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First published online 21 April 2009
doi: 10.1242/jcs.046284

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Lamin B1 maintains the functional plasticity of nucleoli

Faculty of Life Sciences, University of Manchester, MIB, 131 Princess Street, Manchester M1 7DN, UK

View larger version (50K): [in this window] [in a new window]   Fig. 1. Nucleoli are reorganized in cells with reduced lamin B1 expression. HeLa cells were co-transfected with the LB1-RNAi vector and vector expressing DsRed-H2B (red), fixed at selected times during lamin B1 depletion and immunoprocessed for fibrillarin (A,C) or lamin A/C (B) using Alexa Fluor 488-conjugated secondary antibody (green). (A) Confocal analysis shows four distinct phenotypes (normal, distorted, peri-nucleolar and cytoplasmic) at 24-72 hours post-transfection (left). Alterations in nucleolar structure are depicted schematically (center) and the distribution of the major forms is shown graphically (right). (B) After staining for lamin A/C, optical sections of two representative nuclei at 48 hours after transfection show abnormal nucleolar stretching and pronounced association of the nucleoli (white asterisks) with the nuclear periphery (see also the z-orthogonal sections). (C) Changes in the texture of fibrillarin were analyzed using confocal microscopy. Compared with controls (normal), two distinct abnormal patterns (diffuse and large foci) appeared progressively between 24-48 hours post-transfection. The relative distribution of these forms is shown graphically (left). The cartoon (right) depicts the sequential disorganization: elongated nucleoli that retain visibly normal active centers become disrupted (blue arrow) and then rearranged (green arrow) to form large peri-nucleolar aggregates. In all experiments, controls (CT) were transfected with the DsRed-H2B vector only. For each time point, 100 nuclei were scored (see supplementary material Table S1). Scale bars: 5 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Three-dimensional ultrastructure of nucleoli in cells with reduced RNA polymerase activity. HeLa cells were treated to inhibit specifically RNA polymerase II (middle column, DRB for 1.5 hours) or RNA polymerase I (right column, actinomycin D for 1.5 hours; Act. D), and the architecture and composition of the nucleoli was assessed by comparison with untreated controls (left column). (A) Optical sections of the predominant structures seen in each population when nuclei were immunostained to visualize fibrillarin (red) and either DNA-counterstained with Sytox green (DNA, green) or co-labeled for centromeres (CENP, green). Cartoons depicting the predominant structures in each population are shown. (B) High-resolution 3D views of fibrillarin (Fib-3D) were constructed from optical scans of whole nuclei. These images were used to measure the separation of individual FCs from the centers of the masses of fibrillarin foci; coordinates generated by imaging software are shown in red on the individual panels. (C) The ultrastructure of the nucleoli revealed by electron microscopy (nucleolus-EM). Active centers (red arrows) displayed a classical structure in controls but were much more heterogeneous in size (generally bigger) and found in lower numbers following DRB treatment. The structural changes seen in cells treated with actinomycin D were more dramatic, with fibrillarin accumulated in dense lentil-shaped aggregates (green arrow) at the periphery of the now spherical and amorphous residual nucleoli. The images shown focus on a central, densely staining nucleolus within a nucleus (N) that is separated from the cytoplasm (C) by a clearly defined nuclear membrane. Scale bars: 5 µm (A,B); 2 µm (C).

