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• W2024160864 abstract "The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96+/− mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression. The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96+/− mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression. Nuclear transport factors and nuclear pore complex proteins (nucleoporins or Nups) mediate nucleocytoplasmic trafficking in interphase. Both transport factors and certain nucleoporins also have additional functions in mitosis, including spindle assembly and checkpoint functions (Harel and Forbes, 2004Harel A. Forbes D.J. Importin beta: conducting a much larger cellular symphony.Mol. Cell. 2004; 16: 319-330Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, Tran and Wente, 2006Tran E.J. Wente S.R. Dynamic nuclear pore complexes: life on the edge.Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Moreover, nuclear transport is subject to multiple levels of regulation, being impacted by signaling pathways, viral infection, and the proximity of active genes to the nuclear pore complex (NPC) (Tran and Wente, 2006Tran E.J. Wente S.R. Dynamic nuclear pore complexes: life on the edge.Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Furthermore, nucleocytoplasmic transport is specifically regulated during closed mitosis in yeast by molecular rearrangements at the NPC (Makhnevych et al., 2003Makhnevych T. Lusk C.P. Anderson A.M. Aitchison J.D. Wozniak R.W. Cell cycle regulated transport controlled by alterations in the nuclear pore complex.Cell. 2003; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). These changes in the NPC occur via the interaction of a specific nucleoporin with a transport receptor thus resulting in enhanced cargo release. Since these interactions are specific to mitosis, it underscores the importance of cell cycle in regulating nucleocytoplasmic trafficking and vice-versa (Makhnevych et al., 2003Makhnevych T. Lusk C.P. Anderson A.M. Aitchison J.D. Wozniak R.W. Cell cycle regulated transport controlled by alterations in the nuclear pore complex.Cell. 2003; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Another level of NPC regulation related to the cell cycle is the progressive increase in the number of nuclear pores from G1 to G2, presumably due to a doubling of nucleoporin concentration in preparation for postmitotic nuclear assembly in the daughter cells (Maul et al., 1972Maul G.G. Maul H.M. Scogna J.E. Lieberman M.W. Stein G.S. Hsu B.Y. Borun T.W. Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.J. Cell Biol. 1972; 55: 433-447Crossref PubMed Scopus (164) Google Scholar, Maul et al., 1980Maul G.G. Deaven L.L. Freed J.J. Campbell G.L. Becak W. Investigation of the determinants of nuclear pore number.Cytogenet. Cell Genet. 1980; 26: 175-190Crossref PubMed Scopus (45) Google Scholar, Winey et al., 1997Winey M. Yarar D. Giddings Jr., T.H. Mastronarde D.N. Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes.Mol. Biol. Cell. 1997; 8: 2119-2132Crossref PubMed Scopus (158) Google Scholar). Studies performed in a Xenopus nuclear reconstitution system demonstrated that the steady increase in nuclear pore numbers can occur by de novo insertion of nucleoporins from both sides of the nuclear envelope to form new nuclear pores (D'Angelo et al., 2006D'Angelo M.A. Anderson D.J. Richard E. Hetzer M.W. Nuclear pores form de novo from both sides of the nuclear envelope.Science. 2006; 312: 440-443Crossref PubMed Scopus (160) Google Scholar). These observations were made with constituents of the Nup107-160 subcomplex of the NPC, which has a key role in nuclear pore assembly (Harel et al., 2003Harel A. Orjalo A.V. Vincent T. Lachish-Zalait A. Vasu S. Shah S. Zimmerman E. Elbaum M. Forbes D.J. Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores.Mol. Cell. 2003; 11: 853-864Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Rasala et al., 2006Rasala B.A. Orjalo A.V. Shen Z. Briggs S. Forbes D.J. ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division.Proc. Natl. Acad. Sci. USA. 2006; 103: 17801-17806Crossref PubMed Scopus (180) Google Scholar, Walther et al., 2003Walther T.C. Alves A. Pickergill H. Loiodice I. Hetzer M. Galy V. Hulsmann B.B. Kocher T. Wilm M. Allen T. et al.The conserved Nup107–160 complex is critical for nuclear pore complex assembly.Cell. 2003; 113: 195-206Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The vertebrate Nup107-160 complex constitutes one third of the proteins of the vertebrate NPC, containing Nup107, Nup160, Nup133, Nup85, Nup96, Sec13, Nup43, Nup37, Seh1, and variably ELYS/MEL-28 (Belgareh et al., 2001Belgareh N. Rabut G. Bai S.W. van Overbeek M. Beaudouin J. Daigle N. Zatsepina O.V. Pasteau F. Labas V. Fromont-Racine M. et al.An evolutionary conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.J. Cell Biol. 2001; 154: 1147-1160Crossref PubMed Scopus (251) Google Scholar, Enninga et al., 2003Enninga J. Levay A. Fontoura B.M. Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex.Mol. Cell. Biol. 2003; 23: 7271-7284Crossref PubMed Scopus (65) Google Scholar, Fontoura et al., 1999Fontoura B.M. Blobel G. Matunis M.J. A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates nup98 and the novel nucleoporin, nup96.J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (185) Google Scholar, Franz et al., 2007Franz C. Walczak R. Yavuz S. Santarella R. Gentzel M. Askjaer P. Galy V. Hetzer M. Mattaj I.W. Antonin W. MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly.EMBO Rep. 2007; 8: 165-172Crossref PubMed Scopus (163) Google Scholar, Harel et al., 2003Harel A. Orjalo A.V. Vincent T. Lachish-Zalait A. Vasu S. Shah S. Zimmerman E. Elbaum M. Forbes D.J. Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores.Mol. Cell. 2003; 11: 853-864Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Loiodice et al., 2004Loiodice I. Alves A. Rabut G. Van Overbeek M. Ellenberg J. Sibarita J.B. Doye V. The entire Nup107–160 complex, including three new members, is targeted as one entity to kinetochores in mitosis.Mol. Biol. Cell. 2004; 15: 3333-3344Crossref PubMed Scopus (199) Google Scholar, Orjalo et al., 2006Orjalo A.V. Arnaoutov A. Shen Z. Boyarchuk Y. Zeitlin S.G. Fontoura B. Briggs S. Dasso M. Forbes D.J. The Nup107–160 nucleoporin complex is required for correct bipolar spindle assembly.Mol. Biol. Cell. 2006; 17: 3806-3818Crossref PubMed Scopus (116) Google Scholar, Rasala et al., 2006Rasala B.A. Orjalo A.V. Shen Z. Briggs S. Forbes D.J. ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division.Proc. Natl. Acad. Sci. USA. 2006; 103: 17801-17806Crossref PubMed Scopus (180) Google Scholar, Vasu et al., 2001Vasu S. Shah S. Orjalo A. Park M. Fischer W.H. Forbes D.J. Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.J. Cell Biol. 2001; 155: 339-353Crossref PubMed Scopus (132) Google Scholar). Once incorporated into the nuclear pore, the Nup107-160 complex, like its yeast counterpart, the Nup84 complex, plays a key role in mediating mRNA export (Aitchison et al., 1995Aitchison J.D. Blobel G. Rout M.P. Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport.J. Cell Biol. 1995; 131: 1659-1675Crossref PubMed Scopus (112) Google Scholar, Boehmer et al., 2003Boehmer T. Enninga J. Dales S. Blobel G. Zhong H. Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex.Proc. Natl. Acad. Sci. USA. 2003; 100: 981-985Crossref PubMed Scopus (112) Google Scholar, Dockendorff et al., 1997Dockendorff T.C. Heath C.V. Goldstein A.L. Snay C.A. Cole C.N. C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure.Mol. Cell. Biol. 1997; 17: 906-920Crossref PubMed Scopus (40) Google Scholar, Emtage et al., 1997Emtage J.L. Bucci M. Watkins J.L. Wente S.R. Defining the essential functional regions of the nucleoporin Nup145p.J. Cell Sci. 1997; 110: 911-925PubMed Google Scholar, Faria et al., 2006Faria A.M. Levay A. Wang Y. Kamphorst A.O. Rosa M.L. Nussenzveig D.R. Balkan W. Chook Y.M. Levy D.E. Fontoura B.M. The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions.Immunity. 2006; 24: 295-304Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, Teixeira et al., 1997Teixeira M.T. Siniossoglou S. Podtelejnikov S. Benichou J.C. Mann M. Dujon B. Hurt E. Fabre E. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.EMBO J. 1997; 16: 5086-5097Crossref PubMed Scopus (88) Google Scholar). In mitosis, the Nup107-160 complex assumes other important functions, being incorporated into both kinetochores and the centrosomes/proximal spindle poles (Enninga et al., 2003Enninga J. Levay A. Fontoura B.M. Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex.Mol. Cell. Biol. 2003; 23: 7271-7284Crossref PubMed Scopus (65) Google Scholar, Loiodice et al., 2004Loiodice I. Alves A. Rabut G. Van Overbeek M. Ellenberg J. Sibarita J.B. Doye V. The entire Nup107–160 complex, including three new members, is targeted as one entity to kinetochores in mitosis.Mol. Biol. Cell. 2004; 15: 3333-3344Crossref PubMed Scopus (199) Google Scholar, Orjalo et al., 2006Orjalo A.V. Arnaoutov A. Shen Z. Boyarchuk Y. Zeitlin S.G. Fontoura B. Briggs S. Dasso M. Forbes D.J. The Nup107–160 nucleoporin complex is required for correct bipolar spindle assembly.Mol. Biol. Cell. 2006; 17: 3806-3818Crossref PubMed Scopus (116) Google Scholar, Rasala et al., 2006Rasala B.A. Orjalo A.V. Shen Z. Briggs S. Forbes D.J. ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division.Proc. Natl. Acad. Sci. USA. 2006; 103: 17801-17806Crossref PubMed Scopus (180) Google Scholar). Indeed, depletion of the Nup107-160 complex disrupts the correct formation of spindle microtubules in mitotic Xenopus egg extracts (Orjalo et al., 2006Orjalo A.V. Arnaoutov A. Shen Z. Boyarchuk Y. Zeitlin S.G. Fontoura B. Briggs S. Dasso M. Forbes D.J. The Nup107–160 nucleoporin complex is required for correct bipolar spindle assembly.Mol. Biol. Cell. 2006; 17: 3806-3818Crossref PubMed Scopus (116) Google Scholar) and compromises kinetochore function (Zuccolo et al., 2007Zuccolo M. Alves A. Galy V. Bolhy S. Formstecher E. Racine V. Sibarita J.B. Fukagawa T. Shiekhattar R. Yen T. et al.The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions.EMBO J. 2007; 26: 1853-1864Crossref PubMed Scopus (150) Google Scholar). Here we report that the Nup107-160 complex is cell cycle-regulated. In particular, Nup96 is preferentially downregulated in mitosis via the ubiquitin-proteasome pathway. We show that this regulation of Nup96 controls cell cycle progression and differential phase-specific gene expression of key cell cycle regulators. To ask whether the constituents of the Nup107-160 complex are differentially regulated in a cell cycle-dependent manner, we synchronized HeLa cells and performed immunoblot analysis on cell extracts obtained from different phases of the cell cycle. We found that the levels of Nup107, Sec13, Nup85, Nup37, Nup43, Nup160, Nup153, and Nup62 increased from G1 to G2/M phases of the cell cycle (Figures 1A and 1B), as would be predicted from the electron microscopic visualization of nuclear pore doubling that occurs at S phase (Maul et al., 1972Maul G.G. Maul H.M. Scogna J.E. Lieberman M.W. Stein G.S. Hsu B.Y. Borun T.W. Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.J. Cell Biol. 1972; 55: 433-447Crossref PubMed Scopus (164) Google Scholar, Maul et al., 1980Maul G.G. Deaven L.L. Freed J.J. Campbell G.L. Becak W. Investigation of the determinants of nuclear pore number.Cytogenet. Cell Genet. 