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The nucleoporin Nup153 maintains nuclear envelope architecture and is required for cell migration in tumor cells
FEBS Letters 584 (2010) 3013–3020
journal homepage: www.FEBSLetters.org
The nucleoporin Nup153 maintains nuclear envelope architecture and is required for cell migration in tumor cells Lixin Zhou, Nelly Panté * Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4
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Article history: Received 25 March 2010 Revised 12 May 2010 Accepted 13 May 2010 Available online 24 May 2010 Edited by Ulrike Kutay Keywords: Nucleoporin Nucleoporin 153 Nuclear pore complex Cell migration Nuclear lamina Cytoskeleton
a b s t r a c t Nucleoporin 153 (Nup153), a component of the nuclear pore complex (NPC), has been implicated in the interaction of the NPC with the nuclear lamina. Here we show that depletion of Nup153 by RNAi results in alteration of the organization of the nuclear lamina and the nuclear lamin-binding protein Sun1. More striking, Nup153 depletion induces a dramatic cytoskeletal rearrangement that impairs cell migration in human breast carcinoma cells. Our results point to a very prominent role of Nup153 in connection to cell motility that could be exploited in order to develop novel anti-cancer therapy. Structured summary: MINT-7893777: Lamin-A/C (uniprotkb:P02545) and NUP153 (uniprotkb:P49790) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7893761: sun1 (uniprotkb:Q9D666) and Lamin-A/C (uniprotkb:P02545) colocalize (MI:0403) by fluorescence microscopy (MI:0416) Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The mammalian nuclear pore complex (NPC) is composed of multiple copies of 30 different nucleoporins [1]. The nucleoporin 153 (Nup153) is strategically positioned at the NPC nuclear basket to interact with the nuclear lamina, and at least in vitro, it interacts with lamin B3 [2]. Recent findings demonstrate that the nuclear lamina is connected with cytoskeletal elements through the linker of nucleoskeleton and cytoskeleton (LINC) complex that spans the double membrane of the nuclear envelope (NE) [3,4]. This complex is formed by outer nuclear membrane Nesprins (actin-binding proteins) and inner nuclear membrane Sun proteins (lamin-binding proteins), which are tethered together at the perinuclear space. To determine whether Nup153 plays a role in the organization of the NE and thereby indirectly impacts the cytoskeletal architecture and mechanical properties of the cell, we employed an siRNA ap-
Abbreviations: cNLS, classical nuclear localization sequence; EdU, ethynyl deoxyuridine; LINC, linker of nucleoskeleton and cytoskeleton; MTOC, microtubule-organizing centre; NE, nuclear envelope; NPC, nuclear pore complex; Nup153, nucleoporin 153 * Corresponding author. Fax: +1 604 822 2416. E-mail address: [email protected] (N. Panté).
proach to reduce the cellular expression of Nup153. Our data suggest that Nup153 plays an essential role in maintaining nucleoskeleton and cytoskeleton architecture, and is required for cell cycle progression and cell migration. 2. Materials and methods 2.1. Cells, antibodies and Nup153 RNAi Cells were cultured at 37 °C and 5% CO2 in complete Dulbecco’s modified Eagle medium supplemented with 10% FBS, penicillin– streptomycin, and 2 mM L-glutamine. Cells used were: HeLa cells (American Type Culture Collection), tetracycline-inducible HeLa cells stably expressing Sun1 carrying a C-terminal GFP tag (Sun1-GFP [5]; courtesy of Dr. B. Burke and Dr. K. Roux, University of Florida), human breast carcinoma MDA231 cells and human fibrosarcoma HT1080 cells (courtesy of Dr. C. Roskelley, University of British Columbia). The following antibodies were used: monoclonal SA1 against human Nup153 [6] (provided by Dr. B. Burke, Institute of Molecular and Cell Biology, Singapore), polyclonal anti-lamin A/C antibody (sc-20681; Santa Cruz Biotechnology), monoclonal anti-actin (AC-40; Sigma), monoclonal anti-a-tubulin (Sigma), polyclonal anti-c-tubulin (Sigma), monoclonal anti-fibrillarin (38F; Abcam),
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.05.038
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and monoclonal Golgi (GM130; BD Transduction Laboratories). Phalloidin-FITC was obtained from Sigma. For indirect immunofluorescence analysis, secondary antibodies were from Invitrogen. For Western blot, the isotype-specific HRP-coupled secondary antibodies were from Amersham Pharmacia. For RNAi, cells were either mock transfected or transfected with siRNA (Dharmacon) against Nup153 at a final concentration of 10 nM using lipofectamine RNAiMAX (Invitrogen) according to the manufacture’s instructions. The sequence used corresponded to nucleotide 2593–2615 of human Nup153 (AAGGCAGACUCUACCAAAUGUTT). Expression of Nup153 was assessed by Western blot and immunofluorescence microscopy one, two and three days after transfection.
2.2. Immunoblotting and immunofluorescence microscopy Whole cells extracts were prepared by cell lysis in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 0.5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 0.5% NP-40, 10 mM PMSF, 1 lm pepstatin, 10 lg/ml aprotinin, and 2 mg/ml leupeptin). The lysates were incubated for 20 min on ice. After centrifugation at 15 000g for 10 min at 4 °C, the supernatants were mixed with SDS–PAGE sample buffer. Protein concentration was determined using the BCA™ protein assay kit (Pierce, Rockford, IL), and aliquots with equal amounts of proteins were loaded on SDS–PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad), and specific proteins were detected with indicated primary antibodies and
Fig. 1. Nup153 depletion by RNAi alters the cell cycle. (A) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected HeLa cells prepared 1, 2 and 3 days after transfection, and probed with the SA1 antibody to detect Nup153, as well as an anti-fibrillarin antibody as an internal standard. (B) Representative flow cytometric profiles of HeLa cells untransfected, mock transfected and Nup153-siRNA transfected, as measured after a 30 min incorporation of EdU. The dot histograms show the fluorescent intensity of the staining for the incorporated EdU (y axis) and the fluorescent DNA probe 7-amino-actinomycin D (7-AAD; x axis). The percentage of cells in the G1, G2/M and S phases of the cell cycle are indicated in the upper left corner of the plots. (C) Percentage of cells in each cell cycle phase, presented as a percentage of the G1 phase of untransfected cells. Shown are the mean values and standard error measured for three independent experiments. (D) Immunofluorescence microscopy of Nup153-siRNAtreated HeLa cells reveals a loss of NE-associated Nup153, 48 h post-transfection. Cells were probed with an anti-Nup153 antibody, and DNA was detected by staining with DAPI.
