Research ArticleCELL BIOLOGY

Nucleolar integrity during interphase supports faithful Cdk1 activation and mitotic entry

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Science Advances  06 Jun 2018:
Vol. 4, no. 6, eaap7777
DOI: 10.1126/sciadv.aap7777
  • Fig. 1 NOL11 depletion delayed mitotic entry irrespective of proper accumulation of cyclin B1.

    (A) The efficiency of NOL11 depletion using siRNA against NOL11. HeLa cells were transfected with luciferase siRNA (siCont.) or two distinct NOL11-specific siRNAs (siNOL11 #1 and #2). Forty-eight hours after transfection, whole-cell extracts were immunoblotted using the indicated antibodies. A dilution series of cell extracts from control cells (siCont.; lanes 1 to 5; 100, 50, 25, 12.5, and 6.3%, respectively) were performed simultaneously to estimate depletion efficiency. β-Actin was used as a loading control. (B) Arrest at the G2/M phase in NOL11-depleted cells. HeLa cells transfected with the indicated siRNAs were synchronized to the G1/S phase. After release from the thymidine block, the cells were collected at the indicated times, and the cell cycle distribution was measured by FACS using PI. (C) Delayed mitotic entry in NOL11-depleted cells. The top panel shows the scheme for siRNA treatment and cell cycle synchronization by double thymidine block. HeLa cells released from the G1/S phase were collected at the indicated times after release. The collected cells were divided into three aliquots, and each aliquot was analyzed by dual-color FACS using a combination of PI and anti–cyclin B1, anti-MPM2, or anti–H3-pS10 antibodies. The populations of 4N–cyclin B1+ cells, 4N-MPM2+ cells, and 4N–H3-pS10+ cells are indicated as colored squares in the representative plots (right panels). Line graphs indicate the percentage of these populations at the indicated time (lower left graphs). Values are shown as the means ± SD, n = 3.

  • Fig. 2 NOL11 depletion impaired Cdk1 activation.

    (A) Delayed Cdk1 activation after release from the G2/M border in NOL11-depleted cells. 4N-MPM2+ cells were identified by dual-color FACS. siRNA-treated HeLa cells synchronized at the G2/M border were released from the block and collected at the indicated times. Colored squares show 4N-MPM2+ cells in the representative plots at 6 hours after release (left panels). The right graph shows the percentage of 4N-MPM2+ cells at the indicated times after release from the G2/M border. Values are expressed as means ± SD, n = 3. (B) Increased Cdk1-pY15 in NOL11-depleted cells. Cells were synchronized and collected as shown in (A). The whole-cell extracts were immunoblotted with the indicated antibodies. (C) Delayed nuclear translocation of cyclin B1 and NEBD in NOL11-depleted cells. HeLa cells were released from RO-3306 synchronization. At the indicated times, cells were fixed and stained with anti–cyclin B1 antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Arrows and arrowheads indicate cyclin B1 translocated into the nucleus and cells with NEBD, respectively. Scale bars, 10 μm. The percentage of cyclin B1 translocated into the nucleus (upper right graph) and NEBD (lower right graph) is shown. Over 200 cells were counted at each time point for each siRNA.

  • Fig. 3 NOL11 depletion caused accumulation of Wee1.

    (A) Increased Wee1 levels caused by NOL11 depletion. HeLa cells were treated with the siRNAs for NOL11 and synchronized at the G2/M border. Whole-cell extracts were immunoblotted with the indicated antibodies. (B) Reduced Wee1 turnover by NOL11 depletion. NOL11-depleted cells were synchronized by single thymidine block. Six hours after release, cells were treated with 10 μM RO-3306 and collected at the indicated times for immunoblotting. The number in the upper immunoblot panels shows hours after the treatment of RO-3306.

  • Fig. 4 Nucleolar disruption by TIF-IA or UBF depletion also delayed mitotic entry.

    (A) Nucleolar disruption caused by TIF-IA or UBF depletion in addition to NOL11 depletion. HeLa cells were transfected with the indicated siRNAs and cultured for 48 hours. The cells were stained for immunofluorescence with anti-nucleolin antibodies (green). DNA was counterstained by DAPI (blue). Scale bars, 5 μm. (B) Delayed mitotic entry in cells with the disrupted nucleolus. HeLa cells were treated with the indicated siRNAs and synchronized at the G1/S boundary by double thymidine as shown in Fig. 1C. Nine, 10.5, and 12 hours after release from G1/S synchronization, the cells were collected for analysis of mitotic index by FACS. Upper histograms showed the results of TIF-IA or UBF depletion. Bottom histograms showed the results of NOL11 depletion, which is from the result in Fig. 1C. Values are shown as means ± SD, n = 3. (C) Increased Cdk1-pY15 in cells with the disrupted nucleolus. HeLa cells were treated with the indicated siRNAs and released from the G2/M border as the same protocol shown in Fig. 2A. The whole-cell extracts from the collected cells at the indicated times were immunoblotted with the indicated antibodies. (D) Delayed nuclear translocation of cyclin B in cells with the disrupted nucleolus. HeLa cells were transfected with the indicated siRNAs. The percentage of the cells in which cyclin B1 translocated into the nucleus is shown. Upper histograms showed the results of TIF-IA or UBF depletion. Bottom histograms showed the results of NOL11, which is from the result in Fig. 2C. Over 200 cells were counted at each time point for each siRNA.

