The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex

Abstract

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.

Figure 1.

Seh1 and the Nup107 complex are required for chromosome alignment and completion of cytokinesis in human cells. (A) Depletion of Nup107 by siRNA causes major chromosome alignment defects and binucleation. Selected maximum intensity projections from time-lapse images show the mitotic and cytokinesis defects. HeLa^EGFP-Seh1 cells (in green) were transfected with mRFP-H2B to label chromatin (in red) and either control or Nup107 siRNAs. Images were collected at 60 h for a period of 120 min. Numbers indicate time in h:min:s. White arrow points to the cleavage furrow ingression and formation of binucleated cell. Scale bar, 10 μm. (B) Mitotic progression plots of metaphase onset (blue bars) and anaphase onset (red bars) from live HeLa^EGFPH2B cells treated with control, Nup107, and Seh1 siRNAs. NEBD is set as T = 0. Data were collected after 50 h of RNAi treatment for a period of 2 h every 2 min. (C) Percentage of HeLa^EGFP-H2B cells treated with control, Nup107 and Seh1 siRNAs having uncongressed chromosomes at T = 24 min (time of anaphase onset of control cells). Images were collected after 50 h of RNAi treatment for a period of 2 h every 2 min. (D) HeLa cells transfected with control or Seh1 siRNAs were incubated for 48 h before phase-contrast imaging for further 16 h. Images were acquired every 20 min. Note the normal cell division of control cells (white arrow) and the binucleated cells forming (red arrow) or already existing (black arrow) in Seh1-depleted cells. Scale bar, 10 μm. (E) The percentage of cells with a clear cytokinesis bridge and two nuclei, in control and Nup107 complex members depleted cells (siRNAs against Nup107, Seh1, Mel28, Nup133, Nup37, Nup43) were determined 72 h after transfection. At least 300 cells were counted in each category. Cells were stained with α-tubulin, α-Nup37, α-Nup43, α-Mel28, α-Nup107, α-Seh1, α-mAb414, and DNA (DAPI). Corresponding immunofluorescence images of control and Seh1-depleted cells stained with antibodies to tubulin (red) and DNA (blue) are shown. Scale bar, 10 μm.

Figure 2.

kMT attachments are maintained in Seh1-depleted cells. (A) Selected 60-nm EM sections from control or Seh1 siRNA-treated cells 65 h after transfection. Note the prominent kinetochore fibers (black arrowheads) that terminated within the trilaminar plate both in control and Seh1-depleted cells. Black scale bar, 1 μm; white scale bar, 500 nm. (B) Quantification of number of kMT fibers attached per kinetochore in EM sections of control (red squares) and Seh1-depleted (blue square) cells. Eighteen kinetochores were counted from six different cells in three different experiments per condition. (C) CENP-E localization is unaffected upon Seh1 depletion. Immunofluorescence images of control and Seh1-depleted cells are shown as maximum intensity projections or individual 3D sections. Microtubules are shown in red, chromosomes in blue, and CENP-E in green. Scale bar, 10 μm. (D and E) Immunofluorescence analysis of control (i–iv) and Seh1-depleted cells (v–viii) after incubation at 37°C (D), or cold stable microtubule assay at 4°C (E), stained with α-tubulin (red) and α-ACA (green). Scale bar, 10 μm.

Figure 2.

kMT attachments are maintained in Seh1-depleted cells. (A) Selected 60-nm EM sections from control or Seh1 siRNA-treated cells 65 h after transfection. Note the prominent kinetochore fibers (black arrowheads) that terminated within the trilaminar plate both in control and Seh1-depleted cells. Black scale bar, 1 μm; white scale bar, 500 nm. (B) Quantification of number of kMT fibers attached per kinetochore in EM sections of control (red squares) and Seh1-depleted (blue square) cells. Eighteen kinetochores were counted from six different cells in three different experiments per condition. (C) CENP-E localization is unaffected upon Seh1 depletion. Immunofluorescence images of control and Seh1-depleted cells are shown as maximum intensity projections or individual 3D sections. Microtubules are shown in red, chromosomes in blue, and CENP-E in green. Scale bar, 10 μm. (D and E) Immunofluorescence analysis of control (i–iv) and Seh1-depleted cells (v–viii) after incubation at 37°C (D), or cold stable microtubule assay at 4°C (E), stained with α-tubulin (red) and α-ACA (green). Scale bar, 10 μm.

Figure 3.