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peroxidase-conjugated secondary antibodies (Amersham Pharmacia) using a chemiluminescence kit (GE Healthcare, UK). For immunofluorescence microscopy, cells were grown on glass coverslips. After RNAi transfection as indicated above, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and incubated with antibodies by standard immuno-fluorescence protocols [5]. Samples were analyzed with an Olympus Fluvial FV1000 confocal microscope. 2.3. Flow cytometry analysis Cell cycle analysis was performed two days post-transfection after labeling the cells with 5-ethynyl-20 -deoxyuridine (EdU) for 30 min at a final concentration of 10 lM, using the Click-iT EdU Alexa Fluor 448 Flow Cytometry Kit (Invitrogen) according to the manufacture’s instructions. Fluorescence-activated cell sorting data were collected on a FACS Calibur (Becton Dickinson Immuno-
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cytometry Systems, San Jose, CA) and analyzed using FlowJo software.
2.4. Cell migration assay Cells were grown on 35 mm glass bottom dishes. After RNAi transfection as indicated above, cells were grown to confluence. Two days post-transfection, confluent monolayers were wounded by scratching with P20 pipette tips. Samples were washed with PBS to remove floating cells, and FBS-free media was added. To assess the extent of wound closure, phase contrast images were obtained with a ZEISS inverted microscope. Quantification of the wound that remained uncovered by the migrating cells at different times after the introduction of the wound was conducted by tracing the edge of the wound, and calculating the remaining wound area using ImageJ software.
Fig. 2. Nup153 depletion by RNAi alters the localization of the nuclear lamina and Sun1. (A) Double indirect immunofluorescence microscopy of HeLa cells mock-transfected or transfected with Nup153-siRNA and labeled with antibodies against Nup153 and lamin A/C. (B) Western blot of extracts from mock-transfected and Nup153-siRNAtransfected Sun1-GFP HeLa cells prepared 48 h post-transfection, and probed with antibodies against Nup153 and fibrillarin. (C) Immunofluorescence microscopy of Nup153siRNA-treated Sun1-GFP labeled with an antibody against lamin A/C.
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For immunofluorescent analysis of the cells moving into the wound, fixation and staining were performed six hours after wounding.
3. Results 3.1. Nup153 depletion leads to changes in the cell cycle and alteration of the nuclear lamina organization To investigate the role of Nup153 beyond nuclear transport and assembly, we suppressed Nup153 expression in HeLa cells by RNAi using the siRNA sequence corresponding to nucleotide 2593–2615 of human Nup153. This has been previously shown to accomplished efficient depletion of Nup153 in HeLa cells [7]. For control, cells were mock transfected without siRNA. Nup153-expression level was monitored over several days, using both Western blotting of HeLa-cell extracts, and immunofluorescence staining of Nup153. As illustrated in Fig. 1A, the expression of Nup153 was already reduced one day post-transfection. The reduction of Nup153 was most obvious two or three days post-transfection. At the microscopic level, however, cell proliferation was significantly reduced at three days post-transfection, compared to two days post-transfection. Therefore, two days post-transfection was chosen for further experiments throughout this study. The decreased proliferation rate in the Nup153-siRNA-treated cells was not due to apoptosis, because we did not observe TUNEL staining in the Nup153-depleted cells (data not shown). Instead, we found the Nup153-depleted cells were arrested in G1, as revealed by cell cycle analyses using ethynyl deoxyuridine (EdU) staining followed by flow cytometry. Compared to untransfected and mock-transfected cells, the Nup153-siRNA-treated HeLa cells exhibited a marked increase in the percentage of cells in G1, and a concomitant decrease in the percentage of cells in S phase (Fig. 1 B and C). Thus, in addition to its role in mitosis [8–10], Nup153 plays a role in cell-cycle-regulation. Indirect immunofluorescence analysis of the Nup153-siRNAtransfected HeLa cells with the Nup153-specific monoclonal antibody SA1 [6] also demonstrated reduction of the Nup153 by RNAi, with 80–90% of the cells showing a significant reduction of the normal nuclear-rim staining of Nup153 (Fig. 1D). In addition to monitoring for RNAi silencing of Nup153, the immunostaining revealed deformation of the nuclei of Nup153-siRNA-transfected cells, which is more evident after staining the nuclei with DAPI (Fig. 1D). Because the nuclear lamina plays an important role in the establishment and maintenance of a regular nuclear morphology, we then performed immunostaining with antibodies against lamin A/C. As illustrated in Fig. 2A, we found that Nup153 depletion resulted in alteration of the nuclear lamina organization. These alterations include multiple lobes or membrane invaginations spanning the nuclei, as well as a dotted pattern for the immunolabeling of the lamin A/C (Fig. 2A). Thus, Nup153 (probably by its interaction with the nuclear lamina) seems to play a role in the plasticity of the NE.
membrane invaginations spanning the nuclei (Fig. 2C), similar to the alterations in the lamin A/C immunostaining of HeLa cells depleted of Nup153. Because the cytoskeleton plays an important role in the establishment and maintenance of regular nuclear morphology, and because the NE (through the LINC complex) directly impacts the cytoskeletal architecture, we hypothesized that depletion of Nup153 induces cytoskeletal rearrangement. To test this hypothesis, we performed different immunostainings with antibodies or toxins directed against cytoskeletal proteins in HeLa cells depleted of Nup153 by RNAi. As documented in Fig. 3A, RNAi reduction of Nup153 induced rearrangement of the actin cytoskeleton. Similar experiments, but immunolabeling the microtubules instead of the actin cytoskeleton, also revealed reorganization of the microtubule cytoskeleton in Nup153-siRNA-transfected cells (Fig. 3B). Western blot analysis,
3.2. Nup153 depletion alters the localization of Sun1 and causes rearrangement of the cytoskeleton Since Sun1 is a lamin A-binding protein [3], we investigated whether the depletion of Nup153 has any effect on the localization of Sun1. For these experiments, we used HeLa cells expressing Sun1 carrying a C-terminal GFP tag (Sun1-GFP [5]). Similar to the RNAi experiments with HeLa cells, Nup153 was significantly reduced in the Sun1-GFP cells (Fig. 2B). This reduction resulted in morphologically defected nuclei, and in immunolabeling defects of Sun1 characterized by a dotted immunolabeling pattern and
Fig. 3. Nup153 depletion by RNAi results in rearrangement of the cytoskeleton. (A) Phalloidin staining of the actin cytoskeleton of mock-transfected and Nup153siRNA-transfected HeLa cells 48 h post-transfection. DNA was detected with DAPI. (B) Immunofluorescence microscopy of mock- and Nup153-siRNA-transfected HeLa cells labeled with an antibody against a-tubulin. (C) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected HeLa cells prepared 48 h after transfection, and probed with antibodies against Nup153, lamin A/C, actin, a-tubulin and fibrillarin. Fibrillarin antibody was used as an internal standard to control for total protein loaded on each lane of the gel.
