Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.
Parmryd
Ingela
I
Division of Membrane Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK. iparmryd@imperial.ac.ukj
Adler
Jeremy
J
Patel
Roopal
R
Magee
Anthony I
AI
D001665:Binding Sites; D004075:Diglycerides / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013223:Statistics as Topic; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D004075:Diglycerides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D005677:G(M1) Ganglioside; D018631:ras Proteins