Seh1 regulates the localization of Aurora B and the chromosome passenger proteins on mitotic chromosomes and centromeres. (A) Aurora B is delocalized in Seh1- and Mel28-depleted cells. Control, Mel28, and Seh1-depleted cells were fixed and immunostained with α-Aurora B (green), α-tubulin (red), and DNA (blue). Note the staining of Aurora B on misaligned chromosomes. Scale bar, 10 μm. (B) Quantification of Aurora B levels from control (blue bars) and Seh1-depleted cells (red bars) on aligned and unaligned chromosomes. Quantification was performed on 3D data sets. Significance: p values from Student's t test are shown; n = 40 from two different experiments. (C) Survivin is delocalized in Seh1-depleted cells. Control and Seh1-depleted cells were immunostained with α-survivin (green), α-tubulin (red), and DNA (blue). (D) Quantification of survivin levels from control (blue bars) and Seh1-depleted cells (red bars) on aligned and unaligned chromosomes. Quantification was performed on 3D data sets. Significance: p values from Student's t test are shown; n = 32 from two different experiments. (E) Hec1 localization is not affected upon Seh1 depletion. Control and Seh1-depleted cells were immunostained with α-Aurora B (green) and α-Hec1 (red) to visualize kinetochores and DNA (blue). Scale bar, 10 μm. (F) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control, Seh1, Nup107, and Mel28 (using α-Seh1, α-Mel28 antibodies), shows efficient depletion of Seh1 and Mel28 but no down-regulation of Aurora B, survivin, or Plk1 (using α-Aurora B, α-survivin, α-Plk1). (G) FRAP analysis of centromeric association of Aurora B. Scatter plot of t_1/2 (horizontal lines) for Aurora B in control and Seh1-depleted cells on unaligned centromeres. Aurora B recovery times on centromeres of Seh1-depleted cells are significantly longer than on centromeres of control cells.

Figure 4.

Nocodazole and taxol treatment do not suppress the Seh1-depletion phenotype. Control and Seh1-depleted cells, without nocodazole treatment (A and B) or with 12-h nocodazole treatment (C and D) were stained for survivin (green), BubR1 (red), and DNA (blue). Control (E) and Seh1-depleted cells (F) were treated with taxol and stained for tubulin (red) and ACA (green). Insets (optical sections), kinetochore-associated microtubules. Control (G and H) and Seh1-depleted cells (I and J) were treated with taxol and stained for Aurora B (green) and DNA (blue). Scale bar, 10 μm.

Figure 5.

MCAK localization and phosphorylation is affected in Seh1-depleted cells. Control and Seh1-depleted cells immunostained with α-Aurora B (red) and α-MCAK (green) for total MCAK population and DNA (blue) are shown in A. Cells stained for phospho-Ser92-MCAK levels shown in C. Aurora B phosphorylation of MCAK is seen on misaligned chromosomes after Seh1 depletion. Arrows, misaligned chromosomes both in control and Seh1-depleted cells. Quantification of MCAK (B) and P-MCAK (D) levels from control (blue bars) and Seh1-depleted cells (red bars) on aligned and unaligned chromosomes. Quantification was performed on 3D data sets. Significance: p values from Student's t test are shown; n = 20 from two different experiments.

Figure 6.

Seh1-depleted cells are able to achieve bipolarization after monastrol release. Control (a and b) and Seh1-depleted cells (c and d) after removal of monastrol were incubated for 1 h with (b and d) or without ZM447439 (a and c) and then processed for immunofluorescence. Tubulin is shown in red, ACA in green, and DNA in blue. Insets (optical sections), individual kinetochores with syntelic attachments. Quantification of syntelic errors after monastrol release is shown in panel e; quantification of cells with merotelic chromosomes after monastrol release in control and Seh1-depleted cells is shown in panel f. Inhibition of Aurora B does not suppress the Seh1 depletion phenotype. Control (g–j) and Seh1-depleted cells (k–n) were incubated with (i, j, m, and n) or without ZM447439 (g, h, k, and l) and stained for Aurora B (red), ACA (green), and DNA (blue). The percentage of control and Seh1-depleted cells containing a bipolar spindle with a recognizable metaphase plate in the presence or absence of ZM447439 was calculated (o). Cells were stained for tubulin, ACA, and DNA. Scale bar, 10 μm.

Figure 7.

The organization of the anaphase spindle midzone and midbody depends on Seh1. Localization of MKLP2 (A) and P-MKLP1 (B) were analyzed by immunofluorescence on control and Seh1-depleted cells. MKLP2 signal was visualized with α-MKLP2 antibody (green, in A), P-MKLP1 with α-PMKLP1 antibody (green, in B), microtubules with α-tubulin (red) and DNA (blue). (C) Control and Seh1-depleted cells at different stages of mitosis were immunostained with α-Aurora B (green), α-tubulin (red), and DNA (blue). (D) Quantification of Aurora B (I), MKLP2 (II), and P-MKLP1 (III) levels from control ■) and Seh1-depleted cells (). Significance: p values from Student's t test are shown; n = 40 from two different experiments. Note the reduction in metaphase, anaphase, and midbody of Aurora B signal and corresponding reduction P-MKLP1. Scale bar, 10 μm.

Figure 7.

The organization of the anaphase spindle midzone and midbody depends on Seh1. Localization of MKLP2 (A) and P-MKLP1 (B) were analyzed by immunofluorescence on control and Seh1-depleted cells. MKLP2 signal was visualized with α-MKLP2 antibody (green, in A), P-MKLP1 with α-PMKLP1 antibody (green, in B), microtubules with α-tubulin (red) and DNA (blue). (C) Control and Seh1-depleted cells at different stages of mitosis were immunostained with α-Aurora B (green), α-tubulin (red), and DNA (blue). (D) Quantification of Aurora B (I), MKLP2 (II), and P-MKLP1 (III) levels from control ■) and Seh1-depleted cells (). Significance: p values from Student's t test are shown; n = 40 from two different experiments. Note the reduction in metaphase, anaphase, and midbody of Aurora B signal and corresponding reduction P-MKLP1. Scale bar, 10 μm.