L. Zhou, N. Panté / FEBS Letters 584 (2010) 3013–3020
however, revealed that there was not a significant difference in the expression of actin or a-tubulin in the Nup153-siRNA-transfected cells compared to mock-transfected cells (Fig. 3C). 3.3. Nup153 depletion affects cell migration and cell polarity in human breast cancer cells Since Nup153 depletion induces cytoskeleton rearrangement, we hypothesize that Nup153-siRNA-transfected cells have defects in
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cytoskeleton-mediated process, including cell migration, microtubule-organizing centre (MTOC) position with respect to the nucleus, and polarization. To test this hypothesis, we conducted Nup153-siRNA-transfection studies in a well-known human breast carcinoma cell line (MDA-231) that exhibits a very fast migration speed [11]. Because directional cell migration is a fundamental feature in the metastatic spread of cancer, we reason that if reduction of the levels of Nup153 in these cells affects their migration, Nup153-RNAi could be developed as a new therapeutic approach against cancer.
Fig. 4. Nup153 depletion by RNAi in MDA231 cells results in altered cellular morphology and rearrangement of the cytoskeleton. (A) Immunofluorescence microscopy of Nup153-siRNA-treated MDA231 cells reveals a loss of NE-associated Nup153, 48 h post-transfection. Cells were probed with an anti-Nup153 antibody, and DNA was detected by staining with DAPI. (B) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected MDA231 cells prepared 48 h after transfection, and probed with antibodies against Nup153, lamin A/C, actin, a-tubulin and fibrillarin. (C) Phase contrast images reveal a dramatic change in the cellular morphology of the MDA231 cells, 48 h after Nup153-siRNA-treatment. (D) Immunofluorescence microscopy of mock- and Nup153-siRNA–transfected MDA231 cells reveals rearrangement of the actin and microtubule cytoskeleton 48 h post-transfection. Cells were probed with Phalloidin-FITC and an anti-a-tubulin antibody. DNA was detected by staining with DAPI.
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We first determined whether Nup153 could be efficiently depleted by RNAi in MDA231 cells. For this purpose, MDA231 cells were transfected with and without the same Nup153-specific siRNA we used for HeLa cells. Two days post-transfection, the cells were analyzed by immunofluorescence microscopy and Western blot using the SA1 antibody against Nup153. As illustrated in Fig. 4A and B, through RNAi, the expression of Nup153 was significantly reduced in MDA231 cells. Similar to HeLa cells, the expression levels of lamin A/C, actin and a-tubulin did not change in Nup153-RNAi-transfected MDA231 cells when compared to mock-transfected MDA231 cells (Fig. 4B). Cellular morphology, however, was significantly altered (Fig. 4C). Similar to HeLa cells, depletion of Nup153 in MDA231 cells induced dramatic rearrangement of the actin and microtubule cytoskeleton (Fig. 4D). Having proved that reduction of Nup153 through RNAi in MDA231 cells induces cytoskeleton rearrangements, we then investigated the migration response of Nup153-RNAi-treated MDA231 cells. For this purpose, cells were grown to a confluent monolayer and subjected to the wound-induced cell migration assay as described in Section 2.4. As expected, wound closure occurred significantly faster in MDA231 cells (Fig. 5A; see also
videos in Supplementary data); however, the rate of wound healing was significantly reduced in the Nup153-RNAi-transfected MDA231 cells, compared to the mock-transfected MDA231 cells (Fig. 5A and B). Similar results were obtained after subjecting human fibrosarcoma cells HT1080 to the wound-induced cell migration assay 48 h after Nup153-siRNA treatment (Fig. S1). In addition to measuring the migration rate, we also examined changes in polarity, determined by the location of the MTOC and the Golgi complex with respect to the wound edge, in MDA231 cells mock transfected or transfected with the siRNA of Nup153. For this purpose, cell monolayers were immunofluorescence labeled with c-tubulin and Golgi antibodies six hours after introduction of the scratch. We found that the MTOC and Golgi complex of mock-transfected MDA231cells were located towards the wound. This is in contrast to the Nup153-RNAi-transfected MDA231 cells, which failed to position their MTOC and Golgi complex towards the wound (Fig. 5C). Quantification of the number of cells that had a polarized MTOC (defined as location of these structures towards the wound and within a 120° area facing the wound edge) showed that 82% of the mock-transfected MDA231 cells had their MTOC oriented towards the wound (Fig. 5D). In contrast, only 42% of
Fig. 5. Nup153 depletion affects cell migration and cell polarity in MDA231 cells. (A) Phase contrast micrographs show the extent of cell migration in the wound of mocktransfected and Nup153-siRNA-transfected MDA231 cells subjected to the wound-induced cell migration assay. The wound was introduced 48 h post-transfection. (B) Quantification of the fraction of the wound that remains uncovered by the migratory cells as a function of time for mock-transfected and Nup153-siRNA-transfected MDA231 cells (n = 3 for each condition). (C) Indirect immunofluorescence microscopy of mock-transfected and Nup153-siRNA-transfected MDA231 cells six hours after the introduction of the wound. Cells were immunolabeled using antibodies against the Golgi complex (GM130), and MTOC (c-tubulin). DNA was stained with DAPI. The broken lines indicate the migrating cell front. Arrows point to the MTOC. (D) Quantification of the cells with polarized Golgi complex (top) and polarized MTOC (bottom). Cells with immunostaining of the Golgi complex and MTOC positioned within a 120° area facing the wound edge were assessed as polarized. Shown are the mean values and standard error measured for three independent experiments. 100 cells were counted for each condition. (E) Phalloidin staining of the actin cytoskeleton of mock-transfected and Nup153-siRNA-transfected MDA231 cells six hours after the introduction of the wound. DNA was stained with DAPI. The broken lines indicate the migrating cell front.
