Nucleolar organizer structure and activity in a nucleolus without fibrillar centres: the nucleolus in an established Drosophila cell line

0 Laboratoire d'Histologie et Embryologie II, Facultd de Midecine , 27 boulevard Jean Moulin, 13385 Marseille Cedex 5 , France 1 Laboratoire de Ginitxque et Biologic Cellulaires, Centre Universitaire de Marseille-Luminy , Case 907, 13288 Marseille Cedex 9 , France ACTIVITY IN A NUCLEOLUS B. KNIBIEHLER1*, C. MIRRE1 AND R. ROSSET1 Classical electron-microscopic techniques (enzymic digestion, EDTA regressive staining) allied with autoradiographic studies after ['H]uridine incorporation or after RNA synthesis initiated by an exogeneous RNA polymerase in the presence of tritiated GTP, enabled us to describe the fine structure and activity of the nucleolus in an established Drosophila cell line. This nucleolus is composed of a large central multilobed core containing proteins, RNA molecules and a DNA-containing component. This core is surrounded by and connected to large clumps of dense fibrillar nucleolus-associated chromatin, which are intermingled with nbrillogranular ramifications extending from the core towards the nuclear envelope. These ramifications are covered by granules of ribosomal ribonucleoprotein. As shown by EDTA regressive staining the nucleolar core contains a ribonucleoprotein network, which unravels and ramifies within a fibrous matrix. RNA synthesis takes place at the level of this network in the internal part of the core. The molecules synthesized are associated with proteins and are exported out of the core in the form of granules. Although it is composed of the same constituents as other nucleoli, the nucleolus of Drosophila cells seems to be less organized, in that it never displays fibrillar centres, which have been referred to as the nucleolar counterparts of the nucleolus-organizers in a wide variety of organisms. - In comparison to other genes, the ribosomal cistrons are unique in that their transcription occurs in a chromosomal complex: the nucleolus (for a review, see Busch & Smetana, 1970). The fine structure and size of the nucleolus are very sensitive to changes in ribosomal activity. The nucleolus can therefore serve as a cytological indicator of ribosomal RNA gene transcription (and in this respect is analogous to the puffs of the polytene chromosomes) (Ghosh, 1976). The nucleoli of mammalian somatic cells can be classified into three types, ' ringshaped', 'nucleolonema-containing' and 'compact nucleoli', according to their appearance in the electron microscope. They are all composed of three main components: nucleolar chromatin, a granular component containing premature ribosomal particles and a fibrillar component composed of ribonucleoprotein material (Busch & Smetana, 1970; Smetana & Busch, 1974). In addition to these classical components, Author to whom correspondence should be sent. in a large variety of cells the nucleoli also contain clear fibrillar zones, which Recher called 'fibrillar centres' (Recher, Whitescarver & Briggs, 1969). These fibrillar centres have been referred to as corresponding to the nucleolar counterparts of the chromosomal nucleolar-organizer regions (Goessens & Lepoint, 1979). In Drosophila melanogaster, the nucleolus-organizing regions (NORs) are located in the proximal centromeric heterochromatin of the X chromosome, and on the short arm of the Y chromosome (Kaufman, 1936; Ritossa & Spiegelman, 1965). According to Ritossa & Spiegelman (1965) about 0-25 % of the DNA in the haploid genome of D. melanogaster is used to transcribe the 18 S and 28 S rRNA molecules. From these data they calculated that there are approximately 130 cistrons for the 18 S and an equal number for the 28 S rRNA in each sex chromosome. Developmental and ultrastructural studies were made on the nucleoli of Drosophila ovarian nurse cells (Dapples & King, 1970) and of Drosophila primary spermatocytes (Meyer & Hennig, 1974). These studies have shown that changes in nucleolar morphology and activity could be used as a rapid means of identifying transient stages of cellular activity. Nevertheless, no precise ultrastructural identification of the nucleolar components was made, rendering it very difficult to interpret the morpho In this paper we report ultrastructural studies on Drosophila KCo cell nucleoli based on a combination of classical electron-microscopic observations allied with autoradiographic studies after tritiated uridine incorporation, or after RNA synthesis initiated in vitro by an exogeneous RNA polymerase of Escherichia coli in the presence of Drosophila KCo cells were grown in D22 medium without foetal calf serum in 25 cm' Falcon flasks at 22 C (Echallier & Ohanessian, 1970). All the electron-microscopic observations were made with a Siemens Elmiskop IOI microscope at 80 kV. The cells were harvested on ice and washed with phosphate-buffered saline (PBS). They were treated by double fixation: (1) 3 % glutaraldehyde in o-i M-phosphate buffer (pH 7-2), containing o-i M-sucrose for 15 min at 6 C. (2) After washing [with the buffered solution, the cells were postfixed in 2 % osmium tetroxide in identical buffer for 20 min. After dehydration in a graded series of acetone the cells were embedded in Epon. Thin sections were collected on copper grids and contrasted with uranyl acetate and lead citrate. Bernhard's (1969) regressive EDTA staining The cells were fixed only in 3 % glutaraldehyde in o-i M-phosphate buffer, without sucrose for 1 h at 4 C. Fixation was followed by washing for at least 1 h in the same buffer. After embedding in Epon, thin sections were collected on grids and contrasted with uranyl acetate. EDTA (0-2 M) was allowed to act for 45 min. The sections were contrasted with lead citrate. After simple fixation in 3 % glutaraldehyde in o-i M-phosphate buffer, the cells were embedded in glycol methacrylate (GMA, Polysciences Inc.) according to Leduc & Bernhard (1967). Sections of gold interference colour were collected on gold grids and treated with the following enzymes: (1) Pronase, 0-3 % aqueous solution, pH 7-4 for 10 min at 40 C. (2) RNase (after Pronase, O'3 %, 2 min), 0 7 % solution in 2 x SSC (SSC is 0-15 M-NaCl, 0-015 M-Na citrate), pH 7-2, 3 h at 37 C. (3) DNase (after Pronase, 0-3 %, 2 min) 0-4% solution in 7 mMMgSO4, pH 6-8 for 3 h at 37 C. Staining was carried out with both uranyl acetate and lead citrate. RNA synthesis initiated by an exogeneous RNA polymerase The technique used was that described by Geuskens (1977). Ultra-thin sections of KCo cells embedded in glycolmethacrylate (GMA) as described above, were incubated for 2 h 30 min at 37 C on the surface of the following medium: 100 /*M-ATP, CTP, UTP; 150 /*M-[3H]GTP (NEN, i7Ci/mmol); 40 mM-Tris. HC1, P H 7 9 ; iomM-MgCla; 0 1 M-EDTA; o i m M dithiothreitol; 0-15 M-KC1; 0'5 fig/ml bovine serum albumin; 10 units/ml E. coli RNA polymerase holoenzyme. After incubation, the sections were washed in 0-15 M-NaCl for 5 min and then in three baths of 5 % trichloracetic acid containing 2 % Na pyrophosphate for 10 min at 4 CC. After washing in dist (...truncated)