Fig 1.
4D evolution of nucleolar volume and topography during AMD treatment in living KB cells.
One hundred Z-stacks were acquired during 8 h and images were generated by Rev4D software. (A–H) Gallery of 3D reconstructions displaying nuclear and nucleolar evolution and showing the whole nucleus (low threshold, transparent yellow), the nucleoli (medium threshold, transparent orange) and the DFC (high threshold, solid red) (same cell as in S8 Fig and S1–S5 Movies). (F–H) Trajectories of the center of mass of each nucleolus, demonstrating that their relative position is fixed, that there is no nucleolar fusion, and that all nucleoli rotate around nucleolus #2 (see traces of nucleoli 1 to 6 on H). The scale bars represent 10 μm in (A); 8 μm in (F). (I, J) Changes of volume and topography of six nucleoli within the same nucleus, shown in different colors. During inhibition, nucleoli decrease in size (I) and are positioned at a constant distance from each other (J).
Fig 2.
4D dynamics of UBF-GFP during inhibition of rRNA synthesis in living KB cells.
(A–D), gallery of fluorescence maximum intensity projections and 3D reconstructions displaying the dynamics of UBF-GFP during nucleolar segregation. Reorganization of UBF-GFP spots was based on their successive gathering, fusion, enlargement, and final grouping into two or three caps localized at the nucleolar periphery. Blue and red arrowheads identify two large nucleoli to demonstrate their rotation within the nucleus during the step-by-step reorganization of UBF-GFP spots and (E1-E4) their 3D visualization at times 0 and 8 h respectively. The scale bars represent 10 μm.
Fig 3.
4D visualization of UBF-GFP dynamics during inhibition of rRNA synthesis in the same living KB cells as presented on Fig 2.
(A–T) An example of 4D dynamics demonstrating successive fusion and gathering of five UBF positive spheroids (N°1 –N°5) contained in a ROI (delimited by the blue dotted line). Between 2 h 45 min and 7 h 30 min, we noticed the following steps: spheroids N°1and N°2 fused to give structure A; the latter gathered with N°3 to give B, and N°4 and N°5 finally gathered to B to give C. Note that the cap resulting from the fusion and gathering of the different spheroids is more compact than the initial region limited by the blue dotted line. The scale bar represents 2 μm.
Fig 4.
ICC network inside the nucleolar volume of one control HeLa cell expressing histone H2B-GFP.
(A) X/Y plane section of the nucleus. (B–D) Three consecutive optical sections (sections 27 (S27), 29 (S29) and 30 (S30) passing through the nucleolus. The ICC network was clearly seen (red arrow on C). (E) Y/Z plane section of the nucleus. (F-I) Two consecutive Y/Z (F, G) and X/Z (H, I) plane sections at higher magnification. Red arrows indicate strands of ICC localized in the depth of the nucleolar volume. The scale bars represent 7 μm in (A); 4.5 μm in (B-D); 5.5 μm in (E); 4.5 μm in (F-I).
Fig 5.
AMD-induced remodeling of ICC as revealed in fixed HeLa cells stably expressing H2B-GFP.
(A–E) Gradual transition of ICC from a network-like organization (red arrows) to coarse clumps during 1.5 h of AMD treatment. (A) After 15 min the ICC network was more prominent than in control cells. (B) After 30 min the ICC network began coarsening. (C) After 45 min gradual shrinkage of the ICC network and fusion of individual ICC areas into large clumps became obvious. (D, E) During the next 45 min, ICC areas transformed into single large spheroids, often in contact with the PCC shell. (F) Nucleolar capping after 3 h; only tiny ICC inclusions (red arrows) attached to the inner margin of the nucleolar cap were observed. The scale bars indicate 6 μm in (A, B); 5 μm in (C); 4.5 μm in (D); 4 μm in (E); 4.5 μm in (F).
Fig 6.
The dynamics of histone H2B-GFP during inhibition of rRNA synthesis in living HeLa cells.
This gallery of optical sections was extracted from z-stacks of corresponding time series and displayed with 15–45 min intervals to fix the main stages of ICC evolution (corresponds to S8 Movie). The nuclei of three cells in the white rectangle on the initial image (“Start of experiment”) were enlarged to visualize the evolution of the ICC in more detail. In the course of AMD treatment we observed a gradual condensation of filamentous structures of the ICC into coarse clumps during 120 min with their migration from the nucleolar interior towards the PCC shell (165 min). During this movement several ICC clumps approached each other and fused just before the coalescence with the PCC. Note that at the end of the experiment (180 min) there was no more chromatin within the nucleolus. The scale bars represent 12 μm.
Fig 7.
