Immunocytochemical detection of the specific association of different PIC 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 cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC β2, γ1 and δ1 isoforms using confocal and electron microscope immunocytochemistry. PIC β1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC γ1 isoform is found in the nucleus and in the cytosol, while PIC δ1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC β1 and PIC γ1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC γ1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC γ1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.