Methods to Assess Changes in the Pattern of Nuclear Phosphoinositides

  • Nullin DivechaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 645)


Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signalling system that is regulated in response to a variety of extra and intra-cellular cues (Curr Opin Genet Dev 14:196–202, 2004). Phosphoinositides and the enzymes that synthesize them are found in many different sub-cellular compartments including the nuclear matrix, heterochromatin, and sites of active RNA splicing, suggesting that phosphoinositides may regulate specific functions within the nuclear compartment (Nat Rev Mol Cell Biol 4:349–360, 2003; Curr Top Microbiol Immunol 282:177–206, 2004; Cell Mol Life Sci 61:1143–1156, 2004). The existence of distinct sub-cellular pools has led to the challenging task of understanding how the different pools are regulated and how changes in the mass of lipids within the nucleus can modulate nuclear specific pathways. Here we describe methods to determine how enzymatic activities that modulate nuclear phosphoinositides are changed in response to extracellular stimuli.

Key words

Nuclear isolation In nuclei labeling Phosphatidylinositol-5-phosphate Phosphati­dylinositol-4-phosphate Phosphoinositides Nuclear lipid signalling 


  1. 1.
    Jones, D.R., and Divecha N. (2004) Linking lipids to chromatin. Curr. Opin. Genet. Dev. 14, 196–202.CrossRefPubMedGoogle Scholar
  2. 2.
    Irvine, R.F. (2003) Nuclear lipid signalling. Nat. Rev. Mol. Cell Biol. 4, 349–360.CrossRefPubMedGoogle Scholar
  3. 3.
    Hammond, G., Thomas, C.L., and Schiavo, G. (2004) Nuclear phosphoinositides and their functions. Curr. Top. Microbiol. Immunol. 282, 177–206.PubMedGoogle Scholar
  4. 4.
    Martelli, A.M., Fala, F., Faenza, I., Billi, A.M., Cappellini, A., Manzoli, L., and Cocco, L. (2004) Metabolism and signaling activities of nuclear lipids. Cell. Mol. Life Sci. 61, 1143–1156.CrossRefPubMedGoogle Scholar
  5. 5.
    Cocco, L., Gilmour, R.S., Ognibene, A., Letcher, A.J., Manzoli, F.A., and Irvine, R.F. (1987) Synthesis of polyphosphoinosi­tides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem. J. 248, 765–770.PubMedGoogle Scholar
  6. 6.
    Cocco, L., Martelli, A.M., Gilmour, R.S., Ognibene, A., Manzoli, F.A., and Irvine RF. (1988) Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulin-like growth factor I. Biochem. Biophys. Res. Commun. 154, 1266–1272.CrossRefPubMedGoogle Scholar
  7. 7.
    Sun, B., Murray, N.R., and Fields, A.P. (1997) A role for nuclear phosphatidylinositol-specific phospholipase C in the G2/M phase transition. J. Biol. Chem. 272, 26313–26317.CrossRefPubMedGoogle Scholar
  8. 8.
    Vann, L.R., Wooding, F.B., Irvine, R.F., and Divecha, N. (1997) Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei. Biochem. J. 327, 569–576.PubMedGoogle Scholar
  9. 9.
    Jones, D.R., Bultsma, Y., Keune, W.J., Halstead, J.R., Elouarrat, D., Mohammed, S., Heck, A.J., D’Santos, C.S., and Divecha, N. (2006) Nuclear PtdIns5P as a transducer of stress signaling: an in vivo role for PIP4Kbeta. Mol. Cell 23, 685–695.CrossRefPubMedGoogle Scholar
  10. 10.
    Clarke, N.G., and Dawson, R.M. (1981) Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids. Biochem. J. 195, 301–306.PubMedGoogle Scholar
  11. 11.
    Martelli, A.M., Cataldi, A., Manzoli, L., Billi, A.M., Rubbini, S., Gilmour, R.S., and Cocco L. (1995) Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation. Cell. Signal. 7, 53–56.CrossRefPubMedGoogle Scholar
  12. 12.
    Divecha, N., Letcher, A.J., Banfic, H.H., Rhee, S.G., and Irvine, R.F. (1995) Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells. Biochem. J. 312, 63–67.PubMedGoogle Scholar
  13. 13.
    Stephens, L.R., Hughes, K.T., and Irvine, R.F. (1991) Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature 351, 33–39.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.CRUK Inositide LaboratoryPaterson Institute for Cancer ResearchManchesterUK

Personalised recommendations