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First published online 29 May 2007
doi: 10.1242/jcs.008771

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Nucleolin functions in nucleolus formation and chromosome congression

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Osaka, Japan

View larger version (50K): [in this window] [in a new window]   Fig. 1. Dynamic localization of nucleolin during the cell cycle. (A) HeLa cells were stained for nucleolin (green) at different phases of the cell cycle. Arrowheads indicate NDFs, arrows show PNBs. (B) Metaphase chromosome spreads stained with nucleolin (green), Aurora-B (red), CREST (red), Mad2 (red) and fibrillarin (red) antibodies. Enlargements show examples of the centromeric regions. The rectangles indicate the planes where the line scans in C were performed. (C) Corresponding fluorescence intensity profiles of line scans. (D) Measurements of the distances between the centers of immunofluorescence signals for nucleolin and those for Aurora-B, CREST, Mad2 and fibrillarin. More than 50 kinetochores were measured in each experiment. Bars, 5 µm; 1.25 µm (Enlargement).

View larger version (42K): [in this window] [in a new window]   Fig. 2. Nucleolin depletion causes disorganization of nucleoli. (A) Immunoblot showing effective depletion of nucleolin by two siRNAs. Fibrillarin, B23, -tubulin and β-actin are shown as controls. (B) Quantification of nucleolin levels in the nucleoli and chromosome periphery evaluated by immunostaining. Values represent the mean of three independent experiments ± s.d. (n=10). (C) Interphase cells stained for nucleolin (green) and fibrillarin (red). (D) Interphase cells stained for B23 (green). (E) Interphase cells stained for Ki-67 (green) and fibrillarin (red).

View larger version (50K): [in this window] [in a new window]   Fig. 3. Nucleolin depletion induces spindle-checkpoint activation. (A) Mitotic indexes of control cells and cells depleted of nucleolin by siRNA1 or siRNA2. Values represent the mean of three independent experiments ± s.d. (n=400). (B) Mitotic indexes of control, nucleolin-depleted, Aurora-B-depleted and both nucleolin- and Aurora-B-depleted cells at 72 hours after control and RNAi transfections. Values represent the means of three independent experiments ± s.d. (n=400). (C) Percentages of control and nucleolin-depleted cells at different stages in the cell cycle. Values represent the mean of four independent experiments ± s.d. (n=400). (D) Mitotic cells stained for Bub1 (green) and -tubulin (red). (E) Mitotic cells stained for BubR1 (green) and -tubulin (red). (F) Mitotic cells stained for Mad2 (green) and -tubulin (red). Arrowheads in D,E and F show misaligned chromosomes and the fluorescence signals for Bub1, BubR1 and Mad2 at the kinetochores, respectively. Bars, 5 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Nucleolin depletion induces defects in chromosome congression. (A) Mitotic cells stained with an anti-nucleolin antibody. The misalignment (i) and non-alignment (ii) phenotypes are shown. (B) Metaphase chromosome spreads stained with nucleolin (green) and fibrillarin (red) antibodies. (C) Mitotic cells stained with -tubulin (green) and CREST (red) antibodies. The misalignment (i) and non-alignment (ii) phenotypes are shown. Arrowheads show misaligned chromosomes and the fluorescence signals for CREST at the kinetochores. Bars, 5 µm. (D) Quantification of the distributions of chromosome congression defects following transfections. Values represent the mean of four independent experiments (n=400). (E) Histogram showing the numbers of chromosomes that fail to align in each cell. Values represent the mean of four independent experiments ± s.d. (n=400).

View larger version (53K): [in this window] [in a new window]   Fig. 5. Nucleolin depletion induces significant reductions in centromere stretching and syntelic kinetochore-microtubule attachments. (A) Analysis of the interkinetochore distances across sister chromatids. Immunostaining for Aurora-B (central) and CENP-A (either side of Aurora-B) is shown in red and green, respectively. Bars, 5 µm; 1.25 µm (enlarged area). (B) Interkinetochore distances of non-aligned and aligned chromosomes following siRNA transfections as indicated. The interkinetochore distances were measured using CENP-A as a marker. Each value was derived by measuring at least 40 kinetochores in more than five cells. Values for non-aligned chromosomes in control cells were obtained at prometaphase. (C) Control metaphase cells stained with CREST (red) and -tubulin (green) antibodies. (D,E) Misalignment (D) and non-alignment (E) phenotypes stained with CREST (red) and -tubulin (green) antibodies. (F) Cells were cold-treated and stained with -tubulin (green) and CREST (red) antibodies.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Nucleolin depletion induces a mitotic block at prometaphase. (A,B) HeLa cells stably expressing histone GFP-H1.2 were imaged by time-lapse fluorescence microscopy after transfection of the indicated siRNAs. Times are given in minutes. DIC, differential interference contrast. (C) Histogram showing the percentages of cells with different durations of mitosis. Fifty-three control cells and 58 nucleolin-depleted cells were analyzed for time-lapse fluorescence microscopy. (D) HeLa cells stably expressing GFP-CENP-A were imaged by time-lapse fluorescence microscopy after transfection of the indicated siRNAs. Times are given in minutes and seconds. Bars, 10 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Nucleolin depletion causes mitotic spindle defects. (A) Immunoblot showing effective depletion of nucleolin and phospho-nucleolin after nucleolin RNAi. -tubulin is shown as a control. (B) Mitotic cells stained for phosphorylated nucleolin (green). (C) Mitotic cells stained for phosphorylated nucleolin (green) and -tubulin (red). (D) Mitotic cells stained for B23 (green). Arrowheads show fluorescence signals for B23 at the spindle poles. (E) Mitotic spindle defects in nucleolin-depleted cells. The majority of cells with chromosome congression defects (i, misalignment; ii, non-alignment) appear to have normal spindles, whereas a small percentage show disorganized (iii and iv) or multipolar (v) spindles. Staining for nucleolin (green) and -tubulin (red) is shown. (F) Histograms showing quantification of the spindle defects after nucleolin depletion. Values represent the mean of four independent experiments ± s.d. (n=400). (G) Nucleolin depletion induces multiple centrosomes. Staining for -tubulin (green) and -tubulin (red) is shown. Bars, 5 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Immunoblot analysis of nucleolin-binding complexes from total cell extracts and synchronized mitotic cell extracts. Nucleolin-binding complexes were pulled down from total cell extracts (A) and synchronized mitotic cell extracts (B) by an anti-nucleolin antibody. To obtain the synchronized mitotic cell extracts, cells under double-thymidine block were released for approximately 10 hours, and then lysed for immunoblotting detection. Protein G beads coupled with mouse IgG were used as a control.