Elsevier

Analytical Biochemistry

Volume 406, Issue 1, 1 November 2010, Pages 41-50
Analytical Biochemistry

Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy

https://doi.org/10.1016/j.ab.2010.06.033Get rights and content

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 5 min 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.

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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