Reorganization of the nuclear machinery after mitosis is a fundamental but poorly understood process. Here, we investigate the recruitment of the nucleolar processing proteins in the nucleolus of living cells at the time of nucleus formation. We question the role of the prenucleolar bodies (PNBs), during migration of the processing proteins from the chromosome periphery to sites of rDNA transcription. Surprisingly, early and late processing proteins pass through the same PNBs as demonstrated by rapid two-color four-dimensional imaging and quantification, whereas a different order of processing protein recruitment into nucleoli is supported by differential sorting. Protein interactions along the recruitment pathway were investigated using a promising time-lapse analysis of fluorescence resonance energy transfer. For the first time, it was possible to detect in living cells the interactions between proteins of the same rRNA processing machinery in nucleoli. Interestingly interactions between such proteins also occur in PNBs but not at the chromosome periphery. The dynamics of these interactions suggests that PNBs are preassembly platforms for rRNA processing complexes. INTRODUCTIONIn higher eukaryotes, the nucleus is disassembled when chromosomes condense at the beginning of mitosis and reassemble at the end of mitosis. During mitosis, there is redistribution and/or inactivation of the nuclear machineries that will be further involved in rebuilding nuclear functions. One of the fundamental features of nuclear organization is that many components of the RNA synthesis and processing machineries are grouped into compartments (Spector, 1993;Lamond and Earnshaw, 1998;Matera, 1999;Gall, 2000;Misteli, 2000;Spector, 2001;Huang, 2002;Lamond and Spector, 2003). This implies that the recruitment of dedicated machineries and formation of discrete nuclear domains are crucial events at the beginning of interphase. In addition, recent data indicate that the entry as well as correct relocation of the nuclear machineries into newly forming nuclei is a step-dependent and ordered process (Savino et al., 2001;Prasanth et al., 2003;Bubulya et al., 2004;Leung et al., 2004). However, recruitment of the nucleolar processing proteins on transcription sites is still a poorly understood process. To better understand the rebuilding of nucleolar functions after mitosis, we chose to investigate the assembly of the nucleolar processing machinery in real time in living cells.The nucleolus is a functional compartment of the nucleus generated by ribosome biogenesis (Hadjiolov, 1985;Mélèse and Xue, 1995;Leung et al., 2003). It is also a plurifunctional domain involved in the three-dimensional (3D) organization of chromatin in the nucleus (Chubb et al., 2002), in the assembly of several ribonucleoprotein complexes (Pederson, 1998;Olson et al., 2000), and in the formation of nuclear speckles (Bubulya et al., 2004). The nucleolus therefore seems to be a key player of the nuclear functional architecture. Its functions depend on the activation and recru...

The method of spreading transcription complexes has been applied to amphibian oocytes of Pleurodeles genus. Complexes of nucleolar origin show a regular and homogeneous organization similar to that described in other materials. The observations add to the interpretation as an amplification of nucleolar DNA and a redundancy of ribosomal cistrons in the two species studied. -- On the other hand, complexes of chromosomal origin display a great diversity. Two main characteristics can be drawn: the existence of several transcription units in a chromosomal organization unit and the possibility to point out a special architecture at the RNP fibril level. Applying a shadowing technique used for isolated molecules is an improvement compared with earlier methods based on PTA coloration.

To elucidate the potential role of the hsp70 gene family in developmental processes in vertebrates, we chose to study the expression of one of these genes in zebrafish. A zebrafish gastrula cDNA library was screened with a Pleurodeles waltl hsp70 cDNA probe. A 2.3-kb cDNA was thus isolated and sequenced. The predicted amino acid sequence contained an open reading frame encoding for a 649-amino acid polypeptide. Sequence analysis showed strong homology with hsp70-related gene sequences in other species; in particular, the strongest homology was found with the cognate members of this family. Tests of heat inducibility revealed that transcripts were expressed at normal temperature, but the level of transcript expression increased after heat shock. Moreover, experiments of the neosynthesis of total proteins in heat shock conditions and corresponding immunoblotting assays showed that 24-h-stage embryos are able to respond to heat shock. The quantity of 70 kDa proteins, recognized by a specific antibody of the HSP/C70 protein family, is expressed in control condition and increased significantly after heat shock. Furthermore, Northern blot analysis of transcript expression showed that the corresponding mRNAs were detected throughout embryonic development in the absence of any heat shock. Our clone, named hsc70, thus corresponded to a cognate member of the hsp70 gene family, expressed under normal conditions during development, but also heat inducible. The spatio-temporal pattern of transcripts during development was determined by in situ hybridization on wholemount embryos at different stages. As a maternal RNA, hsc70 mRNA was uniformly present in the embryo, up to the end of gastrulation. Later, a tissue-specific enrichment of hsc70 transcripts was detected in the central nervous system (CNS) and in a fraction of the somites. These results suggest that the hsc70 gene may be involved in developmental differentiation events.