ULTRASTRUCTURAL LOCALIZATION OF NUCLEOLAR MATERIAL BY A SIMPLE SILVER STAINING TECHNIQUE DEVISED FOR PLANT CELLS

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1 J. Cell Sd. 79, (1985) 259 Printed in Great Britain The Company of Biologists Limited 1985 ULTRASTRUCTURAL LOCALIZATION OF NUCLEOLAR MATERIAL BY A SIMPLE SILVER STAINING TECHNIQUE DEVISED FOR PLANT CELLS SEIICHI SATO Department of Biology, Faculty of Science, Ehime University, Bunkyo-Macki 2-5, Matsuyama, Ehime 790, Japan SUMMARY A simple silver staining technique for use at the electron microscopic level, consisting only of treatment with aqueous silver nitrate at high temperature for a prolonged time, was applied to thin sections of root tip meristems of Viciafaba. This technique contrasted the fibrillar component and the granular component in interphase nucleoli as a reflection of the degree of packing. In contrast, silver impregnation was scarcely discerned in chromosomes. A comparison of silver staining and conventional double staining showed that the fibrillar centres did not always respond positively to silver. During the course from metaphase to late anaphase the nucleolus organizing secondary constriction was always seen as a heavily impregnated region and the electron density of the cytoplasm increased, probably due to dispersed nucleolar material. An argyrophilic substance began to accumulate on chromosomes in late anaphase. In the beginning of telophase a uniformly impregnated nucleolus was formed at the secondary constriction. It is concluded from these results that argyrophilic substance is associated with RNA-containing structures rather than DNAcontaining structures. The silver staining technique presented here is very convenient and favourable, especially for plant cells, to detect specifically the nucleolus organizing region and to survey the nucleolar material during mitosis at the electron microscopic level. INTRODUCTION The silver staining technique has been developed as a method for specific detection of the nucleolus organizing regions (NORs) on mitotic chromosomes (Goodpasture & Bloom, 1975; Howell, Denton & Diamond, 1975). The strong affinity of silver for both the NORs and the nucleolus has been attributed to acidic protein involved in the transcriptional activity of ribosomal rrna genes (Schwarzacher, Mikelsaar & Schnedl, 1978; Schmiady, Munke & Sperling, 1979). More recently, it has been proposed that silver impregnation does not always reflect the actual transcription of rrna genes because inactive NORs are also argyrophilic (Dimova et al. 1982; Medina, Risuefio, Sdnchez-Pina & Ferndndez-G6mez, 1983). The argyrophilic protein can be localized in a fibrillar centre and/or a fibrillar nucleolar component but not in a granular component (Bourgeois, Hernandez-Verdun, Hubert & Bouteille, 1979; Daskal, Smetana & Busch, 1980; Hernandez-Verdun, Hubert, Bourgeois & Bouteille, 1980; P6busque & Selte, 1981; Dimova et al. 1982; Paweletz & Risueno, 1982; Medina et al. 1983; Boloukhere, 1984). Cytochemical and autoradiographic techniques have shown that the fibrillar centres contain protein as their major component along with a small amount of DNA (Goessens, 1976; Mirre & Key words: argyrophilic protein, silver staining, RNA-containing structures, thin section.

