sion and scanning electron microscopy using different methods of fixation. ... electron microscope as single units or as compound structures consisting of two to ...
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Scanning electron microscopy of the nuclei and nucleoli in Tetrahymena V. LEICK,' B. BRO' and A. LIMA-DE-FARIA* Biochemical Institute B, Panum Institute, Copenhagen, Denmark Institute of Molecular Cytogenetics, University of Lund, Sweden
LEICK.V., BRO. B. and LIMA-DE-FARIA, A. 1979. Scanning electron microscopy of the nuclei and nucleoli in Tetrahymena. - Hereditas 90: 219-226. Lund, Sweden. ISSN 0018-0661. Received November 20, 1978 The macronucleus and the micronucleus of Terrahyrnena thermophila have been analysed by transmission and scanning electron microscopy using different methods of fixation. The micronucleus is frequently found located in a concave depression of the macronucleus. There is good agreement between the observations made with transmission and scanning electron microscopy with respect to the size and shape of the two types of nuclei. The nucleoli have been isolated from the macronucleus and analysed in the scanning electron microscope. Three types of fixatives have been used; osmium, formaldehyde and acetic-alcohol. All three produced similar results. There is also good agreement between the size and shape of the nucleoli observed with the transmission and scanning electron microscopy. The nucleoli appear in the scanning electron microscope as single units or as compound structures consisting of two to three elements. They have a structure with indentations similar to those found in Acheta. In both Terrahyrnena and Achefa there is amplification of ribosomal DNA. The arrangement of the DNA copies within the nucleolus of the two species is compared and correlated with the amplification process.
A . Lima-de-Faria, Insriture of Molecular Cyrogenelics, S-223 63 Lund, Sweden
Of all eukaryotic genes the cistrons for 18s and 28s ribosomal RNA are among those that have been the most extensively studied. Their number and location have been determined in many species (BIRNSTIEL et al. 1971). The order and length of these DNA sequences have been established (DAWID et al. 1970; BROWand BLACKL E R 1972) and the same sequences have been observed in the transmission electron microscope during transcription (MILLERand BEATTY1969; TRENDELENBURG et al. 1976). The scanning electron microscope has mainly been used to study the cell surfaces of animal and plant species and, in a few cases, structures localized inside the cell. Amplification of the genes for 28s and 18s ribosomal RNA occurs in the cricket Acheta. The degree of amplification and the location of these genes have been established by RNA-DNA hybridization (LIMA-DE-FARIA et al. 1969; PERO et al. 1973; ULLMAN et al. 1973). The amplified ribosomal genes together with the ribosomal RNA and protein form a major chromomere on chromosome 6 of Acheta. A technique was developed which
permitted to isolate this gene complex and to analyse it by means of the scanning electron microscope. The ribosomal DNA gene complex forms a spherical structure. The periphery of this structure consists of polygonal plates formed by RNA and protein. The DNA copies terminate at the vertices of the polygonal plates as revealed by a comparison of the results obtained by transmission and scanning electron microscopy (LIMA-DEFARIA 1974a and b). The ribosomal genes of the protozoan Tetrahymena are located in numerous nucleoli which are arranged on the periphery of the macronucleus (NILSSON and LEICK1970; LEicK 1973). This occurs during the exponential growth phase. These genes appear as free ribosomal DNA molecules which form giant palindromes containing two genes for pre-ribosomal RNA per ribosomal DNA molecule (ENGBERG et al. 1976; KARRER and GALL1976). The ribosomal DNA can be labelled preferentially during a nutrional shift-up (ENGBERG et al. 1974). Furthermore, the number of palindromic ribosomal DNA molecules is about lo4 per macronucleus yielding an average of 20