L. Zhou, N. Panté / FEBS Letters 584 (2010) 3013–3020
Nup153-RNAi-transfected MDA231 cells had a polarized MTOC. Similarly, the number of Nup153-RNAi-transfected MDA231 cells with a polarized Golgi complex was significantly lower than the mock-transfected MDA231 cells (Fig. 5D). In addition, immunostaining of the actin filaments of these cells showed that Nup153RNAi-transfected MDA231 cells had defects in forming lamellipodia at their leading edge that moves towards the wound (Fig. 5E). 4. Discussion Nucleoporins, once though to be exclusively structural blocks of the NPC with roles only in nuclear transport, are emerging as regulators of diverse cellular functions. This is the case for Nup153, which in addition to have a structural role in the formation of NPCs [12,13] and a functional role in nuclear import and export [14–17], is involved in facilitating the process of NE disassembly during mitosis [8–10]. The latter function has recently been shown to be mediated through the association of Nup153 with the spindle assembly checkpoint protein Mad1 [18]. Our findings suggest additional roles for Nup153 in cell cycle progression, nuclear lamina organization, cell morphology and cell migration. Given the role of Nup153 in nuclear transport [14–17], one could argue that the phenotypes observed in this study might be a consequence of nuclear import defects in cells depleted of Nup153. A recent study, however, demonstrated that HeLa cells depleted of Nup153 by RNAi can still carry out efficient global nucleocytoplasmic transport, which included nuclear import of the glucocorticoid receptor, nuclear export of a Rev-chimera protein and nuclear export of poly (A)+ RNA [10]. Consistent with this study, our nuclear import assays in digitonin-permeabilized cells show that Nup153-depleted cells are able to nuclear import proteins with classical nuclear localization sequences (cNLSs). Compared to the nuclear import in the mock-transfected cells, however, the cells transfected with Nup153-siRNA exhibited a 37% reduction in the nuclear concentration of the cNLS-protein (Fig. S2). Similar nuclear import deficiency (about 25% reduction) has been reported for the nuclear import of GFP fused to a cNLS in Drosophila cells depleted of Nup153 by RNAi [19]. The slight differences of nuclear transport efficiency between our study and those reported previously, may be due to the extend of Nup153 depletion or the assay used to measured nuclear import. Nevertheless, we can conclude that the defects we found for the Nup153-depleted cells in cell cycle progression, nuclear lamina organization, cell morphology and cell migration might not be a direct consequence of impaired nuclear transport. The defects in cell cycle progression could be explained by a direct role of Nup153 in regulating cell-cycle gene expression, as it has been recently demonstrated for other nucleoporins [20,21]. Because Nup153 is mobile and found at the NPC and inside the nucleoplasm [22], this gene expression control could be achieved by Nup153-chromatin interaction at the NPC or within the nucleoplasm. Alternatively, the defects in cell cycle progression we detected in the Nup153-RNAi treated cells (Fig. 1B) may be a consequence of the alteration of the nuclear lamina of these cells, as nuclear lamina organization plays an important role in the regulation of DNA synthesis, chromatin organization, and gene transcription (reviewed by [23,24]). For example, siRNA knock down of lamin A/C leads to cell cycle G1 arrest [25]. In addition to the cell cycle progression defect, we have found that depletion of Nup153 resulted in alteration of the immunolabeling of the nuclear lamina and the A-type lamin-binding protein Sun1. These alterations are not surprising due to the location of Nup153 at the nuclear periphery of the NPC. Because the nuclear lamina is connected to cytoskeletal elements through the LINC complex [3,4], it is expected that conditions that affect the nuclear lamina will influence cytoskeletal arrangements and cytoskeleton-
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mediated process. Thus, depletion of Nup153 by RNAi resulted in alteration of the nuclear lamina, and consequently in a rearrangement of the cytoskeleton and defects in cytoskeleton-based processes. Further work is necessary to address how the cytoskeletal dynamic is affected when NE components, including the Nup153, are manipulated. This will have highly relevance in understanding the molecular basis underlying certain human diseases (e.g. lipodystrophy, muscular dystrophy, neuropathy, and progeria) that are linked to defects in NE proteins [26,27]. As expected, alterations of the cytoskeleton resulted in defects in several processes that are governed by the cytoskeletal network. Because cell migration is a fundamental feature in the metastatic spread of cancer, we then studied the effect of depleting Nup153 in a human breast carcinoma cell line. We found that depletion of Nup153 significantly reduced the speed of directional cell migration in the human breast carcinoma MDA231 cell line. These cells also had polarization defects. Similar migration and polarization defects have been found in cells lacking lamin A/C [28] and in Nesprin-2-deficient fibroblasts [29]. Our results with MDA231 cells suggests that manipulation of the NPC could be used to regulate cell growth and progression of cancer invasion, and opens the exciting possibility that RNAi of Nup153 could be developed as an anti-cancer therapy. Acknowledgements We thank Dr. Sebastian Witt for contributing preliminary experiments, Dr. Brian Burke (Institute of Molecular and Cell Biology, Singapore) and Dr. K. Roux (University of Florida) for providing the Sun1-GFP cells and the SA1 antibody, and Dr. Calvin Roskelley (Department of Cellular and Physiological Sciences, University of British Columbia) for providing the MDA231, GFPMDA231and HT1080 cell lines. Pamela Austin is acknowledged for help with the cell migration assay. This work was supported by a Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to N.P. N.P. is a Michael Smith Foundation for Health Research (MSFHR) Senior Scholar. Appendix A. Supplementary data Fig. S1 shows that Nup153-siRNA treatment of the human fibrosarcoma cell line, HT1080, also affects the migration speed of these cells. Fig. S2 shows that depletion of Nup153 does not impair the nuclear import of proteins with cNLSs. Videos 1 and 2 show live cell time-lapse imaging of mock-transfected and Nup153-siRNAtransfected GFP-MDA231 cells that have been subjected to wound-induced cell migration assay respectively. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.05.038. References [1] Cronshaw, J.M., Krutchinsky, A.N., Zhang, W., Chait, B.T. and Matunis, M.J. (2002) Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915–927. [2] Smythe, C., Jenkins, H.E. and Hutchison, C.J. (2000) Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs. EMBO J. 19, 3918–3931. [3] Haque, F., Lloyd, D.J., Smallwood, D.T., Dent, C.L., Shanahan, C.M., Fry, A.M., Trembath, R.C. and Shackleton, S. (2006) SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol. Cell. Biol. 26, 3738–3751. [4] Crisp, M., Liu, Q., Roux, K., Rattner, J.B., Shanahan, C., Burke, B., Stahl, P.D. and Hodzic, D. (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J. Cell Biol. 172, 41–53. [5] Liu, Q., Pante, N., Misteli, T., Elsagga, M., Crisp, M., Hodzic, D., Burke, B. and Roux, K.J. (2007) Functional association of Sun1 with nuclear pore complexes. J. Cell Biol. 178, 785–798.