2 Sato Stahl, 1978). It is generally accepted that the fibrillar component consists of a socalled 'Christmas-tree like transcriptional unit' (Jordan, 1984). Both the nucleolar structures contain DNA. It is therefore assumed that argyrophilic protein is associated with DNA-containing structures (Mirre & Stahl, 1981; Spector, Ochs & Busch, 1984). Compared with the original procedure for silver staining, a simpler and more highly reproducible technique at the light microscopic level has been devised for plant materials: the technique consists of chromosome spreading after enzyme maceration and treatment with a silver nitrate solution at high temperature (Hizume, Sato & Tanaka, 1980). Application of this technique to semi-thin sections of specimens embedded in resin led to good preparations for identifying the NOR and for pursuing the nucleolar material during mitosis at the light microscopic level (Sato & Shigematsu, 1985). This paper deau with the first application of this silver staining technique to thin sections at the electron microscopic level. The main aims of this study were as follows: (1) to examine the specificity of silver impregnation into the structural components of the nucleolus; (2) to survey the argyrophilic substance during mitosis; and (3) to evaluate the silver staining technique presented here as a new technique for study of the NORs and the nucleolar material. MATERIALS AND METHODS Plant cultivation Seeds of Viciafaba L. obtained from commercial sources were germinated in moist sawdust and the plants were grown in a constant-temperature incubator under dim light at 22 C. Actively growing lateral roots of the plants were used in the present study. Light microscopy Semi-thin sections of the root tips were made with an LKB Ultrotome from samples embedded in resin for electron microscopy as described below. These sections floating on water were dropped onto glass slides, then the water was removed by placing the slides on a hot-plate. The sections were then stained with silver as previously described (Sato & Shigematsu, 1985). Electron microscopy The root tips were fixed for 2 h in 4 % glutaraldehyde in 1/15 M-phosphate buffer (ph 7-0) but post-fixation in OsO* was omitted. They were embedded in resin according to Spurr's method (Spurr, 1969). Thin sections were subjected to the silver impregnation technique as follows. (1) Thin sections trapped on gold grids were floated on an aqueous AgNOj solution (1 gml~ ); (2) they were placed in a moist chamber for a predeterminate time (4 24 h) at 60 C; then (3) the grids were rinsed thoroughly in distilled water. Some root tips were post-fixed in OsC>4 after glutaraldehyde fixation and before they were embedded in Spurr's resin. Thin sections made as above were double stained with uranyl acetate and Reynold's lead citrate. All the thin sections were examined with a Hitachi HU-12 electron microscope at 100 kv. RESULTS When semi-thin sections were subjected to silver staining, the interphase nucleoli were found to consist of a heavily impregnated area surrounded by moderately impregnated area (Fig. 1). Silver-impregnated dots or masses were very frequently seen

3 Ultrastructural Ag-staining for plant cells 261 in the nucleoplasm, especially around the nucleoli. In metaphase the secondary constrictions were strongly impregnated with silver but no other structures showed strong affinity for silver (Fig. 2). An excessively prolonged treatment with silver gradually stained even the chromosomes so that neither the nucleolus nor the NOR could be differentiated from the chromosomes. The treatment time, therefore, is a critical factor in the silver staining technique presented here for specific detection of the NOR and the nucleolus. Thin sections were treated for 4, 8, 16 and 24 h at 60 C to determine an optimum treatment time for identification of the nucleolus and the NOR. In the specimens treated for both 4 and 8 h the nucleolus and the NOR developed only faint contrast. After 24 h incubation both the nucleolus and the NOR contrasted well with other cell structures, but appreciable numbers of silver grains were also distributed on the chromosomes. The most favourable pictures were obtained when the specimens were treated for 16 h. The following observations, therefore, were obtained from specimens treated for 16 h. Three ultrastructural elements, the fibrillar centre, the granular component and the fibrillar component, were easily discriminated in an interphase nucleolus that was double stained with uranyl acetate and lead citrate (Fig. 3). The adjacent section treated with silver staining showed that some of the fibrillar centres did not respond to silver so they appeared as electron-lucent holes, but others were clearly labelled with silver grains (Fig. 4). Each of these structures was enclosed by a strongly ". ' " : -"* ft. * <*3 ^ k Fig. 1. Silver stained, semi-thin section of root tip meristems of Vkiafaba. The nucleoli are found to consist of a heavily impregnated area surrounded by a moderately impregnated area. X2000. Fig. 2. Silver stained, semi-thin section showing metaphase chromosomes. The nucleolar organizing secondary constrictions are specifically impregnated with silver (arrows). X2000.

