Methods to Assess Changes in the Pattern of Nuclear Phosphoinositides
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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 wordsNuclear isolation In nuclei labeling Phosphatidylinositol-5-phosphate Phosphatidylinositol-4-phosphate Phosphoinositides Nuclear lipid signalling
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