Ptd(4,5)P2 is thought to promote and organize a wide range of cellular functions, including vesicular membrane traffic and cytoskeletal dynamics, by recruiting functional protein complexes to restricted locations in cellular membranes. However, little is known about the distribution of PtdIns(4,5)P2 in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase δ1 (PLCδ1), narrowly specific for PtdIns(4,5)P2, to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCδ1 PH domain (PLCδ1PH—GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P2 labelling in the plasma membrane, and also in intracellular membranes, including Golgi (mainly stack), endosomes and endoplasmic reticulum, as well as in electron-dense structures within the nucleus. At the plasma membrane, labelling was more concentrated over lamellipodia, but not in caveolae, which contained less than 10% of the total cell-surface labelling. A dramatic decrease in signal over labelled compartments was observed on preincubation with the cognate headgroup [Ins(1,4,5)P3], and plasma-membrane labelling was substantially decreased after stimulation with thrombin-receptor-activating peptide (SFLLRN in the one-letter amino acid code), a treatment which markedly diminishes PtdIns(4,5)P2 levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P2 distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.
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Research Article|
April 24 2002
Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C δ1
Stephen A. WATT;
Stephen A. WATT
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Gursant KULAR;
Gursant KULAR
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Ian N. FLEMING;
Ian N. FLEMING
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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C. Peter DOWNES;
C. Peter DOWNES
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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John M. LUCOCQ
John M. LUCOCQ
1
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
1To whom correspondence should be addressed (e-mail j.m.lucocq@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
October 03 2001
Revision Received:
January 24 2002
Accepted:
February 28 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 363 (3): 657–666.
Article history
Received:
October 03 2001
Revision Received:
January 24 2002
Accepted:
February 28 2002
Citation
Stephen A. WATT, Gursant KULAR, Ian N. FLEMING, C. Peter DOWNES, John M. LUCOCQ; Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C δ1. Biochem J 1 May 2002; 363 (3): 657–666. doi: https://doi.org/10.1042/bj3630657
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