To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein–tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.

No abstract

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in a-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the a-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB.

Using confocal and immunofluorescence microscopy the relative distribution of the ribosomal chromatin and some proteins of the RNA polymerase I transcription machinery such as upstream binding factor (UBF), RNA polymerase I and DNA topoisomerase I was analyzed on chromosomal nucleolus organizer regions (NORs) of PtK1 cells. Staining with various DNA fluorochromes revealed that the ribosomal chromatin may be found at the axial region of the NOR and also at lateral expansions around the axis that can also be detected by in situ hybridization. It was observed that the transcription machinery shows a crescent-shaped distribution around the axial ribosomal chromatin at the NOR of metaphase and anaphase chromatids. An ultrastructural analysis of serially sectioned NORs supports this crescent-shape organization. Taking into account previous and present results and the loop/scaffold model of chromosome structure, we propose a model of NOR organization. The model proposes that ribosomal genes that were inactive in the preceding interphase would be present as condensed short Q-loops occupying the axial region of the NOR. Ribosomal genes previously active during interphase would be undercondensed as large R-loops associated with the transcription machinery, which is distributed in a crescent-shaped fashion around the previously active ribosomal DNA.

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.