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First published online October 30, 2006
doi: 10.1242/10.1242/jcs.03207

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Cell-cycle-dependent dynamics of nuclear pores: pore-free islands and lamins

¹ Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
² Department of Molecular Biology, BIKEN, Osaka University, Osaka, Japan
³ Laboratory for Neural Architecture, Brain Research Institute, RIKEN, Wako-shi, Japan
⁴ Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan

View larger version (112K): [in a new window]   Fig. 1. Heterogeneous distribution of nuclear pores. (A1) Low-magnification image of asynchronous HeLa S3 nuclear surfaces stained for nuclear pores with anti-p62 antibody. Note that the nuclear pore distributions are surprisingly heterogeneous from nucleus to nucleus. Inset, visualization of single nuclear pores at high magnification. (A2) Nucleus with an almost uniform pore distribution. (A3) Nucleus with a large `island' that is devoid of nuclear pores. (B) Direct observation of nuclear pores and pore-free islands by scanning electron microscopy. A nuclear surface of freeze-fractured early G1 HeLaS3 cells (inset) and its enlarged image. Many nuclear pores are clearly visible. A large pore-free island is spread on the smooth inner nuclear envelope, demonstrating the integrity of the nuclear envelope.

View larger version (35K): [in a new window]   Fig. 2. (A) Cell-cycle dependency of nuclear pore distribution in HeLaS3 cells. Nuclei were co-stained for nuclear pores (center column) and PCNA (top panels) or G2 marker CENP-F (bottom panels). The paired nuclei without PCNA signal, probably in G1 phase, possess large pore-lacking islands (top panels, small arrows), whereas the nucleus with early-S PCNA foci (large arrows) has a smaller pore-free island. The two nuclei with large and few PCNA foci (top panels, arrowheads), characteristics of mid-late S-phase nuclei, have a much more uniform pore distribution. The nucleus with CENP-F signal has uniform pore distributions (bottom panels, arrowheads). Nuclei without CENP-F have large pore-free islands on the nuclear surface. Asynchronous cells were used in these experiments. (B) Dispersal of pore-free island with cell-cycle progression. Synchronized mitotic HeLaS3 cells were collected and released at 0 hour. After 3, 6, 9, 12, 15, 18, 21, 24 hours, the number of nuclei in which more than 30% of the surface is comprised of pore-free islands, was counted and shown as a percentage. Cell-cycle profiles of the released cells at the indicated time-points were obtained with an Olympus LCS2, demonstrating cell-cycle progress in a time-dependent manner (histograms).

View larger version (68K): [in a new window]   Fig. 3. Structural organization of pore-free islands. (A) Accumulation of inner nuclear membrane proteins emerin and lamin A/C in the pore-free islands. Nuclei co-stained for nuclear pores and inner nuclear membrane proteins: emerin (top two rows), lamin A/C (third row) and lap2ß (fourth row). Emerin and lamin A/C are enriched in the pore-free islands of G1 (first and third rows) and telophase (second row) nuclei. Note that lap2ß is almost uniformly distributed on the surface of the G1 nucleus (fourth row). (B) Lamin B is excluded from the pore-free island. Surface image of the G1 nuclei (top panels) and middle section of the telophase nuclei (bottom panels), co-stained with DAPI, nuclear pores and lamin B. Merged images of nuclear pores and lamin B are shown on the right. Note that nuclear pores colocalized well with lamin B both in telophase and G1 nuclei (arrowheads).

View larger version (32K): [in a new window]   Fig. 4. Expression levels of lamin A/C affect the nuclear pore dynamics. (A) Low-magnification image of nuclear pore staining of asynchronous HeLa nuclear surfaces. The nuclear pores in most of the cells show more uniform distributions, compared with those of HeLa S3 cells shown in Fig. 1A and Fig. 3A. HeLa telophase nuclei co-stained for emerin and nuclear pores, demonstrated notable pore-free islands enriched with emerin (`core' regions) (right). (B) A-type lamins are upregulated by 4.25 times in HeLaS3 cells that contain distinct pore-free islands. Expression levels of lamin A/C in HeLaS3 cells (left two lanes) and the HeLa cells (right two lanes) shown in A were examined by western blotting. Cell lysate, equivalent to 0.5x104 (left) or 1x104 (right) cells, was loaded per lane for each cell type and verified by Coomassie Brilliant Blue (CBB) staining (bottom panel).

View larger version (69K): [in a new window]   Fig. 5. Depletion of lamin A/C specifically disperses the pore-free island. (A) Knockdown of lamin A/C by siRNA results in dispersal of the pore-free islands in HeLaS3 cells. Top panels, control cells (no RNA transfection) co-stained with nuclear pores (center) and lamin A/C (left). Bottom panels, siRNA-transfected cells co-stained with the same set of antibodies. Note that the pore-free islands in lamin A/C-depleted cells are significantly reduced. Lower images are enlarged images of a lamin A/C-reduced nucleus, showing a narrow region where residual lamin A/C has accumulated. This narrow region is almost completely devoid of nuclear pores. The histograms show DNA content of the control and knockdown cells, suggesting no significant difference in cell-cycle profile. (B) Knockdown of emerin in HeLaS3 cells. In contrast to the lamin A/C-depleted cells (A), the emerin-reduced cells still contain the large pore-free islands. This indicates that emerin itself is not directly involved in determining nuclear pore distribution.

View larger version (23K): [in a new window]   Fig. 6. Ectopic expression of lamin A induces formation of the pore-free islands. Transient expression of Venus-lamin-A in HeLa cells with uniform pore distributions. Nuclear surface images of HeLa cells (Fig. 4A) expressing Venus-lamin-A and stained for nuclear pores. Enrichment of expressed Venus-lamin-A induces distinct pore-free islands (arrows).

View larger version (43K): [in a new window]   Fig. 7. Conserved structure and dynamics of pore-free islands in human normal diploid fibroblast IMR90 cells. (A) Dynamic changes in nuclear pore distributions during the cell cycle of human normal diploid IMR90 fibroblasts. In the G1 nucleus (center), the pore distribution displayed a patched pattern (see also supplementary material Fig. S3), but upon cell-cycle progression, pore distribution becomes uniform, coupled with an increase in pore number (top left). Quiescent cells induced by serum depletion still have patched pore distribution (bottom left), whereas the senescent cell nucleus shows a uniform pore distribution with a high density of pores (right). Note that DAPI staining of the senescent nucleus shows typical heterochromatic foci in the nucleoplasm (SAHF). (B) Lamin A/C is enriched in the pore-free islands of G1 nuclei of IMR90 cells (arrows), suggesting the structural conservation of the pore-free islands. 1, DAPI; 2, nuclear pores; 3, lamin A/C; 4, merged images of nuclear pores and lamin A/C.

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