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.
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) 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 PIP(2) 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 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 and instead reports dynamic changes in levels of plasma membrane PIP(2). In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCdelta1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C1(2) domain of protein kinase C-gamma [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling 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.
Nelson
Carl P
CP
Department of Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.
Nahorski
Stefan R
SR
Challiss
R A John
RA
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D004075:Diglycerides; D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D011506:Proteins; C508878:TUB protein, human; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium