Nucleolus Behaviour During the Cell Cycle of a Primitive Dinofiageilate Eukaryote, Prorocentrum Micans Ehr., Seen by Light Microscopy and Electron Microscopy

Journal of Cell Science 102, 475-485 (1992) 475 Printed in Great Britain © The Company of Biologists Limited 1992

Nucleolus behaviour during the cell cycle of a primitive dinofiageilate eukaryote, Prorocentrum micans Ehr., seen by light microscopy and electron microscopy

MARIE-ODILE SOYER-GOBILLARD* and MARIE-LINE GERAUD

Dipartemenl de Biologie Cellulaire, Observatoire Oceanologique de Banyuls, Universite P. et M. Curie, Laboraloire Arago, URA CNRS 117, 66650 Banyuls-sur-mer, France

*To whom correspondence should be addressed

Summary

Light-microscopy observation of the dinofiageilate Pro- nucleoli remain functional, whereas in late prophase rocentrum micans after silver-staining of the argyrophi- they contain only a NOR and the granular component, lic proteins of the nucleolar organizing region (Ag-NOR and the chromosomes are surrounded by many protein staining) showed the presence of nucleolar material masses. In early telophase, the nucleolar material throughout the vegetative cell cycle, and in particular coating the chromosomes migrates along with the during all the mitotic stages. This contrasts with the case chromosomes. Nucleologenesis occurs through the for- in most higher eukaryotes, in which nucleoli disappear mation of prenucleolar bodies around lateral or telo- at the end of prophase and are reconstituted in daughter meric nucleofilaments extruding from the chromosomes. cells during telophase. Several chromosomes can contribute to the formation of Electron-microscope (EM) observations after conven- one nucleolus. The behaviour of these 'persistent tional or fast-freeze fixation revealed that during nucleoli' in a closed-nucleus model such as that of the interphase several functional nucleoli with three dinoflagellates is discussed with regard to the higher compartments (NORs, the fibrillogranular and the eukaryotes. preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early pro- Key words: nucleolus, nuclear cycle, mitosis, Prorocentrum phase, when chromosomes are beginning to split, the micans, dinofiageilate.

Introduction sidered that among the species studied so far, a few dinoflagellates have autonomous nucleoli. Eukaryotes are characterized by the presence of a well- Dinoflagellates differ from the other eukaryotic differentiated nucleolus, and by a distinct envelope protists in their distinctive nuclear organization; in around the nucleus. The nucleolus, which is involved in particular, their chromosomes are permanently con- ribosome formation, is present in eukaryotic protists, densed throughout the cell cycle and the nuclear plants, and metazoa. Although its structural organiz- envelope persists even during the distinctive closed ation into three basic compartments has been clearly mitosis ("dinomitosis") (Triemer and Fritz, 1984; seen by light and electron microscopy (EM), the Schnepf et al., 1990; Perret et al., 1991). The chromo- functioning of these compartments in molecular terms somal nucleofilaments are twisted and helicoid (Herzog is still controversial (Jordan, 1991; Hernandez-Verdun, et al., 1984) and their chromatin is devoid of the 1991). During the cell cycle, RNA synthesis is discon- histones and nucleosomes that characterize prokaryotes tinuous, and the nucleoli almost always disappear (for a review see Rizzo, 1987). However, a small during somatic division (Goessens, 1984). However, in amount of DNA-binding basic protein is associated certain animal cells (Sheldon et al., 1981) and in some with the chromosomal and nucleolar DNA (Vernet et species of plant (Gimenez-Martin et al., 1977; Risueno al. 1990; Sala-Rovira et al., 1991; Geraud et al., 1991b). and Medina, 1986), nucleoli persist during mitosis. Recent comparison of rRNA conserved sequences Risueno and Medina (1986) classified plant nucleoli into suggested that dinoflagellates are phylogenetically close four types, according to their behaviour - autonomous, to the typical eukaryotes yeasts and ciliates (Lenaers et persistent, semi-persistent and dispersive - and con- al., 1991). 476 M.-O. Soyer-Gobillard and M.-L. Geraud