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and 10 h at 20°C). To the pellet obtained after gentle centrifugation 70% alcohol was added and the nuclei were stored at 4°C. Preparations for the scanning microscope were made by directly pipetting the nuclei on to a circular cover-slip (diameter 12 mm) which rested on a slide. A second circular cover-slip was laid over the nuclei and the material squashed between the two cover-slips. The slide was placed over dry ice and after a few minutes the upper cover-slip was removed and discarded. The lower cover-slip was immersed in 95% alcohol, and later in 100% alcohol and finally dried in air. This cover-slip was cemented to the microscope holder. The preparations were coated with gold-palladium and the rest of the procedure was as described by L1MA-m FARIA (1974b). For transmission electron microscopy isolated Cultures of Tetrahymena [hermophila, strain BIV, nuclei or whole cells were fixed at room temperawere grown in a complex peptone medium (NILS- ture in 4 % glutaldehyde in 0.1 M cacodylate SON and LEICK 1970). Exponentially growing cells buffer (pH 7.4) for 10 min followed by post-fixawere harvested by centrifugation at 500 xg for 5 tion in I % osmium tetroxide in the same buffer for min, resuspended in a buffer mixture containing 1 h, or with the omission of the last step. After a 0.01 M Tris pH 7.5,O.OOl M MgClz, 0.003 M CaCI, brief rinse in the buffer, the cells or nulei were and 0.25 M sucrose and lysed in 0.15% Triton-X- dehydrated in a graded series of ethanols followed 100 (final concentration). Nuclei in the lysate were by propylene oxide and were embedded in epon pelleted at 5 0 0 xg for 5 min and immediately fixed according to LUFT(1961). After sectioning, the in the appropriate fixation solution. Three types of double-fixed material was post-stained for 40 min fixatives were used for scanning electron in zinc-uranyl acetate (WtiNsIFIN et al. 1963) and microscopy: L C COGGFSHAI I for 3 min in lead citrate ( V ~ N A B and ( I ) Concentrated acetic acid - absolute alcohol 1965). 1:3, (2) Palade’s buffered osmium tetroxide fixative, and (3) formaldehyde fixation (PEASE 1960). After fixation in acetic acid - alcohol the nulei were left in the fixative for 6 days at 4°C to harden the nuclei. Fixation in osmium was carried out by adding an equal volume of the osmium fixative to When using scanning electron microscopy it is the solution containing the nuclei and leaving for advantageous to have a previous knowledge of the structures studied in the transmission electron 10 min at 4°C. A pellet was obtained after gentle centrifugation and the osmium fixative was added microscope. This is a valuable control as it gives to the pellet to fix the nuclei completely (on ice). the natural position of the different cell and nuclear components and their structural arrangeThe nuclei were kept at 4°C. In a variant of this last method, the nuclei were ment. The size and location of the nucleoli in the fixed for 2 h in Palade’s fixative, and were then macronucleus of T . thermophilo is seen in Fig. 1 partly dehydrated (alcohol series) and kept in 70% and 3. From previous studies it is known that the macronucleus is 6 to I I microns in diameter and alcohol at 4°C. Fixation in formaldehyde took 12 h (2 h at 4°C the micronucleus I .5 to 1.8 microns.
ribosomal DNA molecules per nucleolus (LEICK unpublished). In this study the nuclei and the nucleoli of Tetrahymena were isolated for analysis in the scanning electron microscope. The location of the micronucleus relative to the macronucleus and the three-dimensional structure of the nucleoli is revealed by this procedure. There are similarities and differences between the nucleolar structures of Acheta and Tetrahymena. The differences are mainly due to the fact that the two species have been studied at different stages of DNA amplification.
Materials and methods
Results
Fig. 1-3. Transmission electron microscopy. -Fig. I . Part of a macronucleus of Terrahymena. The nucleoli are in contact with the nuclear envelope. Note the typical irregular profile of the nuclear membrane. x 34,000. - Fig. 2. Part of macronucleus showing the micronucleus located in a depression of the macronuclear membrane. x 25,000. -Fig. 3. Macronucleus of Tetrahymena rhermophilo in the exponential growth phase. Numerous small nucleoli are situated in contact with the nuclear envelope. The central part of the nucleus is occupied by the chromatin granules which form small dark dots. Note the undulated nature of the nuclear envelope. x 14,000.
