Nuclear pore formation but not nuclear growth is governed by cyclin-dependent kinases (Cdks) during interphase

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
1065–1071
Year published:
DOI:
doi:10.1038/nsmb.1878
Received
Accepted
Published online

Abstract

Nuclear volume and the number of nuclear pore complexes (NPCs) on the nucleus almost double during interphase in dividing cells. How these events are coordinated with the cell cycle is poorly understood, particularly in mammalian cells. We report here, based on newly developed techniques for visualizing NPC formation, that cyclin-dependent kinases (Cdks), especially Cdk1 and Cdk2, promote interphase NPC formation in human dividing cells. Cdks seem to drive an early step of NPC formation because Cdk inhibition suppressed generation of 'nascent pores', which we argue are immature NPCs under the formation process. Consistent with this, Cdk inhibition disturbed proper expression and localization of some nucleoporins, including Elys/Mel-28, which triggers postmitotic NPC assembly. Strikingly, Cdk suppression did not notably affect nuclear growth, suggesting that interphase NPC formation and nuclear growth have distinct regulation mechanisms.

At a glance

Figures

  1. NPC density and nuclear volume increase during the cell cycle.
    Figure 1: NPC density and nuclear volume increase during the cell cycle.

    (a) Nuclear surface image of a HeLa cell stained with anti-Nup153 antibody. In the boxed area, individual NPCs are circled. Scale bar, 5 μm. (b) Nuclear regions were segmented and extracted to measure nuclear volumes using a newly developed software package (see Supplementary Methods). Iso-surface models of the extracted regions are shown. (c) Bar graph of NPC density on the nuclear surface of HeLa cells at various intervals after mitotic release (N = 10 for each time point). Error bars, s.d. (d) Bar graph of measured nuclear volumes in HeLa cells at various intervals after mitotic release (for each point, N = 16). Error bars, s.d. (e) Bar graph of NPC density in aphidicolin- (Aphi) and roscovitine (Ros)-treated cells and untreated G2 HeLa cells (for each condition, N = 12). Error bars, s.d. (f) Bar graph of measured nuclear volumes in the indicated HeLa cell-cycle phases or after drug treatment (N = 16 for each condition). PD, PD98059-treated cells; error bars, s.d. (g) Bar graph of NPC density in human normal fibroblasts (TIG-1). NPC density in G0 (serum starvation), early G1, G2 (untreated cells) and Aphi- or Ros-treated TIG-1 cells is shown (N = 10 for each condition). Error bars, s.d. (h) Bar graph of measured nuclear volumes of TIG-1 cells in the indicated cell-cycle phases or after drug treatment (N = 20 for each condition). Error bars, s.d.

  2. Heterokaryon technique for visualization of NPC formation.
    Figure 2: Heterokaryon technique for visualization of NPC formation.

    (a) Schematic representation of the heterokaryon procedure. HeLa cells expressing Nup133-YFP or YFP-Nup107 are used as donor cells, and HeLa cells expressing CFP-H2B are used as acceptor cells. G1-synchronized donor and acceptor cells are treated with 50% (w/v) PEG to make heterokaryons. Many bright fluorescent dots should appear on the acceptor nuclear surface, which has the brighter CFP-H2B signal, of Aphi-treated heterokaryon cells. (b) Immediately after fusion (0 h), donor nuclei derived from Nup133-YFP–expressing cells show many bright fluorescent dots representing NPCs (panel 1), whereas no detectable dots are observed on the acceptor nuclei (panel 2). Relative mean intensity in the rim regions (acceptor/donor) is shown in the brackets on the image (panel 2). Scale bar, 10 μm. (c) Eighteen hours after fusion (panels 4 and 5), many bright fluorescent dots are present on the acceptor nuclear surfaces of Aphi-treated heterokaryon cells. Acceptor nuclei in Ros-treated heterokaryon cells (panel 6) show fewer fluorescent dots. The numbers in the brackets are calculated dot densities on the nuclear surfaces (panels 4–6). Scale bar, 10 μm. Images of middle optical section are also shown (panels 10–12). The relative rim intensity (acceptor/donor) is shown in the brackets on the image (panels 10, 11 and 12). (d) Quantitative analysis on the relative rim intensity in Aphi- and Ros-treated HeLa cells (N = 10 for each condition).

  3. Cdk-dependent NPC formation during interphase.
    Figure 3: Cdk-dependent NPC formation during interphase.

    (a) Top, schematic representation of the photobleaching method. Nuclear surface regions in G1-phase cells expressing Nup133-YFP or YFP-Nup107 are photobleached by a 488-nm laser. Sixteen hours after bleaching, many bright YFP dots should appear in the bleached areas. Bright Nup133-YFP dots are observed in G1-phase cells treated with aphidicolin (panels 11 and 12) but not in those treated with roscovitine (panels 13 and 14). In G1-phase cells treated with purvalanol A at 30 μM, bright Nup133-YFP dots are not observed (panel 15). Relative mean intensity (bleached area/unbleached area) is shown in the brackets on the image (panels 11–15). Scale bar, 10 μm. (b) Roscovitine affects NPC formation in S-phase cells. YFP-Nup107–expressing donor cells and CFP-H2B–expressing acceptor cells were synchronized at G1/S by a 24-h exposure to thymidine and then fused using PEG. The cells were then incubated for 18 h with aphidicolin or roscovitine. Brighter YFP-Nup107 signals are present on the acceptor rims in the Aphi-treated cells (panels 5 and 6). By contrast, the acceptor rims of the Ros-treated heterokaryons have weaker or no detectable YFP-Nup107 signals (panels 7 and 8). Relative mean intensity (acceptor rim/donor rim) is shown in the brackets on the image (panels 5–8). Scale bar, 10 μm. (c) Bar graph of the relative mean intensity in Aphi- and Ros-treated cells is shown (N = 11 for each condition). Error bars, s.d.

  4. Cdk-dependent NPC distribution and direct observation of NPC structures.
    Figure 4: Cdk-dependent NPC distribution and direct observation of NPC structures.

    (a) The distribution of NPCs and lamin B, but not of lamin A/C, is governed by Cdk activity. HeLa S3 cells, which have prominent pore-free islands, were synchronized at G1 phase and treated with either roscovitine or aphidicolin. The cells were then immunostained using antibodies against Nup62 (left column) and lamin A/C or laminB1 (center column). No notable accumulation of lamin A/C was observed in the pore-free island (arrowheads) of the Cdks-inhibited cells. The lamin B1 staining signal was colocalized with the pore regions (arrowheads) of Cdk-inhibited cells (center panel). Scale bar, 20 μm. (b) Direct observation of NPC structures. Aphi-treated HeLa cells were freeze-fractured and observed by cryo-SEM. A number of pores much smaller than usual (nascent pores) are present on the fractured nuclear surfaces of Aphi-treated cells (arrows). Left inset, image of entire nucleus; right inset, pore size distribution on the fractured nuclear surfaces. Size of NPCs, including the nascent pores, was manually measured (number of NPCs counted, N = 1,000) and shown as a histogram. We defined NPCs with a diameter less than 90 nm as the nascent pores. The nascent pores that we observed are considered to be a rate-limiting state of immature NPCs under construction. (c) High-resolution images of nascent pores (arrowheads) are shown, suggesting a structure with four-fold symmetry (asterisks in insets).

  5. Cdks control expression and localization of several nucleoporins.
    Figure 5: Cdks control expression and localization of several nucleoporins.