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5 Ultrastructural Ag-staining for plant cells 263 impregnated area, which was judged to correspond to the area consisting of the fibrillar component. On the other hand, the area consisting of the fibrillar component, probably due to its loose packing. The silver impregnation, therefore, seems simply to reflect packing density of the components. In prophase the nucleoplasm began to increase in electron density and the contour of the nucleolus became wavy, apparently as a sign of nucleolar disintegration. The dense area and the less dense area were still discernible in the nucleolus, especially in early and mid prophase (Fig. 5). In some instances the dense area was found to be directly combined with chromosomes. With the progress of prophase, this area became less dense and reduced in size as a result of its loosening and dispersion. When the mitotic chromosomes were stained with the conventional double staining, the nucleolus organizing secondary constriction appeared less dense than the chromosomes and had a fibrillar texture (Fig. 6). The secondary constriction was specifically impregnated with silver (Fig. 7). Magnification revealed that the silverimpregnated region contained a large number of silver grains (Fig. 8). To determine the size and appearance of the silver grains, the picture was further magnified. Most silver grains in the cytoplasm and on the chromosomes were less than 5 nm in diameter while those on the secondary constriction mostly fell into the range from 5 to 10 nm. Accordingly, it is apparent that specific impregnation with silver reflects both high density and enlargement of silver grains. In early anaphase no specific silver impregnation was discerned in the spindle except for the secondary constrictions. But the electron density of the cytoplasm was appreciably higher than in interphase. This increase in electron density of the cytoplasm was apparently due to the nucleolar material. A silver-impregnated substance gradually accumulated on the surfaces of the chromosomes during the course of late anaphase (Fig. 9). It is very probable that this argyrophilic substance came from the previously dispersed nucleolar material within the spindle. At the beginning of telophase the argyrophilic substance filled the spaces between the chromosomes (Fig. 10). This substance usually had a mesh-like appearance due to the peripheral dispersion of the chromosomes. The new nucleolus became visible in the secondary constriction. However, no differentiation of the three nucleolar components was discerned in this nucleolus, which was instead uniformly impregnated with silver. DISCUSSION Recently, data on the localization of argyrophilic protein at the electron microscopic level have been accumulated but no general agreement on the nature of Fig. 3. An interphase nucleolus double stained with uranyl acetate and lead citrate. Three nucleolar components, a granular component (g), a fibrillar component (/) and fibrillar centres (arrows), are seen. X Fig. 4. Silver stained thin-section adjacent to the section of Fig. 3. Both the granular component (g) and the fibrillar component (/) show a strong affinity for silver, while silver impregnation scarcely occurs at all in the fibrillar centres and chromosomes. X50000.