There have been few previous studies of the silver nitrate aqueous solution for 20 min. The cells were ultrastructure of the nucleolus functioning in relation to rinsed well in water and fixed for 10 min in 5% sodium permanently condensed chromosomes in a nucleus thiosulfate. surrounded by a persistent nuclear envelope (Rae, Nuclei were stained with 0.1 jug/ml DAPI (4',6-diamidino- 1970; Soyer and Haapala, 1974a; Spector, 1984). 2-phenylindole, from Sigma, St Louis, USA), mounted in glycerol with 5% N-propyl gallate, and observed with a Recent immunocytochemical and in situ hybridization Reichert Polyvar photomicroscope either in epifluorescence studies (Geraud et al., 1991a) have shed light on the with a 330-380 nni/LP 418 nm filter or with an interference contents and functions of the various nucleolar com- contrast system. Photographs were taken on Kodak TMax partments: (1) The nucleolar organizing region (NOR), 400-ASA films. formed by the unwound part of the nucleolar chromosome, contains DNA in B and Z configurations (Soyer- EM procedures Gobillard et al., 1990), a DNA-binding protein (Sala- Conventional preparations Rovira et al., 1991), and, in particular, rRNA coding P. micans pellets were fixed for 1 h with 0.2 M PIPES- sequences in its periphery (Geraud et al., 1991a). (2) buffered 2% paraformaldehyde and 1.25% glutaraldehyde The fibrillogranular region, surrounding the NOR, (pH 6.4), in accordance with Karnovsky's procedure as contains a few rDNAs (Geraud et al., 1991a) in the B modified by Soyer (1977). The pellets were washed in 0.2 M configuration (Soyer-Gobillard et al., 1990) located in PIPES buffer, postfixed for 1 h in similarly buffered 2% OsO4 the region adjoining the NOR and argyrophilic (Ag- at room temperature, and embedded in epoxy resin (Epon). NOR) proteins (Salamin Michel et al., 1990). (3) The EM was performed using a Hitachi H-600 electron microscope granular compartment, which is devoid of rDNA and of (Hitachi Ltd., Tokyo). DNA- binding protein, contains preribosomes (Rae, Fast-freeze fixation and freeze-substitution 1970, Geraud et al., 1991b; Sala-Rovira et al., 1991). A portion (20 n\) of a P. micans pellet was transferred to filter 2 Moreover the cell cycle and the pattern of DNA paper (10 mm ) and mounted on a specimen holder, by the synthesis are eukaryotic in type, as shown in Prorocen- method of Escaig et al. (1977). The sample was slammed onto trum micans (Bhaud and Soyer-Gobillard, 1986). This a metal-mirror block of pure copper cooled by liquid helium at —269°C on a cryovacublock (Reichert-Yung, Leica) autotrophic phytoplankton species, used as a model in (Escaig, 1982), transferred to liquid nitrogen, and stored until the present work, has 100 chromosomes (Herzog and freeze-substitution. Freeze-substitution was in acetone and Soyer, 1981) and a DNA content of 42 pg per nucleus 2% OsO4 in the presence of molecular sieves (0.4 nm, (Haapala and Soyer, 1974). Asynchronous populations Perlform; Merck) to absorb the water extracted from the are composed of vegetative cells and gametes (Bhaud et sample. Substitution was in a CryoCool apparatus (RUA) for al., 1988). 