Claude Masson

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Fate of specific nucleolar perichromosomal proteins during mitosis: cellular distribution and association with U3 snoRNA.

Summary— In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of allchromosomes forming a novel class of chromosomal passenger proteins. The nucleolar components which participate in the formation of this perichromosomal layer have been investigated to elucidate the role of these perichromosomal proteins in the assembly and disassembly of the nucleoli. i) Electron microscopy immunolabelling reveals that these proteins are predominantly located in the granular component of the nucleoli during interphase,. ii) Immunoprecipitation data suggest that they are distributed at the chromosome periphery in association with U3 small nucleolar RNA (snoRNA). In addition, the distribution of U3 snoRNA visualized by in situ hybridization, is similar to that observed for the perichromosomal proteins. iii) In cells which possess a nucleolar remnant during mitosis, U3 snoRNA and perichromosomal proteins were found both in the perichromosomal layer and in the nucleolar remnant. iv) Some of these proteins are conserved from yeast to man such as fibrillarin and a protein of 52 kDa. v) The location of these proteins observed in yeast by confocal microscopy shows that they are not dispersed during mitosis. Their partition between the two daughter cells is performed by scission of nucleolar structures forming a rod during the budding process. Therefore RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2 and associate with the chromosome periphery until late telophase. They associate in the perichromosomal layer in human and PtK1 cells and both in the perichromosomal layer and the nucleolar remnant in CHO cells.

The ultrastructure of the chromosome periphery in human cell lines

We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled interchromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.

Cryofixation, cryosubstitution, cryo‐embedding for visualizing of nuclear ultrastructure and for immunodetection HeLa cells

Summary— We investigated the nuclear ultrastructure of HeLa cells using cryotechniques. The cells were cultured as monolayers, quick‐frozen and cryosubstituted in acetone without chemical fixatives. Half the samples were then treated with 1% osmium tetroxide in acetone and embedded in Epon, protocol A. The other half was cryo‐embedded in Lowicryl K11M at −60°C, protocol B. The efficiency of cryofixation was observed with both types of procedure. There was no fixation gradient detectable within the monolayer. The nuclei were well preserved, showing no reticulation of the chromatin. The different nuclear structures which have been observed after chemical fixation were also visible in these preparations. We noted improved visualization of the lamina and nucleolar chromatin, a very sharp limit between the fibrillar center and the dense fibrillar component, and variability in the appearance of the nuclear pores. Immunolabeling with an auto‐immune serum was possible on sections processed by protocol A and protocol B, but not on sections obtained by chemical fixation and cryo‐embedding procedures. Therefore, in the present work we showed that cryofixation, cryosubstitution and cryo‐embedding of cell monolayers can ensure a good preservation of the nuclear structures. Furthermore, detection of nucleolar antigenic determinants is facilitated and rapid nuclear events can easily be monitored.

Autoimmune serum containing an antibody against a 94 kDa nucleolar protein

Little information exists on how various nucleolar proteins function in ribosome biogenesis. Of special interest is that group of nucleolar proteins which are not incorporated into mature ribosomes because they are candidates for a role in the regulation of ribosome construction. Non‐ribosomal nucleolar proteins can be analyzed using autoimmune sera from scleroderma patients which often contain antinuclear antibodies. One such serum, designated ScBr, is shown by indirect immunofluorescence to react specifically with nucleoli in cells of 3 different mammalian species, indicating that the antigen is at least partly conserved evolutionarily. It is not RNase‐sensitive, but is completely eliminated after incubation with pronase and 2 M NaCl. Immuno‐electron microscopy was carried out on Lowicryl ultrathin sections to localize the antigen. The labeling was observed over both the granular and the dense fibrillar component but not the fibrillar centers, indicating that the antigen is associated with ribosomal RNA transcription sites and ribosome assembly into precursor particles. In addition, the antibody was localized to small nucleoplasmic entities, termed dense nuclear bodies. This could indicate a relationship between nucleoli and dense nuclear bodies. By immunoprecipitation, only a single protein of 94 kDa molecular weight was revealed. By immunoblotting, the band at 94 kDa was found to be the only positive band for high ScBr dilutions. Observation of the behavior of the antigen during mitosis revealed that it became dispersed into the cytoplasm after breakdown of the nuclear envelope, lining most of the chromosomes rather that remaining associated with the NOR‐chromosomes. The antigen appeared to be restored to nucleoli only in late telophase; phase‐dense prenucleolar bodies of early telophase cells did not show positive staining for the antigen. During actinomycin‐D RNA synthesis inhibition as well as in non‐stimulated lymphocytes the positive staining is greatly decreased. These results were consistent with a role for the 94 kDa nucleolar protein in the process of preribosome assembly.

CONFORMATION OF RIBOSOMAL GENES AND DISTRIBUTION OF THE TRANSCRIPTION FACTOR UBF ARE CELL CYCLE DEPENDENT

The amount of Ag-NOR proteins has a prugnostic value m human cancers. Ag-NOR proteins are a set of argyrophihc nucleolar proteins that accumulate in highly proliferating celis whereas their expression is very low in non-proliferating cells. To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, tht) relationship between cell cycle and amaunt of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins (Roussel P, Belenguer P, Amairic F ;G Hernandez. Verdun D (1992), Ely. CEI[ Res. 203: 259-269; Roussel P Pr Hernandez-Verdun D (19Y4) Erp. C?II iies 274: 465-4721, nucleolin and protein B23 was performed m exponentially growing, serum-deprived and cell-cycle stimulated cells. The quantification was performed on Western blots as already described (Sirri V, Roussel I’, TrerP D, Derenzini M Br HernandezVerdun D (1995) 1. Histochen~ Cytochrn~ 43: 887-893). Expression of nucleolin was low in serum-deprived cells dnd increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serurndeprived cells, and increased progressively until C2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein 823 in G2 compared to CI was demonstrated using sorted phascspecific cells. In Cg cells, sorted according tcl their very low RNA content, the amount of Ag-NOR proteirts was half elf that founil in Gt cells, nucieolin being only weaklv detectable. Therefore the expression of nucleolin Increased between G()-G [ and G 1-S phases. These data support the hypothesis that quantification of Ag-NOR reflects the percentage of cells at each cell cycle phase, it IS high iz S-C2 and low in G1 phases. STEIMBERG Natha& THENET-GAUCI Sophie, ADOLPHE Monique hborutoire de Phatmacologie CeNuLaire de I’EPHE 1.5 rue de I’ Ecole de hf&ecine 75006 Pot-is France