The fine structure of the nucleoli of normal and actinomycin D-treated Amoeba proteus

© 1968 by Academic Press Inc.

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The Fine Structure of the Nucleoli of Normal and Actinomycin D-Treated Amoeba proteus CHARLES J. FLICKINGER

Institute for Developmental Biology, University of Colorado, Boulder, Colorado 80302 Received February 8, 1968 The fine structure of the nucleoli of normal and actinomycin D-treated Amoeba proteus was studied utilizing fixation in Karnovsky's glutaraldehyde-formaldehyde mixture. The multiple nucleoli of normal cells had a predominately granular substructure. Although some regions appeared to possess a fibrillar component as well, discrete areas of differentiated substructure were not identified. Nucleoli of actinomycin-treated cells first became more dense and regularly spherical and then exhibited segregation of nucleolar components. The center of the nucleoli was composed of ~ 50 A fibrils and was surrounded by a rim of 100-150 A dense granules. Peripheral regions contained low density material mainly of an amorphous character. In cells treated with actinomycin for 3-4 days, nucleolar vacuoles became common. The similarity in the response of the presumed nucleoli of amoebae to that reported for many higher cells is discussed in terms of possible structural and functional analogies between the nucleoli of amoebae and other cells. It is suggested that normal amoeba nucleoli possess fine fibrils and granules characteristic of other nucleoli, and that the unusually uniform fine structure commonly observed is due to the close packing and intermixing of these components. The nuclei of many protozoa contain multiple, roughly spherical, electron-dense bodies with a predominately granular substructure, which have been referred to generally as nucleoli (22). It has been uncertain, however, if they are functionally analogous to the nucleoli of higher cells (16, 31). This uncertainty is due, at least in part, to morphological differences between the structures presumed to be nucleoli in protozoa and the typical organelle of most higher cells. In Amoeba, for example, the presumed nucleoli differ in several respects from the nucleoli of multicellular organisms. First, nucleoli are more numerous than in most other cells (6, 19, 22, 27), with the notable exception of amphibian oocytes (20). Second, typical nucleoli of both animals (1, 8) and plants (17, 18) possess distinct regions occupied by 150 granules and 50-80 A fibrils. In some cases, a variable amount of amorphous material and/or intranucleolar vacuoles are present as well. Amoeba nucleoli, on the other hand, have usually been described as mainly granular, lacking definite areas

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o~ differentiated substructure (6, 19, 27, 31), although such regions have been reported in a few other species of protozoa (7, 28). Third, the components of the nucleoli in mammals (8) and possibly plants (5) are organized into a system of anastomosing cords, the nucleolonema. Amoeba nueleoli, however, are usually solid spheres (6, 19, 27, 31). Cytochemical and radioautographic evidence suggests that, in spite of these morphological differences, the nucleoli in amoebae have chemical and functional similarities to those of higher cells. They are Feulgen negative (4), sensitive to ribonuclease (3, 31), stain cytochemically for R N A (11), and incorporate tritiated uridine (31). These characteristics are common to the typical nucleoli of higher cells (1, 2,

32). Actinomycin D is known to alter significantly the fine structure of the nucleolus in a variety of cells (9, 10, 12-14, 25, 29, 30, 31). If the structures presumed to be nucleoli in Amoeba proteus are analogous to the nucleoli of other organisms, they would be expected to respond to actinomycin D in a similar fashion. The results of the present study indicate that this is the case. In addition, the ultrastructural changes following actinomycin treatment elucidate some details of the fine structure of normal amoeba nucleoli. MATERIALS AND METHODS Cultures of Amoeba proteus were maintained in Prescott's medium (23) with feedings of washed Tetrahymena. Cultures did not receive food organisms for the 2 days preceding the experiment. Some cells from such a stock culture were prepared for electron microscopy and are designated normal or untreated amoebae. The remaining cells were divided into two groups. One was placed in amoeba medium containing 100 ~g of actinomycin D per milliliter (courtesy of Merck, Sharp and Dohme). The second control group was placed in normal amoeba medium. The concentration of actinomycin is greater than is used with most other cell types, but this has been found necessary to suppress completely RNA synthesis in Amoeba proteus (31). Both actinomycin-treated and control organisms were maintained at 21°C without food organisms. Samples of cells were prepared for electron microscopy after 1, 2, 3, and 4 days. The experiment was discontinued on the fifth day because most of the actinomycin-treated amoebae had died. Samples were fixed for 1 hour at room temperature in Karnovsky's glutaraldehydeformaldehyde mixture (15) at pH 7.3. Cells were washed overnight in distilled water and postfixed for 30 minutes in 1% OsO4 in 0.1 M cacodylate buffer at pH 7.3. The amoebae were then briefly rinsed, dehydrated in a graded series of ethanols followed by propylene oxide, and embedded in Araldite. Cells were collected by centrifugation between steps. Sections showing silver to pale gold interference colors were cut on a Porter-Blum-MTq microtome equipped with a diamond knife. The specimens were mounted on uncoated copper grids and stained with lead citrate. (33). Micrographs were obtained with a Philips EM-300 electron microscope operated at 60 kV.

