Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). II. The oocyte nucleus of Blaps lethifera and Gnaptor spinimanus. Light optical data]

Changes in the nuclear structures and their participation in RNA synthesis in the growing oocytes were followed in two species of beetles Blaps lethifera and Gnaptor spinimanus. In the oocytes of both the species, the chromosomes join into the karyosphere following the short-term lampbrush stage. A large capsule appears around the karayosphere which consists of the fibrous substance, granules and karyosphere nucleoli. The latter form in the karyosphere and contain RNP but they are not true nucleoli since they do not include 3H-uridine. RNA synthesis on the chromosomes, active at the lampbrush stage, falls markedly following their joining into the karyosphere. The oocyte nuclei of these beetles are, thus, characterized by the absence of RNA synthesizing nucleolar system and, as compared with the trophocytes, by the low level of RNA synthesis on the chromosomes.     

Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.     

Characteristics of karyosphere formation in the lake frog. Light microscopy data

Morphological peculiarities of the oocyte nuclear organization were examined in R. ridibunda during winter and spring (February-March). Numerous nucleoli were seen to be assembled around regressive lampbrush chromosomes in the centre of the nucleus, and a central body was formed to which the chromosomes were attached. As result, a structural complex is constituted that involves a karyosphere and a capsule. Nucleoli are characterized by segregation and intensive fragmentation of their material. In result, a considerable part of nucleolar DNA is eliminated in the form of ring and polymorphous structures (micronucleoli). Besides the membranous component of nucleoli (nucleolar threads or tails) is lost. Towards the end of this period, nucleoli with complicated morphology become spherical again. The formation of the central body is started from the appearance of some small optically-light protein structures 5-20 nm in diameter (central body precursors-CBP). CBP are closely surrounded with ring micronucleoli to make intimate contact with the chromosomes and nucleolar threads. CBP commonly lie in one region of the nucleus not far from each other. The formation of a definitive central body obviously occurs due to a fusion of some small CBP. A conclusion is made of the nucleolar origin of the ring and polymorphous structures and of their essential role in the central body formation. The participation of chromosomal and eliminated nucleolar DNA in this process is discussed.     

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Argyrophilic proteins in Dinoflagellates: An ultrastructural,cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.     

Electron-microscopic study of silver staining of nucleoli in growing oocytes of rat ovaries

The nucleoli of dictyate-stage growing oocytes in rat ovaries were examined both with routine electron microscopy and electron microscopy after silver nitrate and ammoniacal silver nitrate (Ag-AS) staining. The nucleoli of the unilaminar follicular oocytes consist of twisted strands of dense fibrillar components, aggregates of granular components, and small fibrillar centers. After Ag-AS staining, silver grains are numerous on the dense fibrillar strands, fewer on the fibrillar centers, and very sporadic on the granular aggregates. The same stainability of three nucleolar components with the Ag-AS method was also confirmed in the nucleoli segregated by actinomycin D. During the transition of growing oocytes from bilaminar to plurilaminar follicle stage, the nucleolar dense fibrillar strands gradually conglomerate and are transformed into large and compact spherules. The stainability of dense fibrillar components with the Ag-AS method was lost along with this nucleolar transformation. These results may provide some new clues on the functional significance of Ag-AS-positive proteins in the nucleoli.     

Staining Plant Cells with Silver. I. The Salt-Nylon Technique

A technique is described for selectively silver staining nucleoli, active nucleolus organizers, nucleolar material attached to chromosomes, kinetochores, synaptonemal complexes, and chromosome cores in plant cells. The technique, called salt-nylon silver staining, involves spreading cells on glass slides, treating the cells with a solution of saline sodium citrate, and incubating the cells in a silver nitrate solution covered with nylon screen. Selected variables important for achieving reliable silver staining are considered.     

Funktionsstrukturen im Oocytenkern von Locusta migratoria

Examination of living oocyte nuclei of Locusta migratoria has revealed the presence of thread-like struktures. They are paired and are thought to be the uncoiled chromosomes since they are broken into fragments by treatment with DNase. The greater part of the threads carries lateral loops like the lampbrush chromosomes of Amphibia (Fig. 14). A smaller part has no loops hut bears a series of conspicious granules with bright appearance under positive phase contrast optics (pearl-string segments) (Fig. 2). — The visibility of the chromosomes has been investigated in solutions with several ions. In hypertonic media the chromosomes contract, the granules fuse, and the pearl-string segments become lumpy (Fig. 21). In nitrogenous atmosphere and if kept at low temperature the pearl-string structures are likewise transformed into a few lumps (Fig. 19). After return to normal conditions they reconstitute their characteristic beaded appearance. — In autoradiographs obtained by injection of H³-uridine into the body cavity and by incubation of isolated nuclei in vitro, a rather uniformly distributed labelling occurs over the oocyte nuclei up to 30 min incubation time (Fig. 23). With prolonged incubation the activity of the pearl-string segments becomes more intense than the labelling of the lampbrush chromosomes (Fig. 24). After treatment with actinomycin RNA synthesis is stopped, the pearl-string axes and the lampbrush chromosomes contract, and the granules disappear more and more (Figs. 25-28). — The pearlstring segments look very much like the nucleoli in the oocytes of Amphibia, where the nucleolar substance is likewise distributed as a series of beads of rather uniform size on an axis. Therefore, the pearl-string structures may have nucleolar function in Locusta too. If so, the only difference to the Amphibian nucleoli would be the continued attachment to the lampbrush chromosomes.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

Nucleolar organizer activity in wheat,rye and derivatives analyzed by a silver-staining procedure

Nucleolar activity was analyzed in wheat (Triticum sp.), rye (Secale cereale) and several types of wheat-rye derivatives using a modified, highly reproducible, silver staining procedure (Lacadena et al. 1984). A comparative analysis of the nucleolar organizer regions (NORs) of somatic metaphase chromosomes was made by phase contrast, C-banding, and silver staining. The frequency distribution of the number of nucleoli visualized at interphase by silver staining was also used to infer the activity of NORs. The results agree quite well with data from in situ hybridization reported by other authors. The behavior of euploid, ditelosomic and nulli-tetrasomic plants of common wheat showed the relative nucleolar activity of the four organizer chromosomes to be: 6B > 1B > 5D > 1A. — Several types of wheat-rye derivatives were analyzed: interspecific hybrid, triticale, addition and substitution lines, and plants with the genome constitutions, AABBDR, ABDR + 5D, ABRR, and ABRRR. In all cases the nucleolar organizer chromosome 1R of rye was suppressed by the presence of wheat chromosomes.