  • Fig. 5 Nucleolar disruption caused aberrant Wee1 accumulation and delayed mitotic entry.

    (A) Increased Wee1 levels caused by nucleolar disruption. HeLa cells were treated with the indicated siRNAs and synchronized at the G2/M border. Whole-cell extracts were immunoblotted with the indicated antibodies. (B) Reduced Wee1 turnover by nucleolar disruption. Cells were treated as Fig. 3B and examined by immunoblotting with the indicated antibodies. The number in the upper immunoblot panels shows hours after the treatment of RO-3306. (C) Reduction of enhanced Cdk1-pY15 by MK1775 treatment. Cells treated with indicated siRNAs were synchronized at the G2/M border. Subsequently, the cells were treated with 100 nM MK1775 for 3 hours and collected for immunoblotting using the indicated antibodies. (D) Restoration of mitotic entry by the Wee1 inhibitor. HeLa cells were treated with or without 100 nM MK1775 at 6 hours after release from the double thymidine block. The cells were collected at 9 hours after release from the double thymidine block, and mitotic index was examined using FACS. Values are shown as mean ± SD, n = 3.

  • Fig. 6 Nucleolar disruption is a leading cause of delayed mitotic entry.

    (A) Knockdown efficiency of siRNA for rRNA transcription factor or processing factors. HeLa cells transfected with the indicated siRNAs were cultured for 48 hours and immunoblotted by the indicated antibodies. (B) The alteration of nucleolar structure by depletion of rRNA transcription factor, not by processing factors. HeLa cells were transfected with the indicated siRNAs and cultured for 48 hours. The cells were stained with anti-nucleolin (green) and anti-MYBBP1A (red) antibodies. DNA was counterstained by DAPI (blue). Scale bars, 5 μm. (C) Increased Wee1 and Cdk1-pY15 in cells with the disrupted nucleolus. HeLa cells were synchronized at the G2/M border after siRNA transfection and immunoblotted using the indicated antibodies. (D) Delayed mitotic entry in cells with the disrupted nucleolus. HeLa cells were treated with the indicated siRNAs and synchronized by double thymidine as the same protocol shown in Fig. 1C. Nine hours after the release from G1/S synchronization, the cells were collected for analysis of mitotic index using flow cytometry. (E) Correlation between nuclear disruption and delayed mitotic entry. HeLa cells were transfected with the indicated siRNAs and cultured for 48 hours. Thereafter, cells were incubated in the presence of HPG for 2 hours and collected for the analysis by flow cytometry. Values are shown as means ± SD, n = 3. In parallel, the nucleolar disruption with reference to (B) and Fig. 4A and the delayed mitotic entry with reference to (D) and Figs. 1C and 4B are shown under the graph [+, nucleolar disruption induced (top); −, nucleolar disruption not induced (top); +, delayed mitotic entry induced (bottom); −, delayed mitotic entry not induced (bottom)]. a.u., arbitrary units; CHX, cycloheximide.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaap7777/DC1

    fig. S1. NOL11 predominantly localized in the DFC region within the nucleolus.

    fig. S2. NOL11 depletion induced down-regulation of rRNA transcription and nucleolar disruption.

    fig. S3. NOL11 depletion induced nucleolar disruption without undergoing mitosis.

    fig. S4. Nucleolar disruption is caused by NOL11 depletion alone.

    fig. S5. Treatment with Act D delayed mitotic entry.

    fig. S6. The treatment with MK1775 restored the levels of Cdk1-pY15 and the nuclear translocation of cyclin B1.

    fig. S7. Depletion of NOL11, TIF-IA, or UBF and Act D treatment did not induce DNA damage nor activate G2/M checkpoint.

    fig. S8. Codepletion of RPL11 suppressed nucleolar disruption and restored the levels of Cdk1-pY15 caused by NOL11 or TIF-IA depletion.

    table S1. List of siRNAs that increased H3-pS10 levels in asynchronous cultures.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. NOL11 predominantly localized in the DFC region within the nucleolus.
    • fig. S2. NOL11 depletion induced down-regulation of rRNA transcription and nucleolar disruption.
    • fig. S3. NOL11 depletion induced nucleolar disruption without undergoing mitosis.
    • fig. S4. Nucleolar disruption is caused by NOL11 depletion alone.
    • fig. S5. Treatment with Act D delayed mitotic entry.
    • fig. S6. The treatment with MK1775 restored the levels of Cdk1-pY15 and the nuclear translocation of cyclin B1.
    • fig. S7. Depletion of NOL11, TIF-IA, or UBF and Act D treatment did not induce DNA damage nor activate G2/M checkpoint.
    • fig. S8. Codepletion of RPL11 suppressed nucleolar disruption and restored the levels of Cdk1-pY15 caused by NOL11 or TIF-IA depletion.
    • table S1. List of siRNAs that increased H3-pS10 levels in asynchronous cultures.

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