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Fig. 4-10. Scanning electron microscopy. -Fig. 4 and 5 . The macronucleus of Tefrahymena in the exponential growth phase flattened by the squash procedure. The mass of chromatin granules form an elevation in the central part of the macronucleus. There is a circular depression around the chromatin followed by an outer elevation in which some of the nucleoli are harboured. The micronucleus is located on a concave depression of the macronucleus. -Fig. 6-9. Isolated micronuclei at two different magnifications. - Fig. 10. Isolated macronucleus not squashed. The undulated surface of the macronucleus and micronucleus corresponds to the irregular waving of the nuclear envelope seen in the transmission electron microscope. .-Fig. 4, 5 and 10: Osmium followed by 70% alcohol; Fig. 6-9: acetic-alcohol fixations. -Fig. 4. 5 . 8, 9 and 10: x 12,000; Fig. 6 and 7: X 24,000.
The multiple nucleoli are located on the periphery of the macronucleus near the nuclear envelope. They vary in size from 0.2 to 0.5 microns. The nuclear envelope of the macronucleus is an undulating structure with many depressions and salient regions. The central part of the nucleus is
occupied by the chromatin granules (Fig. 1 and 3). Between the large central mass of chromatin and the peripheral nucleoli a free space is frequently observed in isolated macronuclei as well as in sections of macronuclei present within intact cells (Fig. 3). This phenomenon is known as the halo-
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Fig. 11-14. Scanningelectron microscopy. Successive steps in the isolation of the nucleoli from the macronucleus. -Fig. I I. Part of a macronucleus showing the spherical elevations at the periphery which contain the nucleoli. -Fig. 13. A macronucleus with an attached micronucleus slightly affected by the squash procedure. A single nucleolus can be seen which has become detached from the peripheral zone. - Fig. 12. The macronucleus has been disrupted by squashing. The central mass of chromatin remains as a block and the numerous nucleoli become isolated. -Fig. 14. Higher magnification of a region of Fig. 13. -Fig. I I and 13: Osmium followed by 70% alcohol; Fig. 12 and 14: acetic-alcohol fixations. -Fig. I I and 14: x 24.000; Fig. 12 and 13: x 12,000.
effect (WORTHINGTON et al. 1975) and can be used, both in vivo and in vitro, to detach the peripheral complex of nucleoli and the nuclear membrane from the central mass of chromatin. The scanning electron microscope micrographs agree well with those obtained by transmission
microscopy. The macronucleus has an outer region consisting of irregular eminences below which the nucleoli are harboured (Fig. 4, 5, 11 and 13). The central part forms an elevated area containing the centrally located chromatin. The micronucleus is frequently found enclosed in an
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Fig. IS-18. Scanning electron microscopy. Isolated nucleoli. The nucleoli appear as single units, as the small ones in Fig. I5 and 17 or as larger compound structures composed of two or three units. Indentations appear in the larger structures separating the various units. Formaldehyde fixation. x 24.000.
outer pocket of the macronuclear membrane (Fig. 4 , 5 and 13). When the macronucleus has not been flattened during squashing it appears as a spherical structure with many protuberances which harbour the peripheral nucleoli (Fig. 10). The micronucleus may also occur as a free structure and it has an uneven surface due to undulations of the nuclear envelope (Fig. 6-9). The position of the micronudeus in an outer pocket and its undulated nuclear envelope are also seen in the transmission electron microscope (Fig. 2). The dimensions of the micronucleus and macronucleus as seen in the scanning electron microscope agree with those observed in the transmission electron miscroscope. The
macronucleus was found to be 7 to 8 microns in diameter and the micronucleus 1.2 to 1.6 microns (Fig. 6-10). To get information on the structural organization of the nucleoli an attempt was made to isolate them from the macronucleus. The acetic-alcohol fixation favours such a procedure as the material is soft enough for the macronucleus to break, but sufficiently hard for the nucleoli to preserve their structure and shape. A similar but not so extreme effect may be obtained by the osmium and formaldehyde fixations. The peripheral region of the macronucleus may be disrupted at a few points, the result being the release of a few nucleoli that become isolated (Fig. 13).
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When the acetic acid - alcohol procedure is used, this disruption of the nuclear membrane is more drastic and most of the nucleoli become isolated. The central chromatin mass remains mainly intact and around it are spread the nucleoli, well separated from one another and free of other material (Fig. 12 and 14). At higher magnifications the nucleoli show indentations and can be seen to be compound structures. They vary in size from 0.3 to 0.8 microns depending on whether they appear singly or in aggregates of two or three units (Fig. 15-18). The nucleoli show the same type of structure with the three fixatives employed. This can be seen by comparing Fig. 12-18. Fig. 13 is from osmium, Fig. 12 and 14 are from acetic-alcohol, and Fig. 15-18 from formaldehyde fixation.