1980; 26: 175-190Crossref PubMed Scopus (45) Google Scholar, Winey et al., 1997Winey M. Yarar D. Giddings Jr., T.H. Mastronarde D.N. Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes.Mol. Biol. Cell. 1997; 8: 2119-2132Crossref PubMed Scopus (158) Google Scholar). Notably, the level of Nup96, a member of the Nup107-160 complex, although increasing from G1 to G2, was preferentially downregulated 50%–60% in early mitosis (Figure 1A). This level of change was not seen for the other nucleoporins at mitosis. Cyclins A and B served as controls. Cyclin A was downregulated in early mitosis, as expected, while cyclin B was upregulated in early mitosis and degraded in anaphase (Figure 1A; Murray, 2004Murray A.W. Recycling the cell cycle: cyclins revisited.Cell. 2004; 116: 221-234Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Upon further analysis, sucrose gradient sedimentation of interphase and mitotic extracts of HeLa cells revealed that a very significant pool of Nup43 is partially dissociated from the Nup107-160 complex at mitosis, indicating that the Nup107-160 complex is not identical in interphase and mitosis (Figure 1C). Since Nup96 levels were downregulated in mitosis, we set out to determine the mechanism involved in this process. We first confirmed the observed reduction in Nup96 levels in early mitosis using multiple methods of cell synchronization. HeLa cells were synchronized at the G1/S border with a double thymidine block, followed by treatment with nocodazole, taxol, or colcimid to block cells in mitosis. All three sets of mitotic cells showed a decrease in Nup96 protein levels compared to control asynchronous cells ( Figures 1A and 2A). We observed a similar reduction in Nup96 levels in HeLa cells after mitotic cell shake-off, which was performed to exclude any potential effect of particular drugs on the expression levels of Nup96 in mitosis (Figure 2B). The decrease in Nup96 levels at mitosis was also observed in other cell types, such as primary mouse embryo fibroblasts (MEF) (Figure 2C) and human 293T cells (data not shown). Furthermore, we found that this mitotic downregulation of Nup96 does not occur at the mRNA level (Figure 2D). Quantification of Nup96 mRNA levels between the G2 and M phases, by real-time PCR, did not show significant differences (Figure 2D). We next asked whether downregulation of Nup96 in mitosis involved proteasome-mediated degradation. HeLa cells were treated with nocodazole in the presence or absence of the proteasome inhibitor lactacystin and the levels of Nup96 in mitosis were analyzed by immunoblot. In the presence of lactacystin, the Nup96 level in mitosis was higher than in the absence of lactacystin (Figure 2E). This result indicated that Nup96 is a substrate of the proteasome. In contrast, the levels of Nup160 or actin did not change to the same extent as Nup96 in the presence of lactacystin (Figure 2E). To further investigate whether Nup96 degradation occurs via the ubiquitin pathway, we transfected 293T cells with plasmids encoding HA-tagged ubiquitin alone (Figure 2F) or myc-tagged Nup96 in combination with HA-tagged ubiquitin (Figures 2G; see Figure S1A and Supplemental Data available online), and performed immunoprecipitation using anti-Nup96 antibodies or anti-HA antibody. Overexpression of HA-ubiquitin should compete with endogenous ubiquitin and become covalently conjugated to target proteins. We were able to immunoprecipitate HA-tagged ubiquitinated forms of endogenous Nup96 (Figure 2F, lanes 3 and 4) or overexpressed Nup96 (Figure 2G and Figure S1A). Moreover, ladders of polyubiquitin conjugates of Nup96 were enriched in the presence of the proteasome inhibitor MG132 (Figure S1A). Thus, both endogenous and