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Three-dimensional organization of active rRNA genes within the nucleolus

¹ Unité MéDIAN, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
² DTI, UMR 6107, UFR de Sciences, BP 1039, 51687 Reims Cedex, France
³ Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, 20 rue de Pitteurs, 4020 Liège, Belgium
⁴ IFR53, 51 rue Cognacq-Jay, 51096 Reims Cedex, France

View larger version (39K): [in a new window]   Fig. 8. Organization of the coils. (A,B) Two successive 1.5 nm thick sections were produced parallel to the long axis of coil number 2. The positions of seven twines, orthogonal to the long axis of coil number 2, are indicated by arrows. (C,D) Two successive 1.5 nm thick sections performed perpendicularly to the long axis of coils numbers 1, 2 and 3. Twines (large circles) are bent and constitute closed or partially open circles. Bar, 100 nm. (E) Stereo-pair showing the twines observed in (A-D). The volume of the cluster was cut in two halves along a plane parallel to the long axis of coil number 2. To simplify the visualization, the rear part was eliminated. Several open and closed circles corresponding to bent twines can be seen.

View larger version (90K): [in a new window]   Fig. 1. Ultrastructural localization of nascent rRNA molecules within the nucleolus. Immunogold labelling detection of BrUTP-labelled rRNAs on ultrathin sections of isolated nucleoli from ELT cells pulse-labelled for 1 minute (A) and 10 minutes (B). The main nucleolar components are clearly identified: fibrillar centers (FC), dense fibrillar component (DFC) and granular component (GC). Arrows in (A) point to gold particles. Bar, 0.25 µm.

View larger version (16K): [in a new window]   Fig. 2. Densities (gold particles/µm2) on the FC, DFC, GC and resin (R) in nucleoli isolated from ELT cells and incubated with BrUTP for 1 to 25 minutes. The results shown represent mean values±s.e.m. 7, 14, 13 and 12 random micrographs were analyzed and 51, 120, 211 and 274 gold particles were counted, respectively. Student's t-test for nucleolar components versus resin (+ P<0.05, ++ P<0.01) and for FC versus DFC in different pulse-labelled nucleoli (^ P<0.05, ^^ P<0.01) was used.

View larger version (13K): [in a new window]   Fig. 3. Effect of actinomycin D upon BrUTP incorporation. Densities (gold particles/µm2) on the FC, DFC, GC and resin (R) were calculated in untreated and actinomycin-D-treated nucleoli incubated with BrUTP for 10 minutes. The results represent mean values±s.e.m. 13 and 8 random micrographs were analyzed and 211 and 47 gold particles were counted, respectively. Student's t-test for nucleolar components versus resin (+ P<0.05, ++ P<0.01) for each nucleolar component in untreated versus treated nucleoli (* P<0.05, ** P<0.01) and for FC versus DFC in untreated or treated nucleoli (^ P<0.05, ^^ P<0.01) was used.

View larger version (12K): [in a new window]   Fig. 4. Immunogold detection of BrUTP-labelled rRNAs on ultrathin sections of isolated nucleoli from ELT cells pulse-labelled for 10 minutes with BrUTP and pulse-labelled for 10 minutes with BrUTP followed by a 20 minute chase with UTP. Densities (gold particles/µm2) on the FC, DFC, GC and resin (R) were calculated in both cases. The results shown represent means±s.e.m. 13 and 25 random micrographs were analyzed, and 306 and 420 gold particles were counted, respectively. Student's t-test for nucleolar components versus resin (+ P<0.05, ++ P<0.01) for each nucleolar component in pulse-labelled versus pulse-labelled and chased nucleoli (* P<0.05, ** P<0.01) and for FC versus DFC in pulse-labelled or pulse-labelled and chased nucleoli (^ P<0.05, ^^ P<0.01) was used.