    (a) Expression of the nucleoporins in Aphi- and Ros-treated cells. For quantitative western blot analysis, the integrated intensity of each signal was measured and normalized to the β-actin signal. The aphidicolin:roscovitine signal ratios are shown. (b) Nup62, Nup107 and Nup133 foci colocalize in the cytoplasm of Ros-treated cells (upper two rows). Scale bar, 10 μm. (c) The fluorescence intensity of Elys/Mel28 at nuclear rim was weaker by a factor of 2.29 in the roscovitine-treated cells (panel 4) than in the aphidicolin-treated cells (panel 3). The middle optical section is shown. Images are shown on the same intensity scale. Scale bar, 10 μm. (d) Surface images of Ros-treated cells with immunofluorescence labeling of Nup62 and Elys/Mel28. Images are shown on the same intensity scale except that signals of Elys/Mel28 immunostaining in Cdk-inhibited cells (center) were enhanced for visualization (lower part). Insets, enlarged images of Elys/Mel28 immunostaining. Scale bar, 10 μm. (e,f) NPC formation is impaired in Cdk1/2-depleted cells. In the cells treated with Cdk1- and Cdk2-specific siRNAs for 72 h, their nuclear surfaces were subjected to photobleaching (e, center panel) and imaged 16 h later to examine NPC formation ability (e, right panel). Relative mean intensity (bleached area / unbleached area) is shown in the brackets on the image. Immunostaining of the same bleached cells as in e (shown by arrows) was performed to verify depletion of Cdk1 and Cdk2 by their antibodies (f). Arrowheads, control cell; scale bars, 10 μm.

  6. Cdk activity is not required for postmitotic NPC assembly.
    Figure 6: Cdk activity is not required for postmitotic NPC assembly.

    Mitotic HeLa cells expressing Nup62-YFP, which were arrested with nocodazol, were treated with roscovitine for 2 h. (a) Control mitotic cells without roscovitine stained with anti-lamin A/C antibody. (b) After the treatment, apparent nuclear rim localizations of lamin A/C (1st row), Elys (2nd row), Nup62 (first three rows) and Nup153 (3rd row) were observed by immunostaining with the indicated antibody. Colocalization of most of the Nup62 and Nup153 signals on the nuclear surface show NPC assembly in the induced postmitotic (i-postmitotic) phase (4th row), showing distinct roles of Cdks in NPC formation between the postmitotic phase and interphase. Scale bar, 10 μm.

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Author information

Affiliations

  1. Cellular Dynamics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan.

    • Kazuhiro Maeshima,
    • Haruki Iino,
    • Saera Hihara,
    • Tomoko Funakoshi,
    • Ai Watanabe &
    • Naoko Imamoto
  2. Biological Macromolecules Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka, Japan.

    • Kazuhiro Maeshima &
    • Saera Hihara
  3. Department of Genetics, School of Life Science, Graduate University for Advanced Studies (Sokendai), Mishima, Shizuoka, Japan.

    • Kazuhiro Maeshima &
    • Saera Hihara
  4. Live-cell Molecular Imaging Research Team, RIKEN Advanced Sciences Institute, Wako, Saitama, Japan.

    • Tomoko Funakoshi
  5. Bio-research Infrastructure Construction Team, VCAD System Research Program RIKEN, Wako, Saitama, Japan.

    • Masaomi Nishimura &
    • Hideo Yokota
  6. Support Unit for Neuromorphological Analysis, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    • Reiko Nakatomi &
    • Tsutomu Hashikawa
  7. Department of Molecular Biology, BIKEN, Osaka University, Suita, Osaka, Japan.

    • Kazuhide Yahata &
    • Fumio Imamoto
  8. Present addresses: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany (H.I.) and Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan (K.Y.).

    • Haruki Iino &
    • Kazuhide Yahata

Contributions

K.M. designed the experiments; K.M., H.I. and S.H. performed most of the experiments; A.W. assisted with some experiments; R.N., K.M. and T.H. performed cryo-SEM observations; M.N., K.M. and H.Y. carried out quantitative analyses; T.F., K.Y. and F.I. made some materials; N.I. advised throughout the study; K.M. and N.I. wrote the paper.

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The authors declare no competing financial interests.

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    Supplementary Figures 1–7 and Supplementary Methods

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    Time-lapse movie of HeLa cells stably expressing H2B–mRFP1 and EGFP

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