3 days at —80°C. Then the temperature was gradually raised In order to clarify the behaviour and functioning of to —30°C and kept there for 2 h. Finally, the samples were the nucleolus during the cell cycle in this closed-nucleus thawed at room temperature for 1 h, washed successively in model, we have studied the nucleoli by light mi- pure acetone, absolute ethanol and propylene oxide, and croscopy, using the end products of the Ag-NOR embedded in Epon. staining reaction as markers, and by EM, observing structural alterations in the various nucleolar compart- Results ments after conventional preparation or fast-freeze fixation followed by freeze-substitution. Nucleoli in an asynchronous population In the nuclei of an asynchronous population of P. micans cells, 0 to 5 nucleoli per nucleus were detected Materials and methods by light microscopy after the Ag-NOR staining end- products reaction. Nucleoli of various sizes, stained Cell cultures brownish after the Ag-NOR reaction, stand out from Prorocentmm micans Ehrenberg strains from the Botany the DAPI-stained chromosome masses (Fig. lb). The School, Cambridge University (UK) were grown in Erdsch- sites of nucleoli are seen as dark areas in the DAPI- reiber's medium (Bhaud et al., 1988) under a 12/12 light (2000 lux)/dark cycle at 20°C. The cell cycle lasts 5.5 days, with a stained nuclei of the same preparation observed in a DNA-synthesis period of 4 h, a G2+M period of 8 h, and a Gi dark field (Fig. la, arrow). Vegetative nuclei are period of 120 h (Bhaud and Soyer-Gobillard, 1986). characterized by chromosomes with a large (1 fim) mean diameter, as seen in Fig. lc, and in Fig. li,j(B), Squashes for optical microscopy where a nucleolus is detectable. About 15% of nuclei Pellets of P. micans obtained at 200 g were fixed for 30 min at did not have detectable nucleoli, but either contained 4°C in a mixture of 2% paraformaldehyde in 0.2 M PIPES very small dispersed brown masses (Fig. lf,h, arrows) buffer, pH 7.0. The pellets were washed for 10 min in PIPES or were apparently devoid of nucleolar material (Fig. buffer and sonicated for 1 min in the same buffer to facilitate li,j(A). Such nuclei generally had many thin chromo- the opening of the two halves of the theca. Cells were somes (Fig. Id). squashed on glass slides, frozen for 1 h on a block of dry ice, and stored at -20°C. Distribution and behaviour of nucleoli during the cell Ag-NOR reactions were processed by the method of Ploton cycle et al. (1982). Postfixation in Carnoy's solution (acetic acid/ethanoI:l/3) for 5 min at 4°C was followed by treatment Nondividing cells with 1 vol of 2% gelatin in 1% formic acid and 2 vol of 50% As shown above by light microscopy, nucleoli of various Fig. 1. Nuclei from an asynchronous population of Prorocentrum micans Ehr. Squashes were observed either after staining of chromosomal DNA with DAPI (a, c, d, e, g, j) or after silver-staining (Ag-NOR staining) of the nucleoli (b, f, h, i). Note the difference between the organization and compactness of the chromosomes of vegetative (a, b, c, iB, jB) and gametic cells (d-h, iA, jA). X3450.

