Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1

Stephen A. Watt, Gursant Kular, Ian N. Fleming, C. Peter Downes, John M. Lucocq

    Research output: Contribution to journalArticle

    Abstract

    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 d1 (PLCd1), 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 PLCd1 PH domain (PLCd1PH–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
    Original languageEnglish
    Pages (from-to)657-666
    Number of pages10
    JournalBiochemical Journal
    Volume363
    Issue number3
    Publication statusPublished - Jan 2002

    Fingerprint

    Phospholipase C delta
    Phosphatidylinositol 4,5-Diphosphate
    Phosphatidylinositols
    Labeling
    Cell membranes
    thrombin receptor peptide SFLLRNP
    Cell Membrane
    Membranes
    Lipids
    Caveolae
    Intracellular Membranes
    Pseudopodia
    Endosomes
    Astrocytoma
    Glutathione Transferase
    Endoplasmic Reticulum
    Transfection
    Pleckstrin Homology Domains
    platelet protein P47
    Electrons

    Keywords

    • Immunoelectron microscopy
    • Immunogold
    • Lipid domains
    • Localization
    • Phosphoinositide

    Cite this

    Watt, S. A., Kular, G., Fleming, I. N., Downes, C. P., & Lucocq, J. M. (2002). Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochemical Journal, 363(3), 657-666.
    Watt, Stephen A. ; Kular, Gursant ; Fleming, Ian N. ; Downes, C. Peter ; Lucocq, John M. / Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. In: Biochemical Journal. 2002 ; Vol. 363, No. 3. pp. 657-666.
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    Watt, SA, Kular, G, Fleming, IN, Downes, CP & Lucocq, JM 2002, 'Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1', Biochemical Journal, vol. 363, no. 3, pp. 657-666.

    Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. / Watt, Stephen A.; Kular, Gursant; Fleming, Ian N.; Downes, C. Peter; Lucocq, John M.

    In: Biochemical Journal, Vol. 363, No. 3, 01.2002, p. 657-666.

    Research output: Contribution to journalArticle

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    AU - Watt, Stephen A.

    AU - Kular, Gursant

    AU - Fleming, Ian N.

    AU - Downes, C. Peter

    AU - Lucocq, John M.

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    AB - 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 d1 (PLCd1), 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 PLCd1 PH domain (PLCd1PH–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

    KW - Immunoelectron microscopy

    KW - Immunogold

    KW - Lipid domains

    KW - Localization

    KW - Phosphoinositide

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    SP - 657

    EP - 666

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