Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signaling in single neurons.
J Neurochem. 2008 Jul 17;
Authors: Nelson CP, Nahorski SR, John Challiss RA
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) fulfils vital signaling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP(2) levels within living cells. GFP-labeled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Galpha(q/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 (IP(3)) and instead reports dynamic changes in levels of plasma membrane PIP(2). In contrast, eGFP-tagged pleckstrin homology domain of PLCdelta1 (eGFP-PH) translocation reports increases in cytosolic IP(3). Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C1(2) domain of PKCgamma (eGFP-C1(2); to detect diacylglycerol) allowed a selective and comprehensive analysis of phospholipase C (PLC)-initiated signaling in living cells. Manipulating intracellular Ca(2+) concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca(2+) up to 100-200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca(2+) concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP(2) in hippocampal neurons.
PMID: 18665913 [PubMed - as supplied by publisher]
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