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RESULTS

Untreated amoebae Sections of nuclei of untreated amoebae (Fig. 1) contain up to 10-15 round or irregularly shaped electron-dense bodies 1-3 # in diameter, which have been referred to as nucleoli by previous authors (22). They are associated with multiple smaller masses of similar material 0.2-0.6 # in diameter. The texture of the normal nucleoli (Fig. 2) is mainly granular, but some regions, especially near the periphery of the nucleolus, have somewhat greater density and exhibit a fibrillogranular substructure (Fig. 2, arrow). However, clearly defined separate areas composed predominatly of either granules or fibrils were not identified. In most cases, the nucleoli are arranged around the periphery of the nucleus, immediately inside the honeycomb layer of the nuclear envelope (Fig. 1). Much of the interior of the nucleus is occupied by ~ 100 A fibrils, identified as chromatin. In addition, the interior of the nucleus contains the characteristic helices (21, 31) and "granular masses" (31) that have been described previously.

Actinomycin-treated amoebae In cells treated with actinomycin for 1 day, the density of the nucleoli is increased and they possess more regular circular profiles (Fig. 3), as has been reported for amoebae treated in a similar fashion for 6 hours (31). The ~ 100 A fibrillar material of the nuclear interior, identified as chromatin, is condensed into clumps of greater density than in untreated cells (Fig. 3). In addition, some nucleoli possess separate areas differing in their substructure (Fig. 4). The center of the nucleolus is most dense and contains ~ 50 A fibrils. This fibrillar region is surrounded by a moderately dense, discontinuous rim composed primarily of 100-150 A granules. At the periphery of the nucleoli there are usually several smaller regions that are less dense than the granular or fibrillar regions. These peripheral patches usually have an amorphous texture, but in some (Fig. 6) there is a suggestion of a fibrillar substructure. Nucleoli and condensed chromatin tend to aggregate in cells treated with actinomycin for two or more days (Fig. 5). They frequently form a band either in the center or toward one side of the nucleus. The rest of the nucleus, in contrast, contains only sparse fibrillar and finely granular material that may represent dispersed chromatin and nuclear sap. As the length of actinomycin treatment is increased, these nuclear changes become FIG. 1. Nucleus of an untreated amoeba. The main nucleoli (N) and many small masses of nucleolar material (n) are distributed along the periphery of the nucleus, immediately inside the prominent honeycomb layer (h) of the nuclear envelope (E). The interior of the nucleus contains chromatin (C) and "granular masses" (m) (31). × 10,000.

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F~G. 2. Nucleolus of an untreated amoeba. The texture of the normal nucleolus is predominately granular, but some denser regions at the periphery (arrow) may contain more of a fine fibrillar component. × 29,500. m o r e p r o n o u n c e d . The s e p a r a t i o n of nucleolar c o m p o n e n t s , especially is accentuated (Figs. 6-8). The fibrillar p o r t i o n continues to o c c u p y the center of the nucleolus a n d is a l m o s t c o m p l e t e l y encircled by a g r a n u l a r r i m (Figs. 6 a n d 7). These two regions are a r r a n g e d in a similar fashion in the small masses of nucleolar m a t e r i a l as well (Figs. 6 a n d 7). M a n y small vacuoles are present in the nucleoli of cells treated with a c t i n o m y c i n for 3-4 days (Fig. 8), a n d some nucleoli display a large central vacuole (Fig. 6). The interior of the vacuoles is occupied b y m a t e r i a l t h a t is indistinguishable f r o m t h a t of the b u l k of the s u r r o u n d i n g nucleoplasm. A l t h o u g h i n t r a n u c l e o l a r vacuoles were occasionally observed in u n t r e a t e d a m e b a e , they are enc o u n t e r e d m o r e frequently in a c t i n o m y c i n - t r e a t e d cells. FIG. 3. A portion of the nucleus of an amoeba treated with actinomycin for 1 day. The nucleoli (N) are more condensed and spherical than those of untreated cells. C, condensed chromatin; m, granular mass. × 20,000. FIG. 4. Nucleoli an amoeba treated with actinomycin for 1 day. Central areas of fine fibrils (f) are partially surrounded by granular regions (g). Peripheral patches of lower density material (p) are present. A portion of the honeycomb layer is visible (h). × 19,000.