6 264 S. Sato JB Figs 5 8. For legends see p. 266

7 Ultrastructural Ag-staining for plant cells Figs 9, 10. For legends see p. 266

8 266 S. Sato the ultrastructural component with an affinity for silver has been obtained. These data can be summarized into three categories as follows: (1) fibrillar centre only, (2) fibrillar component only, and (3) fibrillar centre and fibrillar component. The fibrillar centre is known to be composed of a large amount of proteins and a small amount of DNA (Goessens, 1976; Mirre & Stahl, 1978), and the fibrillar component is believed to contain the transcriptional unit. The data above suggest that the argyrophilic substance is associated with DNA-containing structures (Mirre & Stahl, 1981; Spector et al. 1984). Under both the light and electron microscope the silver staining technique presented here could differentiate the fibrillar component from the granular component according to the degree of silver impregnation: the fibrillar component was most heavily impregnated and the granular component was moderately impregnated, but the chromosomes did not show any affinity for silver. The preliminary examination with the light microscope revealed that when the specimens were treated for an excessively prolonged time even the chromosomes gradually became impregnated with silver; eventually they became completely black as did the nucleolus. The staining specificity of the present technique, therefore, depends on the treatment time at the desired temperature. This implies that the rate of the silver staining reaction is faster in the fibrillar component than in the granular component. However, comparison of conventional double staining with silver staining suggests that silver impregnation of the fibrillar and granular components reflects their degree of packing instead of the rate of the silver staining reaction because the area consisting of the granular component is rather rough in texture. The present study suggests that the fibrillar centres can be classified into two categories because one responds to silver but the other does not. The unsettled response to silver on these structures may be due to another reason. The fibrillar centres very frequently located within the electron-lucent spaces. When silver staining was applied, these spaces were actually revealed as transparent holes. The location of the fibrillar centres in the control section may not always ensure that Fig. 5. Silver stained thin-section of a prophase nucleolus. The nucleolus has an irregular shape and the nucleoplasm is electron dense. Note that a strongly impregnated region is directly attached to a chromosome (ch) at the site between the arrows. X Fig. 6. Secondary constriction revealed by conventional double staining. The secondary constriction consists of fibrillar material. X Fig. 7. Silver stained thin-section showing a part of a metaphase cell. The secondary constriction appears as a silver impregnated region, but specific impregnation was not discerned in any other structures. X7800. Fig. 8. Magnified picture of the secondary constriction in Fig. 7. Specific silver impregnation in the secondary constriction proves to result from a great number of silver grains. X Fig. 9. Silver stained thin-section of a cell in late anaphase. An argyrophilic substance has begun to accumulate on the chromosomes. The secondary constriction (sc) is also argyrophilic. X Fig. 10. Silver stained thin-section of a cell in early telophase. The new nucleolus (nu), which is uniformly impregnated, has become visible in the secondary constriction. X

9 Ultrastructural Ag-staining for plant cells 267 they are also detected in the adjacent section. It is possible that the adjacent section goes across the electron-lucent spaces. A substance responding positively to preferential ribonucleoprotein (RNP) staining begins to accumulate on the surface of chromosomes at late anaphase in the mitotic meristems of this plant (Sato & Shigematsu, 1985). This substance was preferentially impregnated with silver by the present technique. The silver staining technique also contrasted the NORs on the mitotic chromosomes as silverimpregnated regions. NORs are also known to stain positively with the RNP preferential staining technique (Sato & Shigematsu, 1985). It is therefore probable from these results that the argyrophilic substance is associated with an RNAcontaining structure rather than with a DNA-containing structure. This conclusion parallels observations in HeLa cells, in which the nucleolar sheath associated with the mitotic chromosomes as well as the NOR is argyrophilic, suggesting that the protein part of the RNP is responsible for the silver impregnation of the nucleolus (Paweletz & Risuefio, 1982). As inhibition of rrna synthesis does not alter the amount of argyrophilic protein compared with that in untreated cells, the argyrophilic protein is probably involved in the processing or packaging of rrna (Dimova et al. 1982; Hubbell, Lau, Brown & Hsu, 1980). If some of this protein is fortuitously trapped in the NOR during chromosome condensation, the NOR will become visible as a silver-impregnated region on the mitotic chromosome. Two argyrophilic proteins, B23 and C23, have been isolated from the nucleoli of Novikoff hepatoma cells (Lischwe, Smetana, Olson & Busch, 1979). Immunofluorescence studies have shown that phosphoprotein C23 is the major silver staining protein of the nucleolus and is associated with DNA-containing structures (Ochs & Busch, 1984; Smetana et al. 1984). A silver staining study on the spread transcriptional unit from nucleoli ascertained that the NOR proteins preferentially located on the DNA axis rather than on the RNP fibrils, suggesting that the silver staining protein may be associated with RNA polymerase I (Angelier, Hernandez- Verdun & Bouteille, 1982). The isolation of an.argyrophilic protein with a molecular weight of , the same as that of RNA polymerase I, would support this concept (Williams, Kleinschmidt, Krohne & Franke, 1984). This protein may remain in contact with the NOR so that the resumption of rrna synthesis can be immediate and efficient (Hubbell et al. 1980). It has been found that other argyrophilic protein, B23, is associated with RNA-containing structures (Smetana et al. 1984; Spector et al. 1984). Comparison of silver staining and immunostaining for protein B23 suggests that the more weakly silver-stained region of the nucleolus might contain protein B23 (Smetana et al. 1984). From these data it is possible to surmise that the proteins C23 and B23 are responsible for silver impregnation of the fibrillar and the granular component, respectively. The molecular weight of protein C23, however, is only , which is appreciably smaller than that of RNA polymerase I (Ochs & Busch, 1984). Thus the relationship of the argyrophilic protein supposed to be associated with DNA-containing structures to protein C23 or to RNA polymerase I still remains unknown. Also, no argyrophilic protein B23 has been identified in an isolated granular component.