478 M.-O. Soyer-Gobillard and M.-L. Geraud

Fig. 2. EM images of P. micans nuclei in interphase either after conventional preparation (a, b) or after fast-freeze fixation and freeze-substitution (c). (a) Three nucleoli (Nu) are seen near compact chromosomes (Ch) 1 f.tm in diameter. Note their three characteristic compartments: fibrillogranular (FG) and granular (G) regions and the NORs, seen in both transverse and longitudinal section. xl8 540. Bar, 1 jtrn. (b, c) Higher magnifications of an interphase nucleolus in which the unwound part of the nucleolar chromosome (UCh), the fibrillar part (F) of the nucleolus, and the granular (G) region are clearly identifiable, (b) X50 200. Bar, 0.5 ^m. (c) Note the extended fibers of the F region and its compact part (arrow), ne, nuclear envelope; arrowhead, ribosomes. x60 000. Bar, 0.5 /zm. Prorocentrum micans nudeolus behaviour 479

Fig. 4. EM images of early (a) and late (b) prophase in P. micans. (a) Some thicker chromosomes are starting to split (white arrows) and others are in close contact with the nuclear envelope (arrow). One of the nucleoli shows the typical three compartments. FG, fibrillogranular region; G, granular region; Nu, nucleolus; NOR, nucleolar organizing region. Xl2 000. Bar, 2 /an. (b) Higher magnification of a nucleus in late prophase. A chromosome is splitting (empty arrows) and, like its neighbours, is surrounded by small, compact masses. The nucleolus (Nu) consists only of the extended NOR and the granular region (G). x27 000. Bar, 1 fjm. vations are summarized in a diagram (Fig. 7) showing magnification of one of these longitudinally sectioned the compartments of a functional dinofiagellate nu- chromosomes, these masses are seen embedding the cleolus in which several chromosomes are contributing extruded lateral unwound chromosomal (UCh) region to the formation of a nucleolus by means of the where the nucleofilaments are uncoiled locally (Fig. telomeric and/or lateral unwinding of specialized nuc- 9b,c, arrows). Several such masses, which are prenuc- leofilaments. leolar bodies (pNu) originating from different chromo- Although the magnification of the light microscope somes, merge to form a large nucleolar mass (Fig. 9a, does not allow perfect observations along squashed and arrow), in which only fibrillar and fibrillogranular silver-stained chromosomes, it is possible to distinguish regions are observable. well black dots along and between some chromosomes in anaphase (Fig. 8a, arrows). In Fig. 8c (anaphase) black dots and big masses (arrow) are simultaneously Discussion visible as well as a nucleolus and in Fig. 8a,b (arrowhead), several chromosomes appear totally black End products of Ag-NOR staining as nucleolar labelled. Another nucleus in early telophase (Fig. 9a), markers in the cell cycle prepared in the usual way for EM, contains many In the light-microscopy observations reported here, randomly orientated chromosomes 1 ^m in diameter, to cytochemical staining of the nucleolar argyrophilic which fibrillogranular masses are attached. At higher proteins (Ag-NOR staining) in the dinofiagellate P. 480 M.-O. Soyer-Gobillard and M.-L. Geraud

Fig. 5. (a, b) Serial ultrathin sections of a fast-freeze-fixed, freeze-substituted P. micans cell in early telophase. Several chromosomes are participating in the formation of nucleoli in various places in the separating nuclei (empty arrows). Note the parallel orientation of some of the stretched chromosomes (filled arrows). xlO 800. Bar, 2 jnn. Prorocentrum micans nucleolus behaviour 481

