Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy
Section snippets
Reagents
The following materials were obtained from the sources indicated: RPMI, Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, and l-glutamine (Gibco/Invitrogen, Carlsbad, CA, USA); glutathione agarose beads, transferrin, albumin, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (Hepes), sodium chloride, tris(hydroxymethyl)aminomethane, and DPX mountant for histology and formaldehyde (Sigma–Aldrich, St. Louis, MO, USA);
Characterization of GST–GRP1PH
GST–GRP1PH was characterized using SDS–PAGE with sensitive Coomassie stain followed by automated in-gel digestion and LC–MS/MS analysis (results not shown) [6], [61]. An 85% peptide coverage of the PH domain was obtained using MS/MS analysis (results not shown).
GST–GRP1PH binding specificity toward PI(3,4,5)P3 was analyzed using both an overlay PIP assay and biosensor analysis (Fig. 2). GST–GRP1PH was found to recognize specifically immobilized PI(3,4,5)P3 when compared with immobilized
Discussion
In this study, we used a combination of GST–GRP1PH, fluorescence confocal microscopy, and image segmentation with cell mask software analysis to develop an immunocytochemistry method for semiquantitative detection of PI(3,4,5)P3 at the cell plasma membrane. GRP1, a guanine nucleotide exchange factor for ARF GTPases, has been commonly used in imaging studies due to the high specificity of its PH domain toward PI(3,4,5)P3 and due to its ability to dock rapidly to PI(3,4,5)P3 at the inner leaflet
Conclusion
Using the PI(3,4,5) reporter protein construct GST–GRP1PH, we have developed a quantitative detection assay of PI(3,4,5)P3 formation at the plasma membranes, cytoplasms, and nuclei of cells on PI3K activation. By measuring the membrane/cytoplasm fluorescence intensity ratio in resting and PI3K activated cells, the fluorescent image segmentation method allowed large-scale analysis of PI(3,4,5)P3 formation without the need to transfect the reporter protein. This assay can be used to directly
Acknowledgments
This work was supported by the Australian Research Council, Discovery Project (Grants DP0770668 and DP1094497), and the National Health and Medical Research Council (NHMRC) Program (Grant 487922). General support was provided by the Commonwealth Scientific and Industrial. Research Organisation (CSIRO) and the Victorian Endowment for Science, Knowledge, and Innovation (VESKI).
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