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[17] Ullman, K.S., Shah, S., Powers, M.A. and Forbes, D.J. (1999) The nucleoporin nup153 plays a critical role in multiple types of nuclear export. Mol. Biol. Cell 10, 649–664. [18] Lussi, C.Y., Shumaker, D.L., Shimi, T. and Fahrenkrog, B. (2010) The nucleoporin Nup153 affects spindle checkpoint activity due to an association wiith Mad1. Nucleus. 1, 71–84. [19] Sabri, N., Roth, P., Xylourgidis, N., Sadeghifar, F., Adler, J. and Samakovlis, C. (2007) Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport. J. Cell Biol. 178, 557–565. [20] Kalverda, B., Pickersgill, H., Shloma, V.V. and Fornerod, M. (2010) Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm. Cell. 140, 360–371. [21] Capelson, M., Liang, Y., Schulte, R., Mair, W., Wagner, U. and Hetzer, M.W. (2010) Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell. 140, 372–383. [22] Daigle, N., Beaudouin, J., Hartnell, L., Imreh, G., Hallberg, E., Lippincott-Schwartz, J. and Ellenberg, J. (2001) Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J. Cell Biol. 154, 71–84. [23] Verstraeten, V.L., Broers, J.L., Ramaekers, F.C. and van Steensel, M.A. (2007) The nuclear envelope, a key structure in cellular integrity and gene expression. Curr. Med. Chem. 14, 1231–1248. [24] Andres, V. and Gonzalez, J.M. (2009) Role of A-type lamins in signaling, transcription, and chromatin organization. J. Cell Biol. 187, 945–957. [25] Pekovic, V. et al. (2007) Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts. J. Cell Biol. 176, 163–172. [26] Wilkie, G.S. and Schirmer, E.C. (2006) Guilt by association: the nuclear envelope proteome and disease. Mol. Cell. Proteomics 5, 1865–1875. [27] Holaska, J.M. (2008) Emerin and the nuclear lamina in muscle and cardiac disease. Circ. Res. 103, 16–23. [28] Lee, J.S. et al. (2007) Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration. Biophys. J. 93, 2542–2552. [29] Luke, Y. et al. (2008) Nesprin-2 Giant (NUANCE) maintains nuclear envelope architecture and composition in skin. J. Cell Sci. 121, 1887–1898.
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The nucleoporin Nup153 maintains nuclear envelope architecture and is required for cell migration in tumor cells Lixin Zhou, Nelly Panté * Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4
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Article history: Received 25 March 2010 Revised 12 May 2010 Accepted 13 May 2010 Available online 24 May 2010 Edited by Ulrike Kutay Keywords: Nucleoporin Nucleoporin 153 Nuclear pore complex Cell migration Nuclear lamina Cytoskeleton
a b s t r a c t Nucleoporin 153 (Nup153), a component of the nuclear pore complex (NPC), has been implicated in the interaction of the NPC with the nuclear lamina. Here we show that depletion of Nup153 by RNAi results in alteration of the organization of the nuclear lamina and the nuclear lamin-binding protein Sun1. More striking, Nup153 depletion induces a dramatic cytoskeletal rearrangement that impairs cell migration in human breast carcinoma cells. Our results point to a very prominent role of Nup153 in connection to cell motility that could be exploited in order to develop novel anti-cancer therapy. Structured summary: MINT-7893777: Lamin-A/C (uniprotkb:P02545) and NUP153 (uniprotkb:P49790) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7893761: sun1 (uniprotkb:Q9D666) and Lamin-A/C (uniprotkb:P02545) colocalize (MI:0403) by fluorescence microscopy (MI:0416) Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The mammalian nuclear pore complex (NPC) is composed of multiple copies of 30 different nucleoporins [1]. The nucleoporin 153 (Nup153) is strategically positioned at the NPC nuclear basket to interact with the nuclear lamina, and at least in vitro, it interacts with lamin B3 [2]. Recent findings demonstrate that the nuclear lamina is connected with cytoskeletal elements through the linker of nucleoskeleton and cytoskeleton (LINC) complex that spans the double membrane of the nuclear envelope (NE) [3,4]. This complex is formed by outer nuclear membrane Nesprins (actin-binding proteins) and inner nuclear membrane Sun proteins (lamin-binding proteins), which are tethered together at the perinuclear space. To determine whether Nup153 plays a role in the organization of the NE and thereby indirectly impacts the cytoskeletal architecture and mechanical properties of the cell, we employed an siRNA ap-
Abbreviations: cNLS, classical nuclear localization sequence; EdU, ethynyl deoxyuridine; LINC, linker of nucleoskeleton and cytoskeleton; MTOC, microtubule-organizing centre; NE, nuclear envelope; NPC, nuclear pore complex; Nup153, nucleoporin 153 * Corresponding author. Fax: +1 604 822 2416. E-mail address: [email protected] (N. Panté).
proach to reduce the cellular expression of Nup153. Our data suggest that Nup153 plays an essential role in maintaining nucleoskeleton and cytoskeleton architecture, and is required for cell cycle progression and cell migration. 2. Materials and methods 2.1. Cells, antibodies and Nup153 RNAi Cells were cultured at 37 °C and 5% CO2 in complete Dulbecco’s modified Eagle medium supplemented with 10% FBS, penicillin– streptomycin, and 2 mM L-glutamine. Cells used were: HeLa cells (American Type Culture Collection), tetracycline-inducible HeLa cells stably expressing Sun1 carrying a C-terminal GFP tag (Sun1-GFP [5]; courtesy of Dr. B. Burke and Dr. K. Roux, University of Florida), human breast carcinoma MDA231 cells and human fibrosarcoma HT1080 cells (courtesy of Dr. C. Roskelley, University of British Columbia). The following antibodies were used: monoclonal SA1 against human Nup153 [6] (provided by Dr. B. Burke, Institute of Molecular and Cell Biology, Singapore), polyclonal anti-lamin A/C antibody (sc-20681; Santa Cruz Biotechnology), monoclonal anti-actin (AC-40; Sigma), monoclonal anti-a-tubulin (Sigma), polyclonal anti-c-tubulin (Sigma), monoclonal anti-fibrillarin (38F; Abcam),
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.05.038
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and monoclonal Golgi (GM130; BD Transduction Laboratories). Phalloidin-FITC was obtained from Sigma. For indirect immunofluorescence analysis, secondary antibodies were from Invitrogen. For Western blot, the isotype-specific HRP-coupled secondary antibodies were from Amersham Pharmacia. For RNAi, cells were either mock transfected or transfected with siRNA (Dharmacon) against Nup153 at a final concentration of 10 nM using lipofectamine RNAiMAX (Invitrogen) according to the manufacture’s instructions. The sequence used corresponded to nucleotide 2593–2615 of human Nup153 (AAGGCAGACUCUACCAAAUGUTT). Expression of Nup153 was assessed by Western blot and immunofluorescence microscopy one, two and three days after transfection.
2.2. Immunoblotting and immunofluorescence microscopy Whole cells extracts were prepared by cell lysis in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 0.5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 0.5% NP-40, 10 mM PMSF, 1 lm pepstatin, 10 lg/ml aprotinin, and 2 mg/ml leupeptin). The lysates were incubated for 20 min on ice. After centrifugation at 15 000g for 10 min at 4 °C, the supernatants were mixed with SDS–PAGE sample buffer. Protein concentration was determined using the BCA™ protein assay kit (Pierce, Rockford, IL), and aliquots with equal amounts of proteins were loaded on SDS–PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad), and specific proteins were detected with indicated primary antibodies and
Fig. 1. Nup153 depletion by RNAi alters the cell cycle. (A) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected HeLa cells prepared 1, 2 and 3 days after transfection, and probed with the SA1 antibody to detect Nup153, as well as an anti-fibrillarin antibody as an internal standard. (B) Representative flow cytometric profiles of HeLa cells untransfected, mock transfected and Nup153-siRNA transfected, as measured after a 30 min incorporation of EdU. The dot histograms show the fluorescent intensity of the staining for the incorporated EdU (y axis) and the fluorescent DNA probe 7-amino-actinomycin D (7-AAD; x axis). The percentage of cells in the G1, G2/M and S phases of the cell cycle are indicated in the upper left corner of the plots. (C) Percentage of cells in each cell cycle phase, presented as a percentage of the G1 phase of untransfected cells. Shown are the mean values and standard error measured for three independent experiments. (D) Immunofluorescence microscopy of Nup153-siRNAtreated HeLa cells reveals a loss of NE-associated Nup153, 48 h post-transfection. Cells were probed with an anti-Nup153 antibody, and DNA was detected by staining with DAPI.