Fig. 6. (a, b) Fast-freeze fixed, freeze-substituted P. micans cell in late telophase. The randomly orientated chromosomes, 1 /im in diameter, are more compact than in the early telophase of Fig. 5, whose highest magnification is shown in (c). xl2 000. Bar, 2 /m\. (b) Higher magnification of (a), showing two chromosomes with their unwound telomeric part (UCh) generating a nucleolus which comprises only fibrillar and fibrillogranular regions. x25 000. Bar, 1 fun. (c) Nucleologenesis from the sides of six nucleolar chromosomes. Note the extruding chromosomal buds (arrowheads). x28 000. Bar, 1 j/m. 482 M.-O. Soyer-Gobillard and M.-L. Geraud NOR CCh which dehydrates the specimen as it thaws and thus prevents the cell structures from collapsing. Behaviour of the nucleolus during mitosis P. micans has a typical eukaryotic cell cycle with a discontinuous S-phase (4 h) and an 8 h G2+M phase (Bhaud and Soyer-Gobillard, 1986). The Gx phase lasts 120 h; during this interphase we observed functional nucleoli with all three characteristic compartments. Bhaud and Soyer-Gobillard (1986) found that the whole vegetative cell cycle lasts 5.5 days, one of the longest UCh cycles known in dinoflagellates. We constructed a picture of dinomitosis in this species from light microscopy and EM observations of several stages from early prophase to late telophase in an asynchronous population. Because there is no metaphase plate and anaphase is transient (Fig. 3d,e) and difficult to identify in this species, no typical anaphase stage was included among our EM observations. However, Ag-NOR staining of squashed nuclei in anaphase (Fig. 3k',1') observed by light microscopy clearly reveals the presence of nucleolar material. Fig. 7. Schematic representation based on EM observations of nucleolar chromosomes of Prorocentrum micans and In early prophase, a three-compartment functional showing the unwinding of nucleofilaments located in their nucleolus is present during chromosomal splitting, telomeric or lateral regions. Several chromosomes are whereas in late prophase the fibrillogranular region is contributing to the formation of the new nucleolus. C Ch, reduced. This region is a counterpart of the dense condensed chromosome; U Ch, unwound chromosome fibrillar component of the higher eukaryotes, which is region; NOR, nucleolar organizing region; F, fibrillar considered to be a transcriptionally active region region; FG, fibrillogranular region; G, granular region. (Hernandez-Verdun, 1991), while the G region contains the matured transcripts. The presence of a predominant micans has revealed the presence of several nucleoli not granular region in late prophase of P. micans indicates only during the vegetative stages of the cell cycle but, in that the transcripts are still maturing, while transcrip- particular, during all the mitotic stages. This technique tion itself has already stopped. At this stage, the small (Ploton et al., 1982) gives highly reproducible detec- protein masses all around the chromosomes (Soyer and tion. Ag-NOR proteins have always previously been Haapala, 1974b) may be the fragmentation residues of detected by EM in the fibrillogranular region of the nucleoli in a nucleus whose envelope is never disrupted. dinoflagellate nucleolus (Spector, 1984; Salamin Michel In higher eukaryotes such protein masses have been et al., 1990). Moreover, in this preliminary work, we identified as silver-staining proteins which in prophase have shown by EM that the distribution of argyrophilic are in close contact with some chromosomes, with proteins in the nucleus of P. micans concerns all fibrillar centres, and with a dense fibrillar component, chromosomes and the nucleolar FG region. Work is in while during interphase such Ag-NOR proteins are progress to study by EM the cytochemistry and the restricted to the latter two compartments (Ploton et al., distribution of argyrophilic proteins in several dino- 1987). flagellates, especially to determine if one or several During anaphase-early telophase, nucleologenesis categories of argyrophilic proteins are present in the occurs. Together with lateral or telomeric rDNA nucleus of P.micans. Nevertheless, it is likely that the chromosomal loops, they form the prenucleolar bodies, partially or totally silver-stained chromosomes visible in which coalesce into new functional nucleoli that can light microscopy (Fig. 8a-c) correspond to nucleolar synthesize rRNA. This process is probably very fast and chromosomes. occurs asynchronously in the various chromosomes of a nucleus (Fig. 9a), accounting for our light-microscopy Conventional and specific EM preparation methods observations of nucleoli in all the vegetative nuclei. Preparation of our specimens by fast-freeze fixation and New nucleoli at first contain only fibrillar and fibrillo- freeze-substitution firstly preserves the fibrillar part of granular regions, with the granular region appearing in the NOR better than conventional preparation does, late telophase, when the new transcripts have matured. keeping the dispersed filaments more identifiable, and, There is already conclusive evidence that in higher secondly, clearly differentiates the unwound but com- eukaryotes prenucleolar material is collected from pact part of the nucleolar chromosome from the various sites along the chromosomes, accumulates in extended part, which has been difficult to see by the prenucleolar bodies, and contributes to nucleolar conventional method even as adapted to dinoflagel- reformation along the NORs in telophase (Lafontaine lates. This better preservation is due to the rapidity of and Lord, 1969; Ploton et al., 1987). However, in a the freeze-fixation (2 ns) and to the freeze-substitution, number of mammalian cell strains, nucleoli that persist Frorocentrum micans nucleolus behaviour 483

