IMMUNOCYTOCHEMICAL DETECTION OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATELOCALIZATION SITES WITHIN THE NUCLEUS

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signa l transduction, being the preferential substrate of specific phospholi pases that produce second messengers such as inositol trisphosphate (I P3) and diacylglycerol (DG). Because PIP2 has been cytochemically iden tified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic imm unocytochemistry to identify the nuclear sites of PIP2 localization an d to exclude any cross-reactivity of the antibody with other nuclear m olecules, The results confirm the specificity of the immunolabeling an d indicate that PIP2 is localized at precise intranuclear sites both i n in situ and in isolated nuclei. They also show that a significant am ount of the phospholipid is retained by the cytoskeleton and by the in ner nuclear matrix in in situ matrix preparations. Moreover the sensit ivity of the immunocytochemical reaction is capable of detecting quant itative variations of PIP2 nuclear content induced by agonists that mo dulate the signal transduction system at the nuclear level,