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 chromo- some, 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.