Discussion There is good agreement between the transmission and the scanning electron microscopy results. The sizes of the macronucleus and micronucleus and of the nucleoli are found to be virtually the same by both methods. Thus the preparation for scanning does not lead to any appreciable deformation. The isolated micronucleus and the macronucleus have an uneven surface in the scanning electron microscope. This agrees well with the lobed and undulating appearance of the nuclear envelope seen in the transmission microscope. The preparation for the scanning has merely accentuated this irregular surface. The location of the micronucleus in a concave depression of the macronucleus also agrees with the previous information from the sectioned maet al. 1976) that terial. I t has been shown (WOLFE the micronucleus is located in a depression of the nuclear membrane of the macronucleus and this location can also be seen in Fig. 2. Such a position may be of physiological significance but this is not yet understood. The micronucleus leaves the pocket in the macronucleus during one of the first stages in the sexual cycle. The micronucleus returns to the pocket late in the conjugation cycle. The three types of fixatives: acetic acid alcohol, osmium and formaldehyde produce similar results. The structure of the nucleoli is the same with the three fixations (Fig. 12, 13 and 15). The same is true for the structure of the
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macronucleus and the micronucleus. The differences are only apparent during squashing, the acetic acid-alcohol fixation favouring an isolation of the nucleoli. In Acheta the scanning electron microscopy revealed that the amplified ribosomal DNA body is a spherical structure covered by polygonal plates which consist of RNA and protein. The multiple DNA copies irradiate from the point of attachment with the chromosome and spread out like the sticks of a fan. The DNA copies terminate at the vertices of the polygonal plates. These polygonal plates differ in size and are separated from one another by deep indentations (LIMA-DEFARIA 1974a and b). The macronuclear nucleoli of Tetrahymena represent a developmental stage, in which the ribosomal DNA copies have become extrachromosomal forming small and numerous nucleoli. During conjugation the new macronucleus must obtain extrachromosomal ribosomal DNA molecules by amplification from the ribosomal DNA integrated into the micronuclear chromosomes (YAOand GALL1977). In the scanning electron microscope the nucleoli of Tetrahymena also show a few indentations and are seen to be compound structures. They are, however, smaller and simpler than those of Acheta. In Acheta hundreds of ribosomal DNA copies are maintained together in the major chromomere of chromosome 6 (LIMA-DE-FARIA 1974a and b). The macronuclear nucleoli are smaller units which during macronuclear development are formed a few hours after the separation of conjugating cells (LEICK,BROand BROMS1979). Moreover, the number of ribosomal DNA copies per nucleolus is of an order of magnitude much lower than the number of genes in the Acheta ribosomal DNA chromomere. At the stage analysed in Acheta the amplified ribosomal DNA copies are over 800 and build a very large nucleolar structure. In Tetrahymena each nucleolus contains only about 20 ribosomal DNA molecules. The indentations seen with the scanning electron microscope in the Terrahymena nucleoli are quite similar to those seen in Acheta and may represent the regions at which the amplified ribosomal DNA molecules reach the surface of the nucleolus. Acknowledgments. - This work was supported by research grants from the Swedish Natural Science Research Council and the Danish Research Council.
