Location of Repository

Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons

By Carl P. Nelson, Stefan R. Nahorski and R.A. John Challiss

Abstract

This paper was published as Journal of Neurochemistry, 2008, 107 (3), pp. 602 - 615. It is available from www.blackwell-synergy.com. Doi: 10.1111/j.1471-4159.2008.05587.xMetadata only entryPhosphatidylinositol 4,5-bisphosphate (PIP2) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP2 levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons

Publisher: Wiley-Blackwell on behalf of the International Society for Neurochemistry.
Year: 2008
DOI identifier: 10.1111/j.1471-4159.2008.05587.x
OAI identifier: oai:lra.le.ac.uk:2381/8084
Journal:
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://hdl.handle.net/2381/808... (external link)
  • http://dx.doi.org/10.1111/j.14... (external link)
  • www.blackwell-synergy.com. (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.