Nuclear Domains Involved in Inositide Signalling

The identification of elements of inositide signalling at nuclear sites has been achieved by immunocytochemical techniques at confocal and electron microscopy level (Maraldi et al., 1999). Each method presents advantages and drawbacks; however, the results obtained are generally consistent and complementary. The main advantage of fluorescent-labelled probes at the confocal microscope is the possibility of examining the whole cell maintaining its three-dimensional organization, while gold-labelled probes at the electron microscope ensure very high resolution and quantitative evaluations. The availability of GFP-tagged probe technique overcomes the limits of traditional cytochemistry based on fixed specimens, and allows one the use of in vivo localization assay. The results till now obtained with this technique confirm those previously obtained with conventional immunocyto-chemistry (Bavelloni et al., 1999).

The use of in situ nuclear matrix preparations allowed one to identify the localization sites of the insoluble versus soluble pools of some elements of the signalling system. The PI(4,5)P2 soluble pool is quite diffused within the nucleus; in fact it is localized at both heterochromatin and interchromatin domains, in agreement with the presence of a large number of nuclear proteins presenting PI-binding motifs. As a consequence of the interactions between polyphosphoinositides and nuclear proteins such as histones, polymerases, and BAF complexes, PI(4,5)P2 localization sites have been detected at the heterochromatin, mainly at its periphery, at some nucleolar components, and at ribonucleoproteins in the interchromatin domains. The insoluble pool, on the other hand, is restricted to interchromatin granules. Other elements of the signalling system were detected to colocalize with PI(4,5)P2 at the same sites (Maraldi et al., 1999). The molecular events occurring at these districts, that is transcription of hnRNA, and pre-mRNA processing, have been widely investigated. Nascent transcripts correspond to perichromatin fibrils, while interchromatin granule clusters, detectable as fluorescent speckles at confocal microscope, contain many of the splicing factors. Splicing can occur both co- and post-transcriptionally, that is at the perichromatin fibres as well as at the interchromatin granules. Activation of splicing factors involves phosphorylation that modulates their shuttling from transcriptional to post-transcriptional sites (Misteli and Spector,

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Fig. 3 Nuclear inositide signalling pathways and nuclear targets. The elements of the signalling system identified by immunocytochemistry within the nucleus are indicated (o). Some of them are constitutively associated to nuclear proteins, such as PI(4,5)P2 and PLCpib, or translocated upon stimuli, such as PI 3-kinase and some PKC isoforms. The targets of lipid second messengers, including PKC isoforms and PKB/Akt, are able to phosphorylate specific transcription factors, or chromatin-associated proteins, or nuclear lamins (grey italics)

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Calmodulin Ca"/ealmodLil!n K

Fig. 3 Nuclear inositide signalling pathways and nuclear targets. The elements of the signalling system identified by immunocytochemistry within the nucleus are indicated (o). Some of them are constitutively associated to nuclear proteins, such as PI(4,5)P2 and PLCpib, or translocated upon stimuli, such as PI 3-kinase and some PKC isoforms. The targets of lipid second messengers, including PKC isoforms and PKB/Akt, are able to phosphorylate specific transcription factors, or chromatin-associated proteins, or nuclear lamins (grey italics)

1997). Therefore, the presence of a signalling system at these sites, colocalized with the protein kinases involved in the phosphorylation of splicing factors (Maraldi et al., 1999), strengths the possibility of a functional involvement of inositides into crucial steps of nuclear metabolism (Fig. 3).

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