Journal
EXPERIMENTAL CELL RESEARCH
Volume 285, Issue 1, Pages 27-38Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0014-4827(02)00048-4
Keywords
colocalisation; cholera toxin; diacylglycerol; GM1; phosphomositides; rafts; Ras
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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 GMI 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. (C) 2003 Elsevier Science (USA). All rights reserved.
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