title: Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy.
abstract: We have developed an immunocytochemistry method for the semiquantitative detection of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the cell plasma membrane. This protocol combines the use of a glutathione S-transferase-tagged pleckstrin homology (PH) domain of the general phosphoinositides-1 receptor (GST-GRP1PH) with fluorescence confocal microscopy and image segmentation using cell mask software analysis. This methodology allows the analysis of PI(3,4,5)P3 subcellular distribution in resting and epidermal growth factor (EGF)-stimulated HEK293T cells and in LIM1215 (wild-type phosphoinositide 3-kinase (PI3K)) and LIM2550 (H1047R mutation in PI3K catalytic domain) colonic carcinoma cells. Formation of PI(3,4,5)P3 was observed 5min following EGF stimulation and resulted in an increase of the membrane/cytoplasm fluorescence ratio from 1.03 to 1.53 for HEK293T cells and from 2.2 to 3.3 for LIM1215 cells. Resting LIM2550 cells stained with GST-GRP1PH had an elevated membrane/cytoplasm fluorescence ratio of 9.8, suggesting constitutive PI3K activation. The increase in the membrane/cytoplasm fluorescent ratio was inhibited in a concentration-dependent manner by the PI3K inhibitor LY294002. This cellular confocal imaging assay can be used to directly assess the effects of PI3K mutations in cancer cell lines and to determine the potential specificity and effectiveness of PI3K inhibitors in cancer cells.
authors.affiliation: Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victsoria, Australia.
authors.forename: Michelle
authors.initials: M
authors.lastname: Palmieri
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Nowell
authors.forename: Melanie
authors.initials: M
authors.lastname: Condron
authors.forename: James
authors.initials: J
authors.lastname: Gardiner
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Holmes
authors.forename: Jayesh
authors.initials: J
authors.lastname: Desai
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: Bruno
authors.initials: B
authors.lastname: Catimel
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism
chemical_list: D002867:Chromones; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one