Guy Goessens

Structure, function and assembly of the nucleolus

Most events of ribosome biogenesis--such as transcription of the ribosomal RNA (rRNA) genes, processing of their primary transcripts into mature rRNAs and assembly with ribosomal and nonribosomal proteins to form the preribosomes--are confined to a special nuclear compartment, the nucleolus. Immunogold labelling and in situ hybridization at the ultrastructural level are providing novel insights into structure-function relationships of the nucleolus, and in vitro systems are beginning to shed light on the molecular mechanisms involved in the reforming of nucleoli after mitosis.

Comparative ultrastructural study of the effects of serum-free medium and dibutyryl-cyclic AMP on newborn rat astroblasts.

SummaryModifications of cell shape induced in cultured newborn rat astroblasts by serum deprivation or dibutyryladenosine 3′–5′ monophosphate (dBcAMP) are described. Serum suppression modifies the adhesivity of the cells to the substrate, but this modification is not consistent with a true differentiation. The main ultrastructural feature of dBcAMP-treated astroblasts is the presence of an extensive system of 90 Å microfilaments, while control cells are relatively devoid of these structures.

Ultrastructural cytochemistry of the mammalian cell nucleolus.

In the present review on the organization of the mammalian cell nucleolus, we report and discuss data obtained during the past 10 years by means of cytochemical and immunocytochemical ultrastructural techniques. Particular emphasis is placed on the following topics: location of the nucleolus organizer regions in interphasic nucleolar components, structure of nucleolar chromatin in situ, and the structure-function relationship of the nucleolar components. The cytochemical and immunocytochemical results are compared and the concordant data are stressed for each topic.

High resolution autoradiographic studies of ehrlich tumour cell nucleoli. Nucleolar labelling after [3h]actinomycin d binding to dna or after [3h]tdr or [3h]uridine incorporation in nucleic acids.

Ehrlich tumour cell nucleoli contain fibrillar and granular components and low electron density areas called “fibrillar centres”. Analysis by high-resolution autoradiography using [3H]actinomycin D or [3H]TdR reveals that a small amount of DNA is present inside the fibrillar centres. Newly synthesized RNA is also present within the fibrillar centres or at their periphery, already 3 min after the precursor is given. According to these results, RNA is synthesized on DNA in the fibrillar centres. It is possible that the latter contain dispersed genetically active chromatin. These observations add further support to the hypothesis that fibrillar centres have a chromosomal origin and are related to the nucleolar organizers.

The fine structure of the nucleolus during interphase and mitosis in Ehrlich tumour cells cultivated in vitro.

Abstract Ehrlich tumour cells have been cultivated in vitro and their nucleoli have been studied by electron microscopy. During the interphase, the nucleoli contain fibrils (pars fibrosa), granules (pars granulosa) and areas of low electron density called ‘fibrillar centres’. The pars fibrosa and the pars granulosa disappear during prophase but the ‘fibrillar centres’ remain visible. The latter, associated with chromosomes, persist throughout the whole mitosis. In late telophase, these nucleolar bodies are situated inside the daughter nuclei, and, progressively become surrounded by the pars fibrosa and the pars granulosa. These morphological observations suggest that some nucleolar material is transmitted from one cell generation to the next. This material could be related to the nucleolar organizers.

Behaviour of nucleolus during mitosis. A comparative ultrastructural study of various cancerous cell lines using the Ag-NOR staining procedure.

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure. Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA topoisomerase I are located together. Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

Localization of DNA within Ehrlich tumour cell nucleoli by immunoelectron microscopy

The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach involving a monoclonal antibody directed against double‐ and single‐stranded DNA. Immunolabelling was performed either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The result seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.

Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors

Abstract: When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16‐fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 ± 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high‐affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low‐affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine‐deficient neuroblastoma cells.

Myosin, parvalbumin and myofibril expression in barbel (Barbus barbus L.) lateral white muscle during development.

Histo- and immunohistochemical techniques have recently been used to study the fibre type and myosin expression in fish muscle during development. In the present work, embryonic, larval and adult myosin isozymes (heavy and light chains) and parvalbumin isotypes were analyzed, from fertization to the adult stage, by polyacrylamide gel electrophoresis of barbel (Barbus barbus L.) trunk muscle extracts. The examined myosins display the sequential transitions from embryonic to larval and adult forms characteristic of higher vertebrates. They are characterized by specific heavy chains but their light chains differ only by the LC1/LC3 stoichiometry with LC3 exceeding LC1 after 10 days. Sarcoplasmic parvalbumins show considerable and unforeseen developmental transitions in their isotype distribution: the PA II isotype first appears after hatching and becomes the predominant form until the length reaches about 6 cm. One month after hatching, the amount of PA II then decreases and the synthesis of PA III and IV further increases to reach the typical adult pattern at a size of 18 cm. These observations show that the distribution of parvalbumin isotypes reflects the stage of development. It suggests a specific role for each isotype in relation to muscle activity. Microscopy illustrates the progressive development of somites, muscles cells, and myofibrils, which accelerates at hatching when movements increase.