The structure and functional organization of the cell nucleus remain the subjects of extensive debate today. Understanding in molecular detail the organizing principles of the nucleus, such as the arrangement of chromosomal DNA, or the coordination and regulation of the synthesis, processing, assembly and transport of macromolecules, are major goals for cell biology. For many years, studies on the cell biology of the nucleus were limited by a relative lack of distinctive substructures revealed by microscopy and amenable to biochemical purification. When a typical mammalian nucleus is observed in the electron microscope, clumps of heterochromatin are visible at the nuclear periphery and the nucleolus is readily identified by virtue of its electrondense appearance, but otherwise the nucleoplasm appears rather featureless and amorphous. However, a very different view is evident when antibody or hybridization probes are used to detect specific nuclear factors or genes. Many nuclear macromolecules are localized to distinct regions and substructures of the nucleus. A specific inquiry into the role of several of these domains and nuclear substructures in RNA metabolism and other nuclear functions is the principal aim of endeavour of the Department of Cell Biology. A useful tool for the investigation of nuclear architecture are human autoantibodies, which are a major source of antinuclear specific probes. Importantly, the Department has a tight collaboration with the Laboratory of Gene Expression of the 1st Faculty of Medicine of Charles University. It is worthy of mention that the Department organized two EMBO nuclear workshops in 1999 and 2002, two EMBO practical electron microscopy courses in 1996 and 1997, and it will coorganize another EMBO course in Ceske Budejovice in 2003.
Current research addresses the following:
- Functional architecture of the nucleolus
- Nuclear architecture associated with extranucleolar RNA synthesis and processing
- Chromatin, transcription, replication and repair
- Impact of autoantibodies

      To clarify the arrangement of rRNA genes within nucleoli, we have generated a cytochemical affinity picture of the nucleolus. Three kinds of affinity probes have been used:
1) probes to nucleolar chromatin, including rDNA sequences,
2) probes to a number of macromolecules that are directly or indirectly involved in the synthesis and processing of rRNA and the formation of preribosomes (including GFP hybrid proteins);
3) antibodies to nucleotide analogues such as bromouridine or biotinylated cytidine, which are used to detect labeled RNA. The results suggest the localization of transcription sites to dense fibrillar components and to the border region between these components and fibrillar centers. Using a permeabilized cell system, we have concluded that the processing factors are assembled on pre-rRNA at the sites of transcription. Our results obtained on living cells show that the early rRNA processing steps take place in the domains of dense fibrillar components that are spatially separated from rDNA transcription sites.

Fig.1: Concomitant light and electron microscopy mapping of transcription signal in a thin sectioned HeLa cell (B). Detailed part of (A) .

      Our results show that transcription takes place in the perichromatin regions of the nucleus and that pre-mRNA processing is not necessarily co-transcriptional. We have provided evidence about the trafficking of released pre-mRNA transcripts to splicing factor reservoirs (nuclear speckles). In another series of experiments, we have shown that the (pre-)spliceosomal complexes on microinjected pre-mRNA are formed inside the nuclear speckles. Their targeting into and accumulation in the speckles are results of the cumulative loading of splicing factors to the pre-mRNA.

      Replication within a replisome is a complex process, and contradictory results have been reported concerning the mutual localization of replication and transcription sites. In order to settle this problem, a straightforward strategy was followed: a pulse with both DNA and RNA base analogues has been detected immunocytochemically. We have developed a new procedure allowing efficient entry of BrUTP and biotinylated dUTP into living cells. Using this approach we have shown that the signals due to the two incorporated analogues are spatially separated one from another. We have been able to expand these findings with regard to specific genes and to show that the replicating ribosomal genes ceased to be transcribed. The obtained results have led us to propose a switching mechanism for coordination of replication/transcription of nucleolar ribosomal genes. In this model, rRNA genes are organized in foci within nucleoli. The foci active in replication are "switched off" for transcription, but can be "switched on" for transcription following replication. Importantly, our data point to dynamical changes in the nucleolar architecture accompanying the switching to replication activity, as documented by the displacement of fibrillarin, a nucleolar protein involved in the processing of rRNA, from the gene environment.
      We have measured the speed of replication fork movement on extended DNA fibers labelled with the 2´-deoxythymidine analogues 5´-chloro-2´deoxyuridine and 5-iodo-2´-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. The availability of 2´-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.
      We have shown that DNA double-stranded breaks (dsb) produced by restriction enzymes induce the formation of foci containing replication protein A and protein Ku on in vitro reconstituted Xenopus sperm nuclei. Such foci are involved in dsb repair and the repair of this type of DNA damage can be specifically studied under conditions of the normal nuclear environment.

Fig. 2: Replication fluorescence pattern seen in a synchronized HeLa cell (A). The corresponding image of the spread DNA fibres allows for the determination of the fork speed (B).

      The investigation of autoantibodies is important in clinical medicine. At the same time, autoantibodies represent a major source of antinuclear probes. Our research aim is oriented to the biological characterization of autoantigens, but the results obtained are also used for diagnostic purposes and may help in the elucidation of the etiopathogenesis of some diseases such as systemic rheumatic diseases. We have identified an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis that reacts with the core proteins of hnRNP particle C1 and C2. Interestingly, these proteins belong to several other autoantigens cleaved by caspases during apoptosis.

Fig. 3.: Localization of spliceable (A) and mutant (B,C) fluorochrome-labeled RNAs microinjected into HeLa cells.