ectopically expressed Nup96 are ubiquitinated in vivo. To determine whether ubiquitination of Nup96 is increased in mitosis, cells were transfected with HA-ubiquitin for 24 hr. Then, the transfected cells were incubated with thymidine for 14 hr to obtain cell cultures enriched at the G1/S border, or incubated with nocodazole for 14 hr to collect cells in mitosis. Cell extracts were subjected to immunoprecipitation with anti-Nup96 antibodies followed by immunoblot analysis with anti-HA antibody. Indeed, increased ubiquitination of Nup96 was observed in mitosis, as compared to cells at the G1/S border (Figure 2H). This increase in Nup96 ubiquitination supports the findings showing downregulation of Nup96 in mitosis at the protein level. We also observed that other Nups, such as Nup107 (Figure S1B) and Sec13 (data not shown), can be ubiquitinated, though their levels do not change significantly in mitosis as in the case of Nup96. These observations indicate that ubiquitin-mediated proteolysis is a mechanism involved in nucleoporin turnover. However, in the case of Nup96, ubiquitination and proteasome-mediated degradation are enhanced in mitosis, which is the mechanism by which Nup96 is preferentially downregulated. To further analyze the proteolysis of Nup96 in mitosis, we carried out experiments with the mitosis-specific ubiquitin-conjugating enzyme UbcH10 to determine if it has a role in the mitotic degradation of Nup96. UbcH10 is known to be involved in mediating degradation of mitotic-specific substrates such as cyclin A and cyclin B (Peters, 2002Peters J.M. The anaphase-promoting complex: proteolysis in mitosis and beyond.Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar). To test whether inhibition of UbcH10 activity in mitosis would diminish Nup96 proteolysis, cotransfection experiments in HeLa cells were performed with plasmids encoding myc-tagged Nup96 and either wild-type UbcH10 or a dominant-negative mutant form of UbcH10, C114-S. UbcH10 (C114-S) inhibits both cyclin A and B degradation, as well as the metaphase to anaphase transition (Townsley et al., 1997Townsley F.M. Aristarkhov A. Beck S. Hershko A. Ruderman J.V. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase.Proc. Natl. Acad. Sci. USA. 1997; 94: 2362-2367Crossref PubMed Scopus (186) Google Scholar). Indeed, overexpression of the C114-S mutant of the mitosis-specific ubiquitin-conjugating enzyme UbcH10 inhibited both Nup96 ubiquitination (Figure 2I) and Nup96 degradation (Figure 2J). On the other hand, C114-S did not significantly affect the levels of Nup43 or hnRNPA1 (Figure 2J). Since UbcH10 is a mitosis-specific E2 enzyme, these results indicate that Nup96 is ubiquitinated and also preferentially degraded in mitosis. To determine the functional significance of Nup96 downregulation in mitosis, we investigated whether Nup96, if present at high levels, has effects on mitotic timing. We generated stably transfected HeLa cell lines expressing high levels of FLAG-tagged-Nup96 (data not shown) and coexpressed histone H2B-YFP to follow live cells as they transitioned through mitosis. In the presence of excess Nup96, we observed a slight delay from metaphase to anaphase and from anaphase to cytokinesis (Table 1). These modest effects of high levels of Nup96 on mitotic timing did not result in any major mitotic defect. We also followed mitotic progression in the presence of high levels of Nup37, another constituent of the Nup107-160 complex, and again did not detect any major effect on mitotic timing or mitotic defects (Table 1). In addition, we tested whether high levels of Nup96 would affect mitotic timing of normal rat kidney (NRK) cells, which have an intact G1 checkpoint. We show, in Table 1, that increased levels of Nup96 had modest effects on mitotic timing in NRK