View larger version (66K): [in this window] [in a new window]   Fig. 3. Changes in nucleolar structure are freely reversible in wild-type and lamin A/C-depleted cells. The plasticity of nucleoli was assessed by evaluating the reversibility of nucleolar organization in HeLa cells treated with DRB. (A) HeLa cells grown on coverslips were treated with DRB for 2 hours to inhibit RNA polymerase II and then grown for 2 hours in medium without DRB. Samples were immunoprocessed for fibrillarin (red) and DNA-counterstained with Sytox green (green) at the times indicated (blue arrows) in the incubation scheme (top). Confocal analysis shows that fibrillarin, which is massively disorganized when RNA polymerase II is inhibited, quickly recovered the normal structure when transcription resumed. The same structural transitions were seen in HeLa cells with reduced expression of A-type lamins (B-E). In contrast to the dramatic loss in nucleolar structure seen when lamin B1 was depleted, a HeLa cell line that constitutively expresses only 5% of the natural level of lamin A/C (LA/C kd; B) maintained normal nucleolar architecture, as shown by immunostaining for fibrillarin (Fib, red; C), and a normal distribution of centromeric repeats (CENP, green; C). Nucleoli with classical morphology (D; red arrows indicate active centers that are embedded with the granular component) were clearly seen in the nuclei (N) of lamin A/C-deficient cells and, when transcription was inhibited using DRB and later allowed to recover, these nucleoli showed the same structural transitions (E) as control cells (A). Scale bars: 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Lamin proteins are retained in purified nucleoli. (A-C) Immunoblotting was used to monitor the relative concentration of the A- and B-type lamin proteins in nuclear fractions isolated from HeLa cells. Different nuclear fractions (N, nucleus; NE, nuclear envelope; NM, nuclear matrix; No, nucleolus) were isolated using established protocols. (A) As a quality control, isolated nucleoli (nucleolus extract) were shown to retain the same size, structure and organization as the nucleoli of untreated cells (whole cell) when analyzed by indirect immunostaining. Bright-field images (left) and optical sections (right) are shown together with a 2x magnification of a typical nucleolus (middle row, from boxed areas in the top row) to show that the fibrillarin-stained active centers (Fib, green) and nucleophosmin-stained granular component (Nucleoph, red) are maintained throughout isolation; the separation of active sites was determined as described (see Fig. 2 legend). (B,C) Following purification, nuclear fractions (7 µg/lane) were separated by gel electrophoresis and the presence of A- and B-type lamins was determined by immunoblotting (left). The same approach was applied to isolated nucleoli after treating cells for 2 hours with DRB or actinomyicn D (Act. D), using untreated controls (Ct) for comparison (right). The changes in the lamin protein concentration (C) were assessed by calculating the intensity ratio of lamin B1 (LB1) relative to lamin A and C (LA/C). Note that although this analysis shows the relative content of A- and B-type lamins in different fractions, it is not designed to provide a quantitative distribution of the proteins during fractionation (see text). Based on the cell numbers used and the product yields, nucleoli contain <2% of the lamin polypeptides in HeLa cells. Scale bars: 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Distribution of lamin B1 in extracted nucleoli. Nucleoli were extracted, deposited on glass slides and immunoprocessed. (A-C) Immunolocalization shows lamin B1 (LB1, green in A and cyan in C) to be dispersed throughout the GC as small discrete foci that localize to the periphery of the FC-DFC complexes that have been stained with fibrillarin (Fib, red). (B) The localization seen by immunostaining was confirmed in nucleoli isolated from cells expressing GFP-conjugated lamin B1. In both cases, the majority of foci have a similar structure, although a small number of bright foci (indicated by arrows in B and C) also localize to the FC-DFC borders. (C) As a staining control, isolated nucleoli and nuclei from GFP–LB1-expressing cells were mixed before immunostaining. In both nucleoli in situ and after isolation, the sites with GFP-LB1 co-localized with lamin B1-containing sites, as defined by immunostaining (LB1, cyan); note the very bright staining of the nuclear lamina in the nuclei, which was never seen in preparations of purified nucleoli. Magnified images (lower panels in B and C show a 2.5x magnification of box regions in the upper panels) emphasize the distribution of labeled sites. Scale bars: 5 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Lamin B1 preferentially associates with nucleophosmin within the nucleolar compartment. HeLa cells were processed for IP and protein complexes linked to lamin B1 or lamin A/C were separated by electrophoresis. Two major GC proteins (nucleolin and nucleophosmin) were then detected by immunoblotting (A). In three separate experiments, nucleophosmin showed a clear association with the nuclear lamin IP fractions, particularly lamin B1 (exposure 40 minutes). No detectable signal was seen when immunoblotting was performed using antibodies to nucleolin (A). IP, IP fraction with either lamin A/C (LA/C) or lamin B1 (LB1); N, nuclear extract (positive control for blotting); (–), IgG (negative control for IP). (B) Proteins from nucleoli isolated after drug treatment to inhibit RNA polymerase I (Act. D) or II (DRB) were inspected after separation by 2D gel electrophoresis. The intensity of the major nucleolar protein nucleophosmin (upper panel, enlargement below) increased by 1.5-fold after DRB and by 2.5-fold after Act. D treatment. As a loading control, actin spots from the same gels do not show any intensity variations (lower panel, actin indicated by the arrows on the corresponding enlargement). Images shown are representative of typical gels from experiments performed on at least three occasions.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Model for a lamin B1-nucleophosmin network maintaining nucleolar architecture. The model depicts a schematic view of nucleolar architecture and changes during transcriptional stress. Under physiological conditions (control), the active sites of rDNA synthesis are contained within fibrillarin-rich FC-DFC complexes (green beads) and surrounded by a granular component (GC) that is rich in nucleophosmin (dotted red). This component interacts with a lamin-containing filament network (blue) through an interaction between nucleophosmin and lamin B1 (blue stars). When RNA polymerase II activity is inhibited and the demand for rRNA falls, the normally highly structured active centers unfold and global nucleolar structure is disrupted. Clusters of rDNA genes that normally occupy a single FC-DFC complex are now distributed throughout the GC. Quite different structural changes are seen when RNA polymerase I activity is inhibited. When rRNA synthesis is completely switched off, the FC-DFC complexes are no longer present and fibrillarin accumulates in large peri-nucleolar aggregates. This structural change correlates with increased retention of nucleophosmin and lamin B1 in residual nucleoli, suggesting that the concentration of these components is normally regulated by the dynamic properties of nucleolar proteins. The model explains how reduced lamin B1 expression leads to loss of nucleolar structure so that fibrillarin can diffuse throughout the cell. Under these conditions, the spherical residual nucleoli seen during RNA polymerase I arrest are not seen, suggesting that this level of organization is dependent on lamin B1-nucleophosmin interactions in the GC. In the presence of NORs that position the rRNA genes, the structural framework formed by the interaction of lamin B1 with nucleophosmin would form an underlying architecture on which the localization and concentration of nucleophosmin is sufficient to drive the assembly of nucleoli as a consequence of the self-organization properties of the major nucleolar proteins. The changes seen when transcription is compromised imply that the underlying architecture is also locally dynamic. Hence, our model supports the dynamic properties of nucleolar components based on their biophysical characteristics, and explains how the nuclear localization and organization of nucleoli respond to any changing demand for RNA synthesis.

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