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LIMA-DE-FARIA. A , , BIRNSTIEL, M. and JAWORSKA. H. 1%9. Amplification of ribosomal cistrons in the heterochromatin of Ac-hera. - Genetics (Suppl.) 61: 145-159 LUFT, J. H. 1%1. Improvements in epoxy resin embedding Literature cited methods. -J. Biophys. Biochem. Cyrol. 9: 40%415 MILLER, 0 . L. and BEATTY.B. R. 1%9. Portrait of a gene. - J . BIRNSIIEL, M . L.. CHIPCHASE. M . and SPEIRS, J. 1971. The Cell. Physiol. 74: 225-232 ribosomal RNA cistrons. - I n Progress in Nucleie Acid Research and Molecular Biology / I (Eds. J . N. DAVIDSON NILSSON, J. R. and LEICK, V. 1970. Nucleolar organization and ribosome formation in Terrahymena pyriformis GL. - Exp. and W. E. COHN),Academic Press, p. 351-389 Cell Res. 60: 361-372 BROWN, D. D. and BLACKLER. A. W. 1972. Gene amplification PEASE, D. C. I%O. Histological Techniques for Electron proceeds by a chromosome copy mechanism. - J . M u / . B i d . Microscopy. -Academic Press, N e w York 63: 75-83 PERO, R., LIMA-DE-FARIA, A,, STAHLF.:,U.,GRANSTROM, H. DAWID. I . B.. BROWN. D. D. and REEDER,R. H. 1970. Comand GHATNEKAR, R. 1973. Amplification of ribosomal DNA position and structure of chromosomal and amplified in Achefa. I V The number of cistrons for 28s and 18s ribosomal DNA's of Xenopus laevis. - J . M o l . Biol. 51: ribosomal RNA. - Hereditas 73: 195-210 341-360 TRENDELENBURG, M. F., SCHEER. U..ZENTGRAF. H. and ENGBERG. 1.. ANDERSSON, P., LEICK. V. and COLLINS, J. F R A N K EW. , W. 1976. Heterogeneity of spacer lengths in 1976. Free ribosomal DNA molecules from Terrahymena circles of amplified ribosomal RNA of two insect species, pyrifurrnis GL are giant palindromes. - J . M o l . Biol. 104: Dytiscus marginalis and Achefa domesricus. - J . M o l . B i d . 455-470 108: 453-470 ENGBERG, J . , NILSSON, J . R., PEARLMAN, R. E. and LEICK, ULLMAN, J. S.. LIMA-DE-FARIA, A ., JAWORSKA,H. and V. 1974. Induction of nucleolar and mitochondria1 DNA BRYNGELSSON. T. 1973. Amplification of ribosomal DNA in replication in Tetrahymena pyriformis. - Proc. N a t . Acud. Acheta. V. Hybridization of RNA complementary to Sci. 71: 894-8898 ribosomal DNA with pachytene chromosomes. - Herediras KARRER. K. M. and GALL, J. G. 1976. The macronuclear 74: 13-24 ribosomal DNA of Tefrahymena pyriformis is a palindrome. VENABLE, J . H. and COGGESHALL. R. 1965. A simplified lead J . M o l . Biol. 104: 421-453 citrate stain for use in electron microscopy. -J. Cell Biol. 25: LEICK, V. 1973. The formation of ribosomes in Tefrahymena
We should like to thank Mr Folke Larsson and Miss Birgitta Sandstrom for their valuable assistance.
pyriformis. - Docfor's thesis. Biochemical Instirufe B , Universify of Copenhagen. Denmark LE ICK . V., BRO, B. and BROMS, P. 1979. Macronuclear
development: A model system to study specific gene expression in nuclear differentiation. - P r o r . A(fred Benzon Symp. N o . 13, Munksgaard. Copenhagen, p. 340-350 LIMA-DE-FARIA, A . 1974a. The three dimensional organization of the amplified ribosomal DNA. - P r o c . N a f . Acad. Sci. 71: 4778-4782
LIMA- I W FA R I A A.. 1974b. Amplification of ribosomal DNA in Acheto. I X . The isolated ribosomal DNA-RNA complex studied in the scanning electron microscope. - Heredifas 78: 255-264
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WEINSTEIN. R.. ABBISS, T. and B U LLI V A N T, S. 1%3. The use of double and triple uranyl salts a s electron stains. - J. Cell B i d . 19: 74A WOLFE. J., HUNTER, 8. and ADAIR, W. S . 1976. A cytological study of micronuclear elongation during conjugation in Tetrahymena.
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WORTHINGTON, D. H., NACHTWEY.D. S . and WA G EN A A R . E. B. 1975. Hydroxyurea: Morphological effects on the macronucleus of Terrahymena. - E r p . Cell Res. 92: 435443 YAO. M. C. and GALL,J. G. 1977. A single integrated gene for ribosomal RNA in a eukaryote. Tefrahymena pyriformis. Cell 12: 121-132