cells causing a slight acceleration from NEBD to metaphase and from anaphase to cytokinesis. Although these minor defects on mitotic timing caused by high levels of Nup96 varied between HeLa cells and NRK cells (Table 1), in both cases they did not result in any major mitotic defect.Table 1High Levels of Nup96 in Mitosis Slightly Alter Mitotic TimingCellsTransfectantNEB-MetaphaseMetaphase-AnaphaseAnaphase-CytokinesisHeLa Tet-On+ control plasmid34 (±6)21 (±4)10 (±2)n = 23+ FLAG-Nup9630 (±4)29 (±4)∗13 (±2)∗HeLa Tet-On+ EGFP29 (±8)19 (±7)10 (±1)n = 28+ EGFP-Nup3726 (±6)19 (±8)12 (±3)NRK+ EGFP21 (±5)14 (±7)10 (±1)n = 14+ myc-Nup9618 (±4)∗12 (±6)7 (±3)∗Tet-On HeLa cells were stably transfected with plasmid alone (control) or plasmid encoding FLAG-Nup96, and Nup96 expression was induced by doxycyclin (n = 23). Tet-On HeLa cells were also transfected with plasmids encoding EGFP (control) or EGFP-Nup37 (n = 28). NRK cells were transfected with myc-Nup96 and EGFP (5:1) or, as control, transfected with EGFP and vector alone (5:1) (n = 14). Mitotic chromosomes labeled with H2B-YFP were followed over time by fluorescence and phase contrast. Time zero was marked by the nuclear envelope breakdown (NEB). ∗p < 0.05. Results are the mean ± SD. Open table in a new tab Tet-On HeLa cells were stably transfected with plasmid alone (control) or plasmid encoding FLAG-Nup96, and Nup96 expression was induced by doxycyclin (n = 23). Tet-On HeLa cells were also transfected with plasmids encoding EGFP (control) or EGFP-Nup37 (n = 28). NRK cells were transfected with myc-Nup96 and EGFP (5:1) or, as control, transfected with EGFP and vector alone (5:1) (n = 14). Mitotic chromosomes labeled with H2B-YFP were followed over time by fluorescence and phase contrast. Time zero was marked by the nuclear envelope breakdown (NEB). ∗p < 0.05. Results are the mean ± SD. However, we observed that high levels of Nup96 resulted in a prominent delay in G1/S progression. In this experiment we also used NRK cells because they have an intact G1 checkpoint. NRK cells were synchronized in S phase with aphidicolin, then transfected 3 hr after release from aphidicolin with plasmids encoding Nup96, Nup37, ALADIN, or EGFP, at a time when the cells were in late G2. Under these conditions, detectable protein expression occurs in G1, after plasmids efficiently enter the nucleus following nuclear envelope breakdown in mitosis. BrdU staining was performed 19 hr after the aphidicolin release and cells were analyzed by immunofluorescence and Apotome microscopy (Figure 3A). Quantification of BrdU-positive cells, indicative of cells having entered S phase, was carried out. The data showed that approximately half of the population of cells expressing high levels of Nup96 was still in G1 at 19 hr (Figure 3A), while the majority of cells expressing high levels of the control EGFP protein had already progressed into S phase, which was set to 100% (Figure 3A). High expression levels of the nucleoporins Nup37 or ALADIN did not result in any inhibition of G1 progression. In fact, high levels of ALADIN showed an acceleration of G1 progression. Thus, we conclude that high levels of Nup96 delay G1 progression or impede the G1/S transition. To further examine the significance of regulating Nup96 levels in vivo, we used our Nup96+/− mouse model, where cells express low levels of Nup96 (Faria et al., 2006Faria A.M. Levay A. Wang Y. Kamphorst A.O. Rosa M.L. Nussenzveig D.R. Balkan W. Chook Y.M. Levy D.E. Fontoura B.M. The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions.Immunity. 2006; 24: 295-304Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We had a previous indication that T cells derived from Nup96+/− mice proliferated faster than those from Nup96+/+ mice, as assessed by thymidine incorporation (Faria et al., 2006Faria A.M. Levay A. Wang Y. Kamphorst A.O. Rosa M.L. Nussenzveig D.R. Balkan W. Chook Y.M. Levy D.E. Fontoura B.M. The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions.Immunity. 