View larger version (91K): [in a new window]   Fig. 5. Ultrastructural identification of initiation sites of rDNA transcription within the nucleolus. (A,B) Immunogold detection of BrUTP-labelled rRNAs on ultrathin sections of isolated nucleoli from ELT cells, pulse-labelled for 15 minutes with BrUTP after a 15 minute incubation with transcription medium (A) or pulse-labelled for 15 minutes with BrUTP after a 15 minute incubation in the presence of an elongation inhibitor, cordycepin, instead of ATP (B). Bar, 0.25 µm. (C) Density (gold particles/µm2) on the FC, DFC, GC and resin (R) in nucleoli isolated from ELT cells. The results represent means±s.e.m. 24, 24 and 14 random micrographs were analyzed and 464, 381 and 141 gold particles were counted, respectively. Student's t-test for nucleolar components versus resin (+ P<0.05, ++ P<0.01) for each nucleolar component in untreated versus treated nucleoli (* P<0.05, ** P<0.01) and for FC versus DFC in untreated or treated nucleoli (^ P<0.05, ^^ P<0.01) was used.

View larger version (143K): [in a new window]   Fig. 6. Ultrastructural localization of RNA polymerase I within A549 cells. Anti-RPI antibodies were revealed with fluoronanogold, followed by silver enhancement. After embedding, ultrathin sections (80 nm) were counterstained and observed with an electron microscope at 100 kV. The main nucleolar components are identified (FC, DFC and GC). A high density of particles is observed within the fibrillar components of the nucleolus. Bar, 0.5 µm.

View larger version (74K): [in a new window]   Fig. 7. Tomographic study of A549 cells immunolabelled with anti-RNA polymerase I antibodies. Contrast was inverted so that silver particles appear white. A 500 nm thick section observed using a STEM working at 250 kV is displayed: several independent clusters, 270 nm in diameter, are seen (A). (B-D) Different projections of the tomogram were calculated after tomographic reconstruction of the cluster framed in (A). At + 15° (D), five 60 nm coils are seen, as indicated by brackets numbers 1 to 5. The large circle shows the area where the coils are fused together. The arrows point to twines, 20 nm in thickness. (E) A stereopair of the tomogram presented in the same orientation as in (D) was calculated thanks to a surfacic rendering mode. (F-I) Four successive 30 nm thick sections were achieved with a coronal orientation within the tomogram presented in (D). Asterisks (G,H) indicate the internal part of the cluster, devoid of labelling, and arrows (G,I) refer to twines. Bar, 200 nm (A) or 100 nm (B-I).

View larger version (71K): [in a new window]   Fig. 9. A model of the three-dimensional organization of an active rDNA gene within a fibrillar center. (A) Spread of a `Christmas tree' drawn at scale. (B) For simplicity, only the rDNA gene covered with 180 RPI molecules is presented; it is folded into four identical loops. (C) Each loop (boxed in B) folds into a separate coil. It consists of two identical rows (200 nm in length) of small loops (60 nm in length) (brackets) that are covered with 3-4 RPI molecules. (D,E) A coil is obtained by bending the small loops on the matrix of a cylinder, 60 nm in diameter and 200 nm in length. In this case, it is composed of a stack of six open rings, 30 nm in thickness (brackets on D). (F,G) Upper and side views of a ring: each ring is composed of two twines, 60 nm long; the arrows in (F) point to the two openings of a ring. (H,I) For convenience, the whole length of a rDNA gene has been wrapped into four identical coils (H, side view; I, perspective view). Experimentally, each cluster was composed of three to five coils, whose lengths were variable. In our model, this disparity could be taken into account by placing more (or less) rings in each coil. (J) A classically stained, ultrathin section was merged with a cross-section of the model shown in (H and I) at the same scale. Cross-sections of the coils, localized within the cortex of FC, appear as open rings without rRNA molecules (on the left) or with elongating rRNAs molecules emerging from the convex face of the coil and entering the surrounding DFC. Bar, 400 nm (A), 100 nm (B) and 50 nm (C-J).