Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Measurement of phosphoinositides in the zebrafish Danio rerio

Nature Protocols volume 8, pages 1058–1072 (2013)Cite this article

Subjects

Abstract

Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [32P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [32P]γATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Phosphoinositide analysis in zebrafish embryos.
Figure 2: TLC analysis of in vivo radiolabeled phospholipids.
Figure 3: HPLC analysis of in vivo radiolabeled deacylated phospholipids.
Figure 4: Quantification of PtdIns5P in zebrafish embryos.
Figure 5: Quantification of PtdIns4P in zebrafish embryos.
Figure 6: Quantification of PtdIns(4,5)P2 in zebrafish embryos.
Figure 7: Quantification of lipid phosphate in zebrafish embryos.

Similar content being viewed by others

References

  1. Di, P.G. & De, C.P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).

    Article  Google Scholar 

  2. Balla, T., Szentpetery, Z. & Kim, Y.J. Phosphoinositide signaling: new tools and insights. Physiology (Bethesda) 24, 231–244 (2009).

    CAS  Google Scholar 

  3. Fiume, R. et al. Nuclear phosphoinositides: location, regulation and function. Subcell. Biochem. 59, 335–361 (2012).

    Article  CAS  Google Scholar 

  4. McCrea, H.J. & DeCamilli, P. Mutations in phosphoinositide metabolizing enzymes and human disease. Physiology (Bethesda) 24, 8–16 (2009).

    CAS  Google Scholar 

  5. Vicinanza, M., D'Angelo, G., Di, C.A. & De Matteis, M.A. Phosphoinositides as regulators of membrane trafficking in health and disease. Cell Mol. Life Sci. 65, 2833–2841 (2008).

    Article  CAS  Google Scholar 

  6. Wenk, M.R. et al. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nat. Biotechnol. 21, 813–817 (2003).

    Article  CAS  Google Scholar 

  7. Pettitt, T.R. Phosphoinositide analysis by liquid chromatography-mass spectrometry. Methods Mol. Biol. 645, 203–217 (2010).

    Article  CAS  Google Scholar 

  8. Clark, J. et al. Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat. Methods 8, 267–272 (2011).

    Article  CAS  Google Scholar 

  9. Morris, J.B., Hinchliffe, K.A., Ciruela, A., Letcher, A.J. & Irvine, R.F. Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay. FEBS Lett. 475, 57–60 (2000).

    Article  CAS  Google Scholar 

  10. Clarke, J.H. et al. Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem. J. 357, 905–910 (2001).

    Article  CAS  Google Scholar 

  11. Guillou, H. et al. Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach. J. Lipid Res. 48, 726–732 (2007).

    Article  CAS  Google Scholar 

  12. Chicanne, G. et al. A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples. Biochem. J. 447, 17–23 (2012).

    Article  CAS  Google Scholar 

  13. Lieschke, G.J. & Currie, P.D. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet. 8, 353–367 (2007).

    Article  CAS  Google Scholar 

  14. Santoriello, C. & Zon, L.I. Hooked! Modeling human disease in zebrafish. J. Clin. Invest. 122, 2337–2343 (2012).

    Article  CAS  Google Scholar 

  15. Montero, J.A., Kilian, B., Chan, J., Bayliss, P.E. & Heisenberg, C.P. Phosphoinositide 3-kinase is required for process outgrowth and cell polarization of gastrulating mesendodermal cells. Curr. Biol. 13, 1279–1289 (2003).

    Article  CAS  Google Scholar 

  16. Van Epps, H.A. et al. The zebrafish nrc mutant reveals a role for the polyphosphoinositide phosphatase synaptojanin 1 in cone photoreceptor ribbon anchoring. J. Neurosci. 24, 8641–8650 (2004).

    Article  CAS  Google Scholar 

  17. Faucherre, A., Taylor, G.S., Overvoorde, J., Dixon, J.E. & Hertog, J. Zebrafish pten genes have overlapping and non-redundant functions in tumorigenesis and embryonic development. Oncogene 27, 1079–1086 (2008).

    Article  CAS  Google Scholar 

  18. Ma, H., Blake, T., Chitnis, A., Liu, P. & Balla, T. Crucial role of phosphatidylinositol 4-kinase IIIα in development of zebrafish pectoral fin is linked to phosphoinositide 3-kinase and FGF signaling. J. Cell. Sci. 122, 4303–4310 (2009).