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peroxidase-conjugated secondary antibodies (Amersham Pharmacia) using a chemiluminescence kit (GE Healthcare, UK). For immunofluorescence microscopy, cells were grown on glass coverslips. After RNAi transfection as indicated above, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and incubated with antibodies by standard immuno-fluorescence protocols [5]. Samples were analyzed with an Olympus Fluvial FV1000 confocal microscope. 2.3. Flow cytometry analysis Cell cycle analysis was performed two days post-transfection after labeling the cells with 5-ethynyl-20 -deoxyuridine (EdU) for 30 min at a final concentration of 10 lM, using the Click-iT EdU Alexa Fluor 448 Flow Cytometry Kit (Invitrogen) according to the manufacture’s instructions. Fluorescence-activated cell sorting data were collected on a FACS Calibur (Becton Dickinson Immuno-
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cytometry Systems, San Jose, CA) and analyzed using FlowJo software.
2.4. Cell migration assay Cells were grown on 35 mm glass bottom dishes. After RNAi transfection as indicated above, cells were grown to confluence. Two days post-transfection, confluent monolayers were wounded by scratching with P20 pipette tips. Samples were washed with PBS to remove floating cells, and FBS-free media was added. To assess the extent of wound closure, phase contrast images were obtained with a ZEISS inverted microscope. Quantification of the wound that remained uncovered by the migrating cells at different times after the introduction of the wound was conducted by tracing the edge of the wound, and calculating the remaining wound area using ImageJ software.
Fig. 2. Nup153 depletion by RNAi alters the localization of the nuclear lamina and Sun1. (A) Double indirect immunofluorescence microscopy of HeLa cells mock-transfected or transfected with Nup153-siRNA and labeled with antibodies against Nup153 and lamin A/C. (B) Western blot of extracts from mock-transfected and Nup153-siRNAtransfected Sun1-GFP HeLa cells prepared 48 h post-transfection, and probed with antibodies against Nup153 and fibrillarin. (C) Immunofluorescence microscopy of Nup153siRNA-treated Sun1-GFP labeled with an antibody against lamin A/C.
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For immunofluorescent analysis of the cells moving into the wound, fixation and staining were performed six hours after wounding.
3. Results 3.1. Nup153 depletion leads to changes in the cell cycle and alteration of the nuclear lamina organization To investigate the role of Nup153 beyond nuclear transport and assembly, we suppressed Nup153 expression in HeLa cells by RNAi using the siRNA sequence corresponding to nucleotide 2593–2615 of human Nup153. This has been previously shown to accomplished efficient depletion of Nup153 in HeLa cells [7]. For control, cells were mock transfected without siRNA. Nup153-expression level was monitored over several days, using both Western blotting of HeLa-cell extracts, and immunofluorescence staining of Nup153. As illustrated in Fig. 1A, the expression of Nup153 was already reduced one day post-transfection. The reduction of Nup153 was most obvious two or three days post-transfection. At the microscopic level, however, cell proliferation was significantly reduced at three days post-transfection, compared to two days post-transfection. Therefore, two days post-transfection was chosen for further experiments throughout this study. The decreased proliferation rate in the Nup153-siRNA-treated cells was not due to apoptosis, because we did not observe TUNEL staining in the Nup153-depleted cells (data not shown). Instead, we found the Nup153-depleted cells were arrested in G1, as revealed by cell cycle analyses using ethynyl deoxyuridine (EdU) staining followed by flow cytometry. Compared to untransfected and mock-transfected cells, the Nup153-siRNA-treated HeLa cells exhibited a marked increase in the percentage of cells in G1, and a concomitant decrease in the percentage of cells in S phase (Fig. 1 B and C). Thus, in addition to its role in mitosis [8–10], Nup153 plays a role in cell-cycle-regulation. Indirect immunofluorescence analysis of the Nup153-siRNAtransfected HeLa cells with the Nup153-specific monoclonal antibody SA1 [6] also demonstrated reduction of the Nup153 by RNAi, with 80–90% of the cells showing a significant reduction of the normal nuclear-rim staining of Nup153 (Fig. 1D). In addition to monitoring for RNAi silencing of Nup153, the immunostaining revealed deformation of the nuclei of Nup153-siRNA-transfected cells, which is more evident after staining the nuclei with DAPI (Fig. 1D). Because the nuclear lamina plays an important role in the establishment and maintenance of a regular nuclear morphology, we then performed immunostaining with antibodies against lamin A/C. As illustrated in Fig. 2A, we found that Nup153 depletion resulted in alteration of the nuclear lamina organization. These alterations include multiple lobes or membrane invaginations spanning the nuclei, as well as a dotted pattern for the immunolabeling of the lamin A/C (Fig. 2A). Thus, Nup153 (probably by its interaction with the nuclear lamina) seems to play a role in the plasticity of the NE.
membrane invaginations spanning the nuclei (Fig. 2C), similar to the alterations in the lamin A/C immunostaining of HeLa cells depleted of Nup153. Because the cytoskeleton plays an important role in the establishment and maintenance of regular nuclear morphology, and because the NE (through the LINC complex) directly impacts the cytoskeletal architecture, we hypothesized that depletion of Nup153 induces cytoskeletal rearrangement. To test this hypothesis, we performed different immunostainings with antibodies or toxins directed against cytoskeletal proteins in HeLa cells depleted of Nup153 by RNAi. As documented in Fig. 3A, RNAi reduction of Nup153 induced rearrangement of the actin cytoskeleton. Similar experiments, but immunolabeling the microtubules instead of the actin cytoskeleton, also revealed reorganization of the microtubule cytoskeleton in Nup153-siRNA-transfected cells (Fig. 3B). Western blot analysis,
3.2. Nup153 depletion alters the localization of Sun1 and causes rearrangement of the cytoskeleton Since Sun1 is a lamin A-binding protein [3], we investigated whether the depletion of Nup153 has any effect on the localization of Sun1. For these experiments, we used HeLa cells expressing Sun1 carrying a C-terminal GFP tag (Sun1-GFP [5]). Similar to the RNAi experiments with HeLa cells, Nup153 was significantly reduced in the Sun1-GFP cells (Fig. 2B). This reduction resulted in morphologically defected nuclei, and in immunolabeling defects of Sun1 characterized by a dotted immunolabeling pattern and
Fig. 3. Nup153 depletion by RNAi results in rearrangement of the cytoskeleton. (A) Phalloidin staining of the actin cytoskeleton of mock-transfected and Nup153siRNA-transfected HeLa cells 48 h post-transfection. DNA was detected with DAPI. (B) Immunofluorescence microscopy of mock- and Nup153-siRNA-transfected HeLa cells labeled with an antibody against a-tubulin. (C) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected HeLa cells prepared 48 h after transfection, and probed with antibodies against Nup153, lamin A/C, actin, a-tubulin and fibrillarin. Fibrillarin antibody was used as an internal standard to control for total protein loaded on each lane of the gel.
