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

272 Citations (Scopus)

Abstract

Ptd(4,5)P(2) 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)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) 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)P(3)], 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)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.
Original languageEnglish
Pages (from-to)657-66
Number of pages10
JournalBiochemical Journal
Volume363
Issue numberPt 3
Publication statusPublished - 2002

Fingerprint

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

Keywords

  • Blood Proteins
  • Cell Line
  • Cell Membrane
  • Humans
  • Intracellular Membranes
  • Isoenzymes
  • Lipid Metabolism
  • Microscopy, Immunoelectron
  • Peptide Fragments
  • Phosphatidylinositol 4,5-Diphosphate
  • Phospholipase C delta
  • Phosphoproteins
  • Sequence Homology, Amino Acid
  • Tumor Cells, Cultured
  • Type C Phospholipases

Cite this

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. Pt 3, 2002, p. 657-66.

Research output: Contribution to journalArticle

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. Pt 3. pp. 657-66.
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T1 - Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1

AU - Watt, Stephen A

AU - Kular, Gursant

AU - Fleming, Ian N

AU - Downes, C Peter

AU - Lucocq, John M

PY - 2002

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N2 - Ptd(4,5)P(2) 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)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) 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)P(3)], 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)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.

AB - Ptd(4,5)P(2) 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)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) 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)P(3)], 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)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.

KW - Blood Proteins

KW - Cell Line

KW - Cell Membrane

KW - Humans

KW - Intracellular Membranes

KW - Isoenzymes

KW - Lipid Metabolism

KW - Microscopy, Immunoelectron

KW - Peptide Fragments

KW - Phosphatidylinositol 4,5-Diphosphate

KW - Phospholipase C delta

KW - Phosphoproteins

KW - Sequence Homology, Amino Acid

KW - Tumor Cells, Cultured

KW - Type C Phospholipases

M3 - Article

VL - 363

SP - 657

EP - 666

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - Pt 3

ER -