The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis.

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.

The SURF-6 protein is a component of the nucleolar matrix and has a high binding capacity for nucleic acids in vitro.

The recently identified novel protein SURF-6 is shown to be a component of the nucleolar matrix. Immunofluorescence analysis demonstrated that SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of biochemical nucleolar subfractions confirmed that SURF-6 was present in the nucleolar matrix fraction, and was absent from the fractions of soluble proteins released by DNase or RNase. The capacity of SURF-6 to bind nucleic acids was investigated in vitro. Both endogenous SURF-6 from nuclear extracts and recombinant SURF-6 exhibited a strong binding capacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by immunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the cell cycle suggesting that it is a structural protein constitutively present in nucleolar substructures. The colocalization of SURF-6 with the major nucleolar proteins B23 and fibrillarin, which are known to be involved in the processing of ribosomal RNA (rRNA), was examined both in interphase and mitosis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cytoplasm and prenucleolar bodies, in mitosis. However, colocalization of SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these results indicate that SURF-6 is a novel nucleolar matrix component and imply that SURF-6 might support nucleolar matrix structure and function(s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteristics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.

Cell cycle-dependent translocations of a major nucleolar phosphoprotein, B23, and some characteristics of its variants.

A major nucleolar phosphoprotein, B23, is thought to play several apparently unrelated roles and appears to be associated with other cell compartments besides the nucleolus. However, characteristic properties of B23 variants still remain to be established. Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issue particularly focusing on the events during mitosis. Also, we made an attempt to generalize the data on the cell cycle-dependent translocations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT) which were found to be immunoreactive for 20B2. In all the cell strains studied, B23 was chiefly located within the nucleolus at interphase, and was associated with a few cellular domains during mitosis. They were: the nucleoplasm (at prophase before the nuclear envelope breakdown), the cytoplasm (from prometaphase until mid telophase), the perichromosomal layer (from prometaphase till early telophase), cytoplasmic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobility of B23 was found to be the same at G1, S and G2 periods of interphase, but became slower at mitosis. In situ and cell extraction experiments showed that like the nucleolar B23, B23 of the perichromosomal layer and that of PNBs was highly resistant to extraction with Triton X-100, but could be released with Triton X-100/RNase A. These findings indicated that these portions of B23 were most likely to be associated with RNA. The cytoplasmic B23 was the major intracellular variant of B23 during mitosis. It had a slightly lower electrophoretic mobility than the perichromosomal B23 and could readily be extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplasmic B23 was mainly in an RNA-free state. Mitosis-like translocations of B23 from the nucleolus to the nucleoplasm induced by actinomycin D increased its extractability but did not affect the electrophoretic mobility. The phosphorylation status of different B23 variants at interphase and mitosis both in controls and following the drug, is discussed.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

News and views on the nucleolus in 1996. Report on the 'Colloquium on the nucleolus', a meeting at Paris-Grignon, July 18-20, 1996.

The new data presented by the participants during the 'Colloquium on the Nucleolus' were reviewed and regrouped in different topics concerning the molecular and functional significance of nucleolar structure. The topics included: rRNA transcription and nucleolar organization, the nucleolus in relation to cell proliferation including nucleolus in pathology during the cell cycle and cell division, nucleologenesis, and nucleolar proteins.

The nucleolar architecture of polymerase I transcription and processing.

The nucleolus, the site of transcription and processing of the major ribosomal genes, generally reveals three distinct ultrastructural components in conventional thin-section electron micrographs (fibrillar centres, dense fibrillar component and granular component). We show here that different parts of the transcription and transcript processing pathway can be mapped to the different nucleolar components in pea root cells. This study shows the full three-dimensional arrangement of the different domains by in situ hybridization and confocal microscopy, and their correspondence with the major ultrastructural components of the nucleolus is revealed by parallel serial section electron microscopy. The active rDNA is widely dispersed in discrete foci, the larger of which, at least, correspond to well-defined fibrillar centres. A probe to the external transcribed spacer (ETS) sequence of the pre-rRNA transcripts labels clearly demarcated regions surrounding the foci of rDNA, and which we show correspond to the dense fibrillar component. Finally, a probe to the entire 45S transcript shows a higher concentration in regions corresponding to the granular component, surrounding the dense fibrillar component labelled by the ETS probe. The changes in structure that occur with heat shock show that nucleolar organization is dynamic and dependent upon transcriptional activity. These results show that the various RNA processing events are spatially highly organized and suggest a vectorial or radial model of transcription and transcript processing, where nascent and newly completed transcripts occupy zones surrounding the genes, which are in turn surrounded by regions containing the older more mature transcripts.

The nucleolus.