2006; 24: 295-304Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). However, the mechanisms underlying this proliferation defect have not been investigated. Here we report, using different methods to assess cell proliferation, that both T cells and bone marrow-derived macrophages (BMDM) from Nup96+/− mice show an acceleration of the cell cycle, with this phenotype being most pronounced in T cells. This was observed after resting T cells (G0 phase), obtained from spleen, were labeled with CSFE and induced to proliferate with anti-CD3 and anti-CD28 antibodies. CSFE, a fluorescent dye, binds covalently to cytoplasmic proteins. Following each cell division, the fluorescence intensity of CSFE is reduced, which is quantified by flow cytometry. Nup96+/− T cells showed a higher reduction in fluorescence intensity between day 1 and day 2 than Nup96+/+ T cells, indicating that Nup96 mutant cells proliferated faster than wild-type cells (Figure 3B). Similar results were obtained when T cells from the spleen, mesenteric lymph nodes (MLN), and peripheral lymph nodes (PLN) of Nup96+/+ and Nup96+/− mice were induced to proliferate with anti-CD3 and anti-CD28 antibodies for 72 hr, and their proliferation was measured by thymidine incorporation. Lastly, we examined growth rates of bone marrow-derived macrophages (BMDM) from Nup96+/+ and Nup96+/− mice. An equal number of cells was incubated with IL-3 conditioned and MEF-conditioned medium. The number of BMDM cells was then calculated after 48 hr by measuring the activity of the mitochondrial reductase enzymes, which correlates to cell number. Proliferation of BMDM was slightly enhanced in Nup96+/− cells as compared to Nup96+/+ cells (Figure 3B). Cell cycle acceleration was, notably, more prominent in T cells than in macrophages, especially in the case of T cells obtained from mesenteric lymph nodes (Figure 3B; Faria et al., 2006Faria A.M. Levay A. Wang Y. Kamphorst A.O. Rosa M.L. Nussenzveig D.R. Balkan W. Chook Y.M. Levy D.E. Fontoura B.M. The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions.Immunity. 2006; 24: 295-304Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). One explanation for the observed effect on cell proliferation might be that inappropriate levels of Nup96 in G1 may interfere with certain key aspects of gene expression related to cell cycle progression. Thus, mRNA abundance, nucleocytoplasmic distribution of mRNAs, and protein expression of key cell cycle regulators were assessed in G1. To test for such effects using a physiological model, we obtained resting T cells (G0) from the spleens of Nup96+/+ or Nup96+/− mice. These cells were then stimulated for 2 hr with anti-CD3 and anti-CD28 antibodies to enter G1. As expected, both Nup96+/+ and Nup96+/− T cells similarly transitioned into G1 after 2 hr stimulation, as demonstrated by the comparable fold-induction of IκBα mRNA levels in both cell types (Figure 4A). Increase in IκBα mRNA levels correlates with entry into G1 as NFκB is imported into the nucleus and activates the transcription of the IκBα gene as part of a feedback mechanism (Hoffmann et al., 2002Hoffmann A. Levchenko A. Scott M.L. Baltimore D. The IκB-NF-κB signaling module: temporal control and selective gene activation.Science. 2002; 298: 1241-1245Crossref PubMed Scopus (1410) Google Scholar). Since mouse T cells, when stimulated, complete one cell cycle within ∼5–6 hr (van Stipdonk et al., 2001van Stipdonk M.J. Lemmens E.E. Schoenberger S.P. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiatio" @default.
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