10 268 S. Sato In the previously reported techniques of silver staining at the electron microscopic level the pieces of tissue or cell aggregates were first subjected to a silver staining procedure consisting of fixation, treatment with silver nitrate and immersion in developing solution; then they were embedded in resin for electron microscopy (Bourgeoisesal. 1979; Daskal e*al. 1980; Hernandez-Verdunetal. 1980; Pe"busque & Seite, 1981; Dimovae* al. 1982; Paweletz & Risuefio, 1982; Medina et al. 1983; Boloukhere, 1984). The silver staining technique presented here differs in that the treatment with silver nitrate is applied to the specimens after they have been embedded and sectioned. This technique seems to be favourable for plant cells since plant tissue does not well absorb reaction solutions such as aqueous silver nitrate and developing solution due to the presence of the cell wall. As described above, the staining specificity of the present silver staining technique is different from that of the previously reported ones. It seems that fixation plays an important role in staining specificity. In the previously reported techniques, post-fixation with acetic ethanol is used after fixation with glutaraldehyde or formaldehyde, while postfixation is not used in the technique presented here. It has been proposed that the silver staining reaction may not proceed until silver staining regions are unmasked by acetic ethanol, and alternatively, some residues may be methylated under the acidic conditions permitting the silver reaction to occur (Daskal et al. 1980). The preliminary experiment at the light microscopic level showed that when the samples were fixed with acetic ethanol the nucleolar material in the interphase nucleoli tended to lose their affinity for silver while the NORs on the mitotic chromosomes were always detected as silver impregnated regions. These results suggest that the specificity of the silver staining is critically influenced by fixation. The effects of pretreatment with various fixatives and other reagents on the specificity of the silver staining technique presented here is now under investigation. I thank Professor N. Sawada for permission to use the electron microscope and Miss Y. Shimokawa for her assistance in operating the electron microscope. REFERENCES ANGELIER, N., HERNANDEZ-VERDUN, D. & BOUTEILLE, M. (1982). Visualization of Ag-NOR proteins on nucleolar transcriptional units in molecular spreads. Chromosoma 86, BOLOUKHERE, M. (1984). Ultrastructural localization of nucleolar organizers during oogenesis in Xenopus laevis using a silver technique, jf. Cell Set. 65, BOURGEOIS, C. A., HERNANDEZ-VERDUN, D., HUBERT, J. & BOUTEILLE, M. (1979). Silver staining of NORs in electron microscopy. Expl Cell Res. 123, DASKAL, Y., SMETANA, K. & BUSCH, H. (1980). Evidence from studies on segregated nucleoli that nucleolar silver-staining proteins C23 and B23 are in the fibrillar component. Expl Cell Res. 127, DMOVA, R. N., MARKOV, D. V., GAJDARDJIEVA, K. C, DABEVA, M. D. & HADJIOLOV, A. A. (1982). Electron microscopic localization of silver staining NOR-proteins in rat liver nucleoli upon D-galactosamine block of transcription. Eur.J. Cell Biol. 28, GOESSENS, G. (1976). High resolution autoradiographic studies of Ehrlich tumour cell nucleoli. Nucleolar labelling after [ 3 H]actinomycin D binding to DNA or after [ 3 H]TdR or [ 3 H]uridine incorporation in nucleic acids. Expl Cell Res. 100,