Spatial proximity of intranucleolar chromatin (ICC), perinucleolar chromatin (PCC), and UBF spots in fixed control cells.
(A) 3D reconstruction of the nucleus in a HeLa cell stably expressing histone H2B-GFP using volume rendering. A virtual cube inserted into the nuclear volume delineates the nucleolar territory (nl) extracted in order to analyze its interior. (B) The nucleolar volume reconstructed using simultaneous volume rendering and surface visualization of intranucleolar H2B-GFP for better visualization of ICC. After volume extraction, the nucleolar interior revealed a network of ICC interconnected with the PCC shell. (C, D) Relationships between ICC (green) and UBF spots (red) within the nucleolus of KB cells cotransfected with H2B-GFP and UBF-dsRed. These 3D views demonstrate, inside the nucleolus, the presence of the ICC network in contact with UBF spots. The scale bar represents 3.5 μm in (A, B); 5 μm in (C) and 3.5 μm in (D).
Fig 8.
Interaction between UBF spots, ICC, and PCC within the pre-segregated nucleoli of HeLa and KB cells.
(A-C) Consecutive optical sections (S.21-23) of nucleoli in HeLa cells stably expressing histone H2B-GFP. Note the prominent ICC clumps within the nucleolar interior after rRNA synthesis inhibition. These cells were immunolabeled for UBF at the end of time-lapse imaging (the corresponding nuclei are marked by red stars on A, D). (D-F) The same optical sections merged with 3D surface rendering of UBF spots (red). Note the direct contact between UBF spots and ICC inclusions. At the same time UBF spots that shifted to the nucleolar periphery became incorporated into the solid PCC shell. (G-I) 3D reconstructions of enlarged UBF spots extracted from the nucleolus of a KB cell doubly transfected with H2B-GFP and UBF-dsRed plasmids, fixed and imaged. A typical pre-segregated nucleolus containing five large UBF spots reconstructed using volume rendering (green) and surface visualization (red). Incorporation of peripherally-located UBF spots into the massive PCC shell as well as their contact with ICC can be readily recognized. Note the ICC clump that is in contact with two UBF spots. After extraction according to the virtual cubes in H, images were rotated at an appropriate angle to exemplify how UBF spots can be linked to each other by ICC. The scale bars represent 10 μm in (A); 5 μm in (G, H); 2 μm in (I).
Fig 9.
Ultrastructural pre-embedding localization of UBF within the nucleolus of control KB cells.
(A) An abundant labeling (silver/gold particles) inside several fibrillar centers (fc) (outlined by a white dotted line). A few particles (black arrows) were visible within the nucleolar dense fibrillary component (dfc), nucleolar granular component (gc), nucleoplasm, or cytoplasm. (B, C) At high magnification, numerous particles were located within fibrillar centers (fc) (outlined by white dotted lines). The dfc is outlined by a black line on C. The scale bars represent 500 nm.
Fig 10.
Ultrastructural pre-embedding localization of UBF within the nucleolus of KB cells treated with AMD for 1 h (A) or 2 h (B).
(A) Abundant labeling (silver/gold particles) inside two large ovoid fcs (outlined by a white dotted line). White arrows point to electron-transparent vacuoles (or interstices) containing small clumps of intranucleolar condensed chromatin (icc). The dfc is outlined by a black line. (B) Numerous silver/gold particles were located within three cap-shaped fcs (outlined by white dotted lines) positioned at the nucleolar periphery. Note the direct contact between large clumps of perinucleolar condensed chromatin (pcc; outlined by a dark dotted line) and two fcs. Very few particles were found on the dfc (outlined by a black line) and the gc. Scale bars represent 500 nm.
Fig 11.
Within cells treated with AMD during 1 h, the contours of nucleolar components identified on serial ultrathin sections were superposed to build 3D models of four different nucleoli (nl). Confocal images of the same nucleoli were used to confirm the identification of ICC (GFP fluorescence) and of FC (UBF fluorescence). The color-code is: nuclear and nucleolar contours—black and light blue lines; FCs—red and brown lines; ICC—black shading; NVs—green lines; DFC—dark blue lines. (A) 3D reconstruction of the equatorial part of the nucleolus in cell N°1(serial ultrathin sections on S10 Fig and serial fluorescent images shown on S14D and S14E Fig). (B, C) Two pre-segregated nucleoli in cell N°2 (serial ultrathin sections on S11 and S12 Figs and serial fluorescent images shown on S14A–S14C Fig). (D) 3D reconstruction of a nucleus with a ring-shaped nucleolus with one FC (serial ultrathin sections on S13 Fig and serial fluorescent images shown on S14F–S14I Fig).