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FIG. 5. Nucleus of a cell treated for 2 days with actinomycin. The nucleoli (N) and chromatin (C) have aggregated leaving only material of low density in the remainder of the nucleus. × 11,000.

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FIG. 6. Amoeba treated for 4 days with actinomycin. A large nucleolus exhibits central fibrillar ( f ) and peripheral granular (g) regions as well as a large central vacuole. (V). Small masses of nucteolar material (n) have a similar regional differentiation, p, peripheral patch; m, granular mass. × 26,000.

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Helices are absent in cells treated with actinomycin, but the "granular masses" (31) are frequently observed in proximity to nucleoli (Figs. 3, 4, and 6) as previously noted in amoebae treated for 6 hours with actinomycin (31).

Control amoebae Amoebae maintained for 1-5 days in normal medium without actinomycin exhibit nuclei that are similar to those of the normal cells from stock cultures. They do not undergo the changes described for amoebae treated with actinomycin. DISCUSSION The bulk of the nucleoli of actinomycin-treated amoebae is made up of two readily differentiated regions, composed respectively of fine fibrils and ~ 150 A dense granules. Similar granular and fibrillar regions in the nucleoli of other cells contain R N A (1, 32). Some difficulty, however, is encountered in the description and identification of the low density peripheral patches. Their density and texture corresponds most closely to that of the amorphous material of other nucleoli, which has been shown by enzymatic digestion to be composed primarily of protein (1). However, in some micrographs, these areas appear to be composed of fibrils similar to those of the chromatin and may represent moderately condensed nucleolus-associated chromatin. Unfortunately, precise identification of this material cannot be made on the basis of the present study. Actinomycin D inhibits DNA-dependent RNA synthesis by binding to guanine in D N A (24). Correlated changes in the fine structure of the nucleolus in different cell types include the segregation of nucleolar components (1, 10, 13, 25, 29) and, in some cases, a decrease in size, increased density, vacuolization, fragmentation, and blebbing or budding (9, 10, 12, 14, 26, 29, 30). The most consistent morphological effect of actinomycin is the separation of nucleolar areas of 70-90 A fibrils, 150 A granules, amorphous material, and chromatin (1). The similarity of the response of the nucleoli in Amoeba proteus to actinomycin D suggests that they are similar in their function to the nucleoli of these other cell types. Under the influence of actinomycin, amoeba nucleoli display to advantage components similar to those of metazoan and plant cells. This suggests that the relatively uniform fine structural appearance of amoeba nucleoli in routine preparations is due FIG. 7. Nucleolus of an amoeba treated with actinomycin for 4 days. A central area of fine fibriUar material (f) is almost completely surrounded by a rim of material containing 100-150 A granules (g). p, peripheral patch. × 46,000. FIG. 8. The nucleolus of a cell treated for 4 days with actinomycin contains several small vacuoles (v). Granular (g) and fibrillar (f) regions can be differentiated. × 36,000.

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to a high degree of intermixing and close packing of granules and fibrils such that the character of the nucleolar substructure is obscured. In favorable micrographs of normal amoeba nucleoli, differences in texture of different parts of the same nucleolus can be detected (Fig. 2). In view of the results of actinomycin treatment, these regional differences may be due to the greater concentration of granules in one region and of fibrils in another, although the components remain intermixed to a greater extent than is the case in most other cells. In particular, the denser regions (Fig. 2) appear to contain a higher concentration of the fibrillar component than the rest of the nucleolus. In summary, the following suggestions are made: (a) normal nucleoli of Amoeba proteus contain fine fibrillar and the granular components similar to those of most higher ceils; (b) these components are not obvious in normal amoebae because they are intermixed and packed together very closely; and (c) the existence of these components is demonstrated by treating ceils with actinomycin, which promotes their segregation into discrete areas. This study was supported by a grant from the American Cancer Society (E-434), D. M. Prescott, principal investigator, and Program project HD-02282 of the National Institutes of Health. The author wishes to thank Dr. A. R. Stevens and Dr. D. M. Prescott for their helpful suggestions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8, 9. 10.

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