IMMUNOCYTOCHEMICAL DETECTION OF THE SPECIFIC ASSOCIATION OF DIFFERENTPIC ISOFORMS WITH CYTOSKELETAL AND NUCLEAR MATRIX COMPARTMENTS IN PC12 CELLS

The increasing evidence of discrete roles of phosphoinositidase C (PIC ) isoforms and the assessment of their localization in the cytoskeleto n and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheoc hromocytoma is one of the few cell lines expressing three immunologica lly distinct isoforms of PIC. We have analyzed the subcellular distrib ution of the PIC beta(1), gamma(1) and delta(1) isoforms using confoca l and electron microscope immunocytochemistry. PIC beta(1) is mainly f ound in the nucleus and is associated with interchromatin domains. On the other hand, the PIC gamma(1) isoform is found in the nucleus and i n the cytosol, while PIC delta(1) is exclusively cytoplasmic. Immunobl ot and immunocytochemical experiments indicate that the various PIC is oforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC beta(1) and PIC gamma(1) isoforms are tightly associated with the nuclear matrix, whi le only about 50 % of PIC gamma(1) is associated with the cytoskeleton after DNase I and high salt extractions. PIC gamma(1) is almost compl etely soluble under these conditions. These results further confirm th e complexity of the inositide signal transduction mechanism, which inv olves several PIC isoforms, specifically localized in different cell c ompartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.