Chem 276, 35900–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Babwah, A.V., Dale L.B. and Ferguson S.S. (2003) Protein kinase C isoform-specific differences in the spatial-temporal regulation and decoding of Metabotropic glutamate receptor1a-stimulated second messenger responses. J Biol Chem 278, 5419–26.PubMedCrossRefGoogle Scholar
  8. 8.
    Collazos, A., Diouf, B., Guérineau, N.C., Quittau-Prévostel, C., Peter, M., Coudane, F., Hollande, F. and Joubert, D. (2006) A spatiotemporally coordinated cascade of protein kinase C activation controls isoform-selective translocation. Mol Cell Biol 26, 224761.PubMedCrossRefGoogle Scholar
  9. 9.
    Uhlén, P. and Fritz, N. (2010) Biochemistry of calcium oscillations. Biochem Biophys Res Commun 396, 2832.PubMedCrossRefGoogle Scholar
  10. 10.
    Paredes, R.M., Etzler, J.C., Watts, L.T., Zheng, W. and Lechleiter, J.D. (2008) Chemical calcium indicators Methods 46, 14351PubMedCrossRefGoogle Scholar
  11. 11.
    Wiedenmann, J., Oswald, F. and Nienhaus, G.U. (2009) Fluorescent proteins for live cell imaging: opportunities, limitations and challenges IUBMB Life 61, 102942.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.The J. Allyn Taylor Centre for Cell Biology, Robarts Research InstituteThe University of Western OntarioLondonCanada
  2. 2.Department of Physiology & PharmacologyThe University of Western OntarioLondonCanada

Personalised recommendations