11 Ultrastructural Ag-staining for plant cells 269 GOODPASTURE, C. & BLOOM, S. E. (1975). Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53, HERNANDEZ-VERDUN, D., HUBERT, J., BOURGEOIS, C. A. & BOUTEILLE, M. (1980). Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures. Chromosoma 79, HIZUME, M., SATO, S. & TANAKA, A. (1980). A highly reproducible method of nucleolus organizing regions staining in plants. Stain Technol. 55, HOWELL, W. M., DENTON, T. E. & DIAMOND, J. R. (1975). Differential staining of the satellite regions of human chromosomes. Experientia 31, HUBBELL, H. R., LAU, Y.-F., BROWN, R. L. & Hsu, T. C. (1980). Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells. Expl Cell Res. 129, JORDAN, E. G. (1984). Nucleolar nomenclature. J. Cell Set. 67, LISCHWE, M. A., SMETANA, K., OLSON, M. O. J. & BUSCH, H. (1979). Proteins C23 and B23 are the major nucleolar silver staining proteins. Life Set. 25, MEDINA, F. J., RISUENO, M. C, SANCHEZ-PINA, M. A., FERNANDEZ-G6MEZ, M. E. (1983). A study on nucleolar silver staining in plant cells. The role of argyrophilic proteins in nucleolar physiology. Chromosoma 88, MiRRE, C. & STAHL, A. (1978). Peripheral RNA synthesis of fibrillar center in nucleoli of Japanese quail oocytes and somatic cells. J. Ultrastruct. Res. 64, MTRRE, C. & STAHL, A. (1981). Ultrastructural organization, sites of transcription and distribution of fibrillar centres in the nucleolu* of the mouse oocytes. jf. Cell Sci. 48, OCHS, R. L. & BUSCH, H. (1984). Further evidence that phosphoprotein C23 (HOKD/pI 5-1) is the nucleolar silver staining proteins. Expl Cell Res. 152, PAWELETZ, N. & RISUENO, M. C. (1982). Transmission electron microscopic studies on the mitotic cycle of nucleolar proteins impregnated with silver. Chromosoma 85, PEBUSQUE, M.-J. & SETTE, R. (1981). Electron microscopic studies of silver-stained proteins in nucleolar organizer regions: Location in nucleoli of rat sympathetic neurons during light and dark periods. J. Cell Sci. 51, SATO, S. & SHIGEMATSU, T. (1985). Light and electron microscopic study on the behavior of the nucleolar material during mitosis in root tip meristems of Vtciafaba. Caryologia 38 (in press). SCHMIADY, H., MUNKE, M. & SPERLING, K. (1979). Ag-staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity. Expl Cell Res. 121, SCHWARZACHER, H. G., MTKELSAAR, A.-V. & SCHNEDL, W. (1978). The nature of the Ag-staining of nucleolus organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis and meiosis. Cytogenet. Cell Genet. 20, SMETANA, K., OCHS, R., LISCHWE, M. A., GYORKEY, F., FREIREICH, E., CHUDOMEL, V. & BUSCH, H. (1984). Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes. Expl Cell Res. 152, SPECTOR, D. L., OCHS, R. L. & BUSCH, H. (1984). Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23. Chromosoma 90, SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, WILLIAMS, M. A., KLEINSCHMIDT, J. A., KROHNE, G. & FRANKE, W. W. (1984). Argyrophilic nuclear and nucleolar proteins of Xenopus laevis oocytes identified by gel electrophoresis. Chromosoma 90, (Received 30 April - Accepted 1 July 1985)

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