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however, revealed that there was not a significant difference in the expression of actin or a-tubulin in the Nup153-siRNA-transfected cells compared to mock-transfected cells (Fig. 3C). 3.3. Nup153 depletion affects cell migration and cell polarity in human breast cancer cells Since Nup153 depletion induces cytoskeleton rearrangement, we hypothesize that Nup153-siRNA-transfected cells have defects in
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cytoskeleton-mediated process, including cell migration, microtubule-organizing centre (MTOC) position with respect to the nucleus, and polarization. To test this hypothesis, we conducted Nup153-siRNA-transfection studies in a well-known human breast carcinoma cell line (MDA-231) that exhibits a very fast migration speed [11]. Because directional cell migration is a fundamental feature in the metastatic spread of cancer, we reason that if reduction of the levels of Nup153 in these cells affects their migration, Nup153-RNAi could be developed as a new therapeutic approach against cancer.
Fig. 4. Nup153 depletion by RNAi in MDA231 cells results in altered cellular morphology and rearrangement of the cytoskeleton. (A) Immunofluorescence microscopy of Nup153-siRNA-treated MDA231 cells reveals a loss of NE-associated Nup153, 48 h post-transfection. Cells were probed with an anti-Nup153 antibody, and DNA was detected by staining with DAPI. (B) Western blot of extracts from mock-transfected and Nup153-siRNA-transfected MDA231 cells prepared 48 h after transfection, and probed with antibodies against Nup153, lamin A/C, actin, a-tubulin and fibrillarin. (C) Phase contrast images reveal a dramatic change in the cellular morphology of the MDA231 cells, 48 h after Nup153-siRNA-treatment. (D) Immunofluorescence microscopy of mock- and Nup153-siRNA–transfected MDA231 cells reveals rearrangement of the actin and microtubule cytoskeleton 48 h post-transfection. Cells were probed with Phalloidin-FITC and an anti-a-tubulin antibody. DNA was detected by staining with DAPI.
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We first determined whether Nup153 could be efficiently depleted by RNAi in MDA231 cells. For this purpose, MDA231 cells were transfected with and without the same Nup153-specific siRNA we used for HeLa cells. Two days post-transfection, the cells were analyzed by immunofluorescence microscopy and Western blot using the SA1 antibody against Nup153. As illustrated in Fig. 4A and B, through RNAi, the expression of Nup153 was significantly reduced in MDA231 cells. Similar to HeLa cells, the expression levels of lamin A/C, actin and a-tubulin did not change in Nup153-RNAi-transfected MDA231 cells when compared to mock-transfected MDA231 cells (Fig. 4B). Cellular morphology, however, was significantly altered (Fig. 4C). Similar to HeLa cells, depletion of Nup153 in MDA231 cells induced dramatic rearrangement of the actin and microtubule cytoskeleton (Fig. 4D). Having proved that reduction of Nup153 through RNAi in MDA231 cells induces cytoskeleton rearrangements, we then investigated the migration response of Nup153-RNAi-treated MDA231 cells. For this purpose, cells were grown to a confluent monolayer and subjected to the wound-induced cell migration assay as described in Section 2.4. As expected, wound closure occurred significantly faster in MDA231 cells (Fig. 5A; see also
videos in Supplementary data); however, the rate of wound healing was significantly reduced in the Nup153-RNAi-transfected MDA231 cells, compared to the mock-transfected MDA231 cells (Fig. 5A and B). Similar results were obtained after subjecting human fibrosarcoma cells HT1080 to the wound-induced cell migration assay 48 h after Nup153-siRNA treatment (Fig. S1). In addition to measuring the migration rate, we also examined changes in polarity, determined by the location of the MTOC and the Golgi complex with respect to the wound edge, in MDA231 cells mock transfected or transfected with the siRNA of Nup153. For this purpose, cell monolayers were immunofluorescence labeled with c-tubulin and Golgi antibodies six hours after introduction of the scratch. We found that the MTOC and Golgi complex of mock-transfected MDA231cells were located towards the wound. This is in contrast to the Nup153-RNAi-transfected MDA231 cells, which failed to position their MTOC and Golgi complex towards the wound (Fig. 5C). Quantification of the number of cells that had a polarized MTOC (defined as location of these structures towards the wound and within a 120° area facing the wound edge) showed that 82% of the mock-transfected MDA231 cells had their MTOC oriented towards the wound (Fig. 5D). In contrast, only 42% of
Fig. 5. Nup153 depletion affects cell migration and cell polarity in MDA231 cells. (A) Phase contrast micrographs show the extent of cell migration in the wound of mocktransfected and Nup153-siRNA-transfected MDA231 cells subjected to the wound-induced cell migration assay. The wound was introduced 48 h post-transfection. (B) Quantification of the fraction of the wound that remains uncovered by the migratory cells as a function of time for mock-transfected and Nup153-siRNA-transfected MDA231 cells (n = 3 for each condition). (C) Indirect immunofluorescence microscopy of mock-transfected and Nup153-siRNA-transfected MDA231 cells six hours after the introduction of the wound. Cells were immunolabeled using antibodies against the Golgi complex (GM130), and MTOC (c-tubulin). DNA was stained with DAPI. The broken lines indicate the migrating cell front. Arrows point to the MTOC. (D) Quantification of the cells with polarized Golgi complex (top) and polarized MTOC (bottom). Cells with immunostaining of the Golgi complex and MTOC positioned within a 120° area facing the wound edge were assessed as polarized. Shown are the mean values and standard error measured for three independent experiments. 100 cells were counted for each condition. (E) Phalloidin staining of the actin cytoskeleton of mock-transfected and Nup153-siRNA-transfected MDA231 cells six hours after the introduction of the wound. DNA was stained with DAPI. The broken lines indicate the migrating cell front.