Fig. 9. Nucleologenesis in a P. micans cell (early telophase). (a) Coalescing prenucleolar bodies are forming a new nucleolus (Nu, arrow), and prenucleolar bodies (pNu) coat several randomly orientated chromosomes. xl6 200. Bar, 1 /an. (b, c) Higher magnifications of (a), showing prenucleolar bodies (pNu) around the sides (arrows), extruded from parts of a partially unwound chromosome (UCh). (b) x62 750. Bar, 0.5 /an. (c) x64 800. Bar, 0.5 ^m. 484 M.-O. Soyer-Gobillard and M.-L. Geraud during mitotic division have also been observed, either (Geraud et al., 1991a). Coding sequences of the attached to the chromosomes or free in the cytoplasm ribosomal cistron were found on the periphery of the (Hsu et al., 1965). NOR and in the adjoining part of the fibrillogranular The processes of nucleologenesis in the dinoflagellate region, suggesting that rRNA transcription is initiated P. micans are comparable to those in higher eukary- on the periphery of the NOR, as shown in the predictive otes, but the persistence of the nuclear envelope, model of Fig. 5 of that paper. The findings of this preventing the dispersion into the cytoplasm of nu- present work lead us to suggest a dinoflagellate model cleolar residues coating the chromosomes, permits rather similar to the eukaryote model recently proposed classification of the P. micans nucleolus as "persistent" by Jordan (1991, Scheme 5 and Fig. 3B) with respect to according to Risueno and Medina's (1986) system of the distinctive characteristics of their nuclei and nucleolus classification. nucleoli. In most other free-living dinoflagellates previously In order to identify the molecular mechanisms of the studied with the electron microscope (for a review see functional structure of the dinoflagellate nucleolus Raikov, 1982), the nucleolus of the vegetative cell during the cell cycle, and in particular during nucleolo- divides in two during mitosis and the two halves become genesis, work is in progress to perform both in situ incorporated into the daughter nuclei, so that Risueno hybridization, to find out whether the rRNA genes are and Medina (1986) classify such nucleoli as "auton- present in the prenucleolar bodies and in prophase and omous". anaphase nucleoli, and an immunocytochemical study of the enzymes specifically involved in ribosomal Nucleolus behaviour during sexual reproduction transcription, such as RNA polymerase I and DNA Some of the many nuclei we studied in an asynchronous topoisomerase I. Moreover, finding in situ the specific population of P. micans did not contain nucleoli. These subunits of rRNA cistron cloned in our laboratory to be looked like large nuclei containing many thin chromo- the external transcribed spacers and the nontranscribed somes. This type of nucleus has been described as spacer would supplement our structural (this work) and containing 2 or 4 qDNA and leading to a tetrad of functional (Geraud et al., 1991a) conclusions and gametes (Bhaud et al., 1988, Figs 2H and 3E,F). models with regard to recent data concerning the higher Moreover, during meiosis in higher eukaryotes, such as eukaryote nucleolus, in which the actual sites of some plant cells, the nucleolus, still visible in the early transcription are still extremely controversial (Jordan, diplotene, disappears totally during diakinesis (Risueno 1991; Hernandez-Verdun, 1991). and Medina, 1986). In other free-living dinoflagellates, such as Noctiluca We are grateful to Mrs Danielle Saint-Hilaire for mainten- scintillans, nucleoli are absent from sporocytes (Soyer, ance of the strains and cultures, to Mrs Marie Albert for excellent technical assistance, to Mrs M. J. Bodiou for 1972), which are considered isogametes (Zingmark, drawing Fig. 7 and to Dr Andr6 Picard and Mrs Suzanne 1970). Nucleoli have not been observed in mature Miller (Wight Scientific, London) for critical reading of the sporocytes of the parasitic dinoflagellates Blastodinium manuscript. This work was supported by Centre National de sp. (Soyer, 1971) or Syndinium sp. (Ris and Kubai, la Recherche Scientifique (URA 117). 1974; Soyer, 1974). Conclusions References The behaviour of the nucleolus in the primitive Bhaud, Y., Salmon, J. M. and Soyer-Gobillard, M. O. (1991). 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