The nucleolus is the site within the eukaryotic nucleus of transcription of rDNA, of processing of the rDNA transcripts, and of the formation of pre-ribosomal particles. We review current ideas for the molecular organization of these processes. The earliest transcriptional events take place near the junction of the fibrillar centers and the dense fibrillar component; nascent transcripts occupy the dense fibrillar component, and the later stages of pre-ribosome formation take place in the granular component. We review current knowledge of non-ribosomal nucleolar proteins. Nucleoli contain a group of proteins that bind RNA and are likely to act as chaperones to facilitate the molecular structural events in the folding and interaction of the many components of ribosomes. Some of these nucleolar proteins are also engaged in a shuttling cycle between the nucleus and the cytoplasm and may serve to transport other proteins.

Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning.

Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.

Estrogens and cell death in murine uterine luminal epithelium.

The luminal epithelium of adult ovariectomized mice responds to estradiol-17 beta with a synchronised wave of DNA synthesis and mitosis. Estriol, however, although producing a similar DNA-synthetic and mitotic response fails to cause an increase in cell number owing to a wave of cell death occurring at mitosis. In the present study it was shown that cells died by two different routes. The majority died by apoptosis but, unusually, a minority also died by necrosis. In the apoptotic cells the cytoplasm became dense, the endoplasmic reticulum and nuclear cisternae dilated; chromatin became marginated the nucleus shrank and became deeply infolded and contorted. Apoptosis, however, was uncharacteristic in that the nucleus failed to fragment, form caps or show disruption before the cells died by membrane rupture. Furthermore, the cells were frequently lost in sheets from the epithelium into the lumen. Part of the biochemical explanation for this onset of cell death comes from the accelerated loss from the tissue of estriol when compared to estradiol-17 beta. This resulted in a decline in protein and rRNA biosynthesis and a failure to complete ribosomal maturation. Evidence in favour of this explanation came from experiments that showed a return to the estradiol-17 beta level of response and an inhibition of cell death when the occupancy of the estriol receptor was maintained.

The effect of soil temperature on the degradation of cis, trans - permethrin in soil.

The degradation of [carbonyl - 14C] - cis, trans-permethrin was studied in Dubbs fine sandy loam soil incubated at 10, 25, and 40 degrees C from 0 to 64 days. The most rapid degradation of permethrin to [carbonyl - 14C] - cis, trans -3- (2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (DCVA) and ultimately 14CO2 occurred at 25 degrees C. Rapid degradation to DCVA also occurred at 40 degrees C, however, further degradation of DCVA to 14CO2 was reduced. Permethrin degradation to 14CO2 was slowest at 10 degrees C. The degradation rate of trans-permethrin was greater than the cis-permethrin at all temperatures tested. The halflives of cis- and trans-permethrin were 55 and 14 days at 10 degrees C, 12 and 5 days at 25 degrees C, and 27 and 4 days at 40 degrees C, respectively. Compounds identified from treated soil were the original [carbonyl - 14C]-cis, trans-permethrin and the major degradation products cis-, and trans-DCVA.

Interrelationship of mitochondria with cytoplasmic 10 nm filaments in tapetal cells of Hyacinthoides non-scripta (L.) Chouard.

An aggregation of 10 nm filamentous structures in a paracrystalline array associated with stacks of mitochondria in the tapetal cells of Hyacinthoides non-scripta (L.) Chouard is reported. The filaments are around 0.4 micrometer long and form two different types of association with mitochondria. They may be in the form of a single layer between adjacent mitochondria or packed into groups surrounded by mitochondria. The former arrangement is reminiscent of that reported for the intercisternal elements of dictyosomes. Various explanations relating to the function of tapetal cells are discussed.

Nuclear pores. Can they expand and contrast to regulate nucleocytoplasmic exchange?

Recent evidence has revived the idea that translocations of macromolecules between nucleus and cytoplasm may be regulated via expansion and contraction of the entire diameter of the nuclear pore complex. The present investigation does not support this hypothesis.

The nucleolus and meiosis during microsporogenesis in Endymion non-scriptus (L.).

Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The segregated components of the nucleolus at meiotic prophase underwent fragmentation and dissolution at pachytene-diplotene. Nucleoli were absent during both meiotic divisions and reformed on the nucleolus organizer into a fibrillar mass from scattered fibrillar components at the dyad and tetrad stages. Ti is argued that the fibrillar region shows continuity through nuclear division though undergoing structural transformations in the process. Nucleolar reformation occurs on condensed nucleolus organizers. Processing of the ribosomal precursors and the resumption of RNA synthesis is discussed in relation to the dispersal of the nucleolus organizer into the fibrillar region of the reformed nucleolus.