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Nup153-RNAi-transfected MDA231 cells had a polarized MTOC. Similarly, the number of Nup153-RNAi-transfected MDA231 cells with a polarized Golgi complex was significantly lower than the mock-transfected MDA231 cells (Fig. 5D). In addition, immunostaining of the actin filaments of these cells showed that Nup153RNAi-transfected MDA231 cells had defects in forming lamellipodia at their leading edge that moves towards the wound (Fig. 5E). 4. Discussion Nucleoporins, once though to be exclusively structural blocks of the NPC with roles only in nuclear transport, are emerging as regulators of diverse cellular functions. This is the case for Nup153, which in addition to have a structural role in the formation of NPCs [12,13] and a functional role in nuclear import and export [14–17], is involved in facilitating the process of NE disassembly during mitosis [8–10]. The latter function has recently been shown to be mediated through the association of Nup153 with the spindle assembly checkpoint protein Mad1 [18]. Our findings suggest additional roles for Nup153 in cell cycle progression, nuclear lamina organization, cell morphology and cell migration. Given the role of Nup153 in nuclear transport [14–17], one could argue that the phenotypes observed in this study might be a consequence of nuclear import defects in cells depleted of Nup153. A recent study, however, demonstrated that HeLa cells depleted of Nup153 by RNAi can still carry out efficient global nucleocytoplasmic transport, which included nuclear import of the glucocorticoid receptor, nuclear export of a Rev-chimera protein and nuclear export of poly (A)+ RNA [10]. Consistent with this study, our nuclear import assays in digitonin-permeabilized cells show that Nup153-depleted cells are able to nuclear import proteins with classical nuclear localization sequences (cNLSs). Compared to the nuclear import in the mock-transfected cells, however, the cells transfected with Nup153-siRNA exhibited a 37% reduction in the nuclear concentration of the cNLS-protein (Fig. S2). Similar nuclear import deficiency (about 25% reduction) has been reported for the nuclear import of GFP fused to a cNLS in Drosophila cells depleted of Nup153 by RNAi [19]. The slight differences of nuclear transport efficiency between our study and those reported previously, may be due to the extend of Nup153 depletion or the assay used to measured nuclear import. Nevertheless, we can conclude that the defects we found for the Nup153-depleted cells in cell cycle progression, nuclear lamina organization, cell morphology and cell migration might not be a direct consequence of impaired nuclear transport. The defects in cell cycle progression could be explained by a direct role of Nup153 in regulating cell-cycle gene expression, as it has been recently demonstrated for other nucleoporins [20,21]. Because Nup153 is mobile and found at the NPC and inside the nucleoplasm [22], this gene expression control could be achieved by Nup153-chromatin interaction at the NPC or within the nucleoplasm. Alternatively, the defects in cell cycle progression we detected in the Nup153-RNAi treated cells (Fig. 1B) may be a consequence of the alteration of the nuclear lamina of these cells, as nuclear lamina organization plays an important role in the regulation of DNA synthesis, chromatin organization, and gene transcription (reviewed by [23,24]). For example, siRNA knock down of lamin A/C leads to cell cycle G1 arrest [25]. In addition to the cell cycle progression defect, we have found that depletion of Nup153 resulted in alteration of the immunolabeling of the nuclear lamina and the A-type lamin-binding protein Sun1. These alterations are not surprising due to the location of Nup153 at the nuclear periphery of the NPC. Because the nuclear lamina is connected to cytoskeletal elements through the LINC complex [3,4], it is expected that conditions that affect the nuclear lamina will influence cytoskeletal arrangements and cytoskeleton-
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mediated process. Thus, depletion of Nup153 by RNAi resulted in alteration of the nuclear lamina, and consequently in a rearrangement of the cytoskeleton and defects in cytoskeleton-based processes. Further work is necessary to address how the cytoskeletal dynamic is affected when NE components, including the Nup153, are manipulated. This will have highly relevance in understanding the molecular basis underlying certain human diseases (e.g. lipodystrophy, muscular dystrophy, neuropathy, and progeria) that are linked to defects in NE proteins [26,27]. As expected, alterations of the cytoskeleton resulted in defects in several processes that are governed by the cytoskeletal network. Because cell migration is a fundamental feature in the metastatic spread of cancer, we then studied the effect of depleting Nup153 in a human breast carcinoma cell line. We found that depletion of Nup153 significantly reduced the speed of directional cell migration in the human breast carcinoma MDA231 cell line. These cells also had polarization defects. Similar migration and polarization defects have been found in cells lacking lamin A/C [28] and in Nesprin-2-deficient fibroblasts [29]. Our results with MDA231 cells suggests that manipulation of the NPC could be used to regulate cell growth and progression of cancer invasion, and opens the exciting possibility that RNAi of Nup153 could be developed as an anti-cancer therapy. Acknowledgements We thank Dr. Sebastian Witt for contributing preliminary experiments, Dr. Brian Burke (Institute of Molecular and Cell Biology, Singapore) and Dr. K. Roux (University of Florida) for providing the Sun1-GFP cells and the SA1 antibody, and Dr. Calvin Roskelley (Department of Cellular and Physiological Sciences, University of British Columbia) for providing the MDA231, GFPMDA231and HT1080 cell lines. Pamela Austin is acknowledged for help with the cell migration assay. This work was supported by a Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to N.P. N.P. is a Michael Smith Foundation for Health Research (MSFHR) Senior Scholar. Appendix A. Supplementary data Fig. S1 shows that Nup153-siRNA treatment of the human fibrosarcoma cell line, HT1080, also affects the migration speed of these cells. Fig. S2 shows that depletion of Nup153 does not impair the nuclear import of proteins with cNLSs. Videos 1 and 2 show live cell time-lapse imaging of mock-transfected and Nup153-siRNAtransfected GFP-MDA231 cells that have been subjected to wound-induced cell migration assay respectively. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.05.038. References [1] Cronshaw, J.M., Krutchinsky, A.N., Zhang, W., Chait, B.T. and Matunis, M.J. (2002) Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915–927. [2] Smythe, C., Jenkins, H.E. and Hutchison, C.J. (2000) Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs. EMBO J. 19, 3918–3931. [3] Haque, F., Lloyd, D.J., Smallwood, D.T., Dent, C.L., Shanahan, C.M., Fry, A.M., Trembath, R.C. and Shackleton, S. (2006) SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol. Cell. Biol. 26, 3738–3751. [4] Crisp, M., Liu, Q., Roux, K., Rattner, J.B., Shanahan, C., Burke, B., Stahl, P.D. and Hodzic, D. (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J. Cell Biol. 172, 41–53. [5] Liu, Q., Pante, N., Misteli, T., Elsagga, M., Crisp, M., Hodzic, D., Burke, B. and Roux, K.J. (2007) Functional association of Sun1 with nuclear pore complexes. J. Cell Biol. 178, 785–798.
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