    Article  CAS  Google Scholar 

  19. Jurynec, M.J. & Grunwald, D.J. SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo. Dis. Model. Mech. 3, 733–742 (2010).

    Article  CAS  Google Scholar 

  20. Rbaibi, Y. et al. OCRL1 modulates cilia length in renal epithelial cells. Traffic 13, 1295–1305 (2012).

    Article  CAS  Google Scholar 

  21. Luo, N. et al. OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome. Hum. Mol. Genet. 21, 3333–3344 (2012).

    Article  CAS  Google Scholar 

  22. Coon, B.G. et al. The Lowe syndrome protein OCRL1 is involved in primary cilia assembly. Hum. Mol. Genet. 21, 1835–1847 (2012).

    Article  CAS  Google Scholar 

  23. Luo, N., Lu, J. & Sun, Y. Evidence of a role of inositol polyphosphate 5-phosphatase INPP5E in cilia formation in zebrafish. Vision Res. 75, 98–107 (2012).

    Article  Google Scholar 

  24. Pan, W. et al. CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis. Blood 120, 489–498 (2012).

    Article  CAS  Google Scholar 

  25. Lambaerts, K. et al. Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish. J. Cell Sci. 125, 1129–1140 (2012).

    Article  CAS  Google Scholar 

  26. Ramirez, I.B. et al. Impaired neural development in a zebrafish model for Lowe syndrome. Hum. Mol. Genet. 21, 1744–1759 (2012).

    Article  CAS  Google Scholar 

  27. Sarkes, D. & Rameh, L.E. A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides. Biochem. J. 428, 375–384 (2010).

    Article  CAS  Google Scholar 

  28. Ndamukong, I., Jones, D.R., Lapko, H., Divecha, N. & Avramova, Z. Phosphatidylinositol 5-phosphate links dehydration stress to the activity of Arabidopsis trithorax-like factor ATX1. PLoS ONE 5, e13396 (2010).

    Article  Google Scholar 

  29. Schacht, J. Purification of polyphosphoinositides by chromatography on immobilized neomycin. J. Lipid Res. 19, 1063–1067 (1978).

    Article  CAS  Google Scholar 

  30. Palmer, F.B. Chromatography of acidic phospholipids on immobilized neomycin. J. Lipid Res. 22, 1296–1300 (1981).

    Article  CAS  Google Scholar 

  31. Fuchs, B., Suss, R., Teuber, K., Eibisch, M. & Schiller, J. Lipid analysis by thin-layer chromatography–a review of the current state. J. Chromatogr. A 1218, 2754–2774 (2011).

    Article  CAS  Google Scholar 

  32. Dove, S.K. et al. Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis. Nature 390, 187–192 (1997).

    Article  CAS  Google Scholar 

  33. Jones, D.R. et al. The identification of phosphatidylinositol 3,5-bisphosphate in T-lymphocytes and its regulation by interleukin-2. J. Biol. Chem. 274, 18407–18413 (1999).

    Article  CAS  Google Scholar 

  34. Halstead, J.R. et al. A role for PtdIns(4,5)P(2) and PIP5Kalpha in regulating stress-induced apoptosis. Curr. Biol. 16, 1850–1856 (2006).

    Article  CAS  Google Scholar 

  35. Divecha, N. Methods to assess changes in the pattern of nuclear phosphoinositides. Methods Mol. Biol. 645, 165–177 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Financial support for the Cancer Research UK Inositide Laboratory is entirely from Cancer Research UK. This work was supported by a Wellcome Trust project grant (079615) and a research grant from the UK Lowe Syndrome Trust awarded to M.L. (ML/MU/DEC07).

Author information

Authors and Affiliations

  1. Cancer Research UK Inositide Laboratory, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK

    David R Jones & Nullin Divecha

  2. Faculty of Life Sciences, University of Manchester, Manchester, UK

    Irene Barinaga-Rementeria Ramirez & Martin Lowe

Authors
  1. David R Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Irene Barinaga-Rementeria Ramirez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Lowe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nullin Divecha
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.R.J. and I.B.-R.R. designed and performed the experiments and analyzed the data. D.R.J., I.B.-R.R., M.L. and N.D. wrote the manuscript.

Corresponding authors

Correspondence to Martin Lowe or Nullin Divecha.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jones, D., Ramirez, IR., Lowe, M. et al. Measurement of phosphoinositides in the zebrafish Danio rerio. Nat Protoc 8, 1058–1072 (2013). https://doi.org/10.1038/nprot.2013.040

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.040

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing