Bernis C , Swift-Taylor B , Nord M , Carmona S , Chook YM , Forbes DJ .

R01 GM033279 NIGMS NIH HHS , R01 GM069909 NIGMS NIH HHS , R01-GM033279 NIGMS NIH HHS , R01-GM069909 NIGMS NIH HHS

	FIGURE 1:. Molecular tools for probing the mechanism of action of transportin in mitosis. (A) M9M is a synthetic hybrid PY-NLS peptide capable of binding to transportin with 200-fold binding strength relative to the M9 NLS (Cansizoglu et al., 2007). M9 is the NLS found in hnRNP A1 (Siomi and Dreyfuss, 1995; Nakielny et al., 1996; Pollard et al., 1996; Iijima et al., 2006; Lee et al., 2006); it is in the class of hydrophobic PY-NLSs. Also shown is the basic PY-NLS found in hnRNP M. M9M is a hybrid constructed from these hydrophobic and basic PY-NLSs. In all experiments, M9M is used in a recombinant form of MBP-M9M, where M9M is fused to MBP for ease of purification and use. (B) Wild-type transportin recognizes and binds cargo containing a PY-NLS; when the transportin:cargo complex encounters and binds RanGTP, the H8 loop moves to displace the cargo. The transportin TLB mutant lacks the H8 loop, and, as such, when RanGTP interacts with the cargo-laden complex, the cargo is not displaced. This allows TLB to be bound to cargo and RanGTP simultaneously. This schematic representation is an adaptation of the description provided in Chook et al. (2002).
	FIGURE 2:. M9M inhibition of transportin in HeLa cells causes defects in mitosis. HeLa cells were transfected for 24 h with mammalian expression vectors containing the myc-tagged MBP constructs MBP, MBP-M9, and MBP-M9M. The transfection of the M9M construct caused clear defects in cytokinesis. Abnormal structures observed included (A) excess midbodies, (B) DNA bridges between cells accompanying said midbodies, and (C) multinucleate cells. Few of these defects were observed in cells that were untransfected or transfected with myc-tagged MBP (see F). In addition, in cells showing mitotic spindles, a large fraction had defective spindles after MBP-M9M plasmid transfection (D), whereas those transfected with MBP constructs showed normal spindles (E). Tubulin was detected with a FITC-labeled anti-tubulin antibody (green), myc-construct transfected cells were visualized with a TRITC-labeled anti-myc antibody (red), and DNA was visualized with DAPI (blue). (F) Quantification of the different aberrant structures observed in HeLa cells transfected with MBP constructs was performed and graphed. In cells transfected with MBP-M9M, 18% showed midbodies (+M9M, red bar), as compared with <3% of nontransfected cells on the same coverslip (+M9M, gray bar) or cells transfected with MBP (CTL, red bar, transfected; gray bar, untransfected on same coverslip). Thirteen percent of the MBP-M9M–transfected cells showing midbodies also showed DNA bridges (DNA bridges, +M9M, red bar), as opposed to no detectable DNA bridges in the MBP-transfected control or the nontransfected cells. Another 6% of the MBP-M9M–transfected cells were multinucleate (multinucleate cells, +M9M, red bar), as opposed to <1% of the MBP-transfected control or nontransfected cells. In all of our transfections, 6% of the total cells had mitotic spindles. (G) However, whereas only ∼1% of the spindles in control myc-MBP–transfected cells and 1% of the spindles in nontransfected cells were abnormal, we found that 80% of the spindles in MBP-M9M– transfected cells were abnormal (see also abnormal spindles in F).
	FIGURE 3:. M9M addition to mitotic extracts mirrors Ran GTP in causing large spindles, free asters, and multipolar spindles. (A–C) Freshly prepared interphase Xenopus egg extract was mixed with sperm chromatin; nuclei were allowed to form and the DNA to replicate for 2 h. Mitotic Xenopus extract was then added to convert it to a mitotic state. Rhodamine-labeled tubulin was added with recombinant MBP, MBP-M9M, or RanGTP (Ran Q69L-GTP) 5 min later. The resulting microtubule structures (red) were examined at 60 min using fluorescence microscopy; chromatin was visualized with Hoechst DNA dye (blue). (A) Control condition: after MBP addition (10 μM), the majority of condensed chromatin structures (77%) showed strong bipolar spindles. Rarer structures included weak bipolar spindles (13%), half-spindles (5%), and chromatin with no associated microtubules (4%). (B) When MBP-M9M (10 μM) was added, 45% of the structures observed were normally shaped bipolar spindles of slightly increased size. However, now we also observed very large bipolar spindles with a larger than normal complement of DNA (15%), clusters of asters with no associated chromatin (26%), multipolar spindles (8%), and chromatin with no associated microtubules (6%; not shown). (C) When RanQ69L-GTP (10 μM) was instead added, we observed normally shaped bipolar spindles of slightly larger size (25%), very large spindles with much more associated DNA (8%), clusters of large asters with no associated chromatin (48%), and multipolar spindles (13%). Finally, 6% were chromatin with no associated microtubules (not shown). We note that the aforementioned M9M-induced asters are smaller than those induced in the RanGTP condition. (D) M9M, like RanGTP, promoted larger spindles in vitro. For A–C, representative images of the observed structures are shown. The percentage indicated on an image indicates the average percentage of that structure under that specific condition summarized from five replicate experiments; ∼150 structures were counted for each condition for each experiment. The surface area of the normally shaped bipolar spindles from the MBP, M9M, and RanGTP conditions in A–C (top) were measured using ImageJ software (∼40 spindles were measured per condition in three independent experiments). Overall, the bipolar spindles were ∼40% larger with MBP-M9M addition and ∼70% larger with RanQ69L-GTP addition than with MBP addition. (E–G) In the absence of chromatin, M9M, like RanGTP, induced aster assembly in vitro. Xenopus mitotic extract plus Energy Mix, but without added chromatin, was incubated with rhodamine-labeled tubulin and 10 μM MBP (control), 10 μM RanQ69L-GTP, or 10 μM MBP-M9M. No microtubule-containing structures were observed in the control (E; +MBP). RanGTP addition induced formation of microtubule asters (F; +Ran). RanGTP releases spindle assembly factors such as NuMa and TPX2 from importin (Zhang and Chook, 2012), causing impromptu microtubule nucleation (Gruss et al., 2001; Nachury et al., 2001). Microtubule asters also are seen here to form when MBP-M9M is added in the absence of chromatin (G; +M9M).
	FIGURE 4:. TLB inhibition of spindle assembly can be reversed by M9M addition but not by RanGTP addition. (A–H) Interphase Xenopus egg extract was mixed with sperm chromatin and rhodamine-labeled tubulin; nuclei were allowed to form, and the DNA was allowed to replicate for 1 h. A portion of this reaction was added to mitotic Xenopus extract to convert it to a mitotic state. Recombinant proteins were added as noted and the resulting structures examined by immunofluorescence microscopy; microtubules are red and chromatin is blue due to Hoechst DNA dye. Representative images. (A, B) Here 20 μM GST and 10 μM MBP are controls and show normal bipolar spindles. (C, D) Addition of 20 μM GST-transportin or 20 μM GST-TLB produced almost no microtubule formation over chromatin. (E) As shown in Lau et al. (2009), 10 μM RanQ69L-GTP plus 20 μM GST-transportin induced rescue of normal bipolar spindle assembly. (F) Use of 10 μM RanQ69L-GTP plus 20 μM GST-TLB produced essentially no microtubule formation over chromatin. (G) Use of 10 μM MBP-M9M plus 20 μM GST-transportin gave robust bipolar spindle assembly. (H) Use of 10 μM MBP-M9M plus 20 μM GST-TLB also produced robust bipolar spindle assembly. (I) Quantitation of the effects of addition of TLB, transportin, M9M, and RanGTP on spindle assembly. Five replicates of the experiment in A–H were done. Coverslips from each condition were examined and the different structures quantified; ∼150 structures were examined for each condition in each experiment. Analysis of the combined results are shown in the graph. As shown by the error bars, little variability was observed between the five different experiments. Control conditions (10 μM MBP or 20 μM GST; gray, green bars) showed almost 80% normal bipolar spindles. Addition of 20 μM GST-TLB or 20 μM GST-transportin to the reaction caused dramatic loss of spindle formation, down to 5% or less in these experimental conditions (yellow, red bars). Addition of 10 μM RanQ69L-GTP with 20 μM GST-transportin (purple bar) rescued this inhibition to ∼55% normal spindles. However, addition of 10 μM RanQ69L-GTP to the 20 μM GST-TLB condition showed no increase in spindle formation (blue bar; <5% normal bipolar spindles). Surprisingly, addition of 10 μM MBP-M9M to 20 μM GST-transportin (light green) rescued normal bipolar spindles >65%. Addition of 10 μM MBP-M9M to 20 μM GST-TLB pink bar) was also rescued to >70% normal bipolar spindles.
	FIGURE 5:. Transportin inhibition of nuclear membrane fusion is rescued by M9M addition. (A–H) High-speed interphase Xenopus egg extract was mixed with sperm chromatin and membranes and allowed to incubate at room temperature for 60 min. Recombinant proteins were added at the concentrations detailed in Materials and Methods. The resulting nuclear structures were examined for integrity using 70-kDa rhodamine–dextran exclusion and for membrane fusion with the membrane dye DHCC (green). Chromatin was stained with Hoechst DNA stain (blue). (A) As a control, addition of 15 μM GST showed smooth nuclear envelope staining and impermeability to 70-kDa dextran. (B, D) The same phenotype was observed when 8 mM BAPTA or 15 μM RanQ69L-GTP was added to the reaction. (C) Addition of 2 mM GTPγS prevented vesicle–vesicle fusion, shown by the rough nuclear envelope staining around the DNA and a permeable nuclear envelope. (E) Addition of 15 μM GST-TRN prevented vesicle–vesicle fusion, inducing a permeable nuclear envelope. (F, G) Addition of 15 μM RanQ69L-GTP or 15 μM MBP-M9M to 15 μM GST-TRN rescued membrane integrity and continuous envelope staining. (H) Vesicle–vesicle fusion was also inhibited by 15 μM GST-TLB. (I) Addition of 15 μM RanQ69L-GTP could not rescue inhibition by 15 μM GST-TLB. (J) Addition of 15 μM MBP-M9M was able to restore the integrity of the nuclear envelope when added with 15 μM GST-TLB. (K) Quantitation of five different experiments; between 70 and 100 nuclei were examined for each condition for each experiment. Little variability was observed between the five experiments for each condition (±1–5%). Blue bars, percentage of nuclei showing dextran diffusion; red bars, percentage of nuclei excluding dextran; representative images in A–J.
	FIGURE 6:. Nuclear pore assembly inhibition by excess transportin is reversed by M9M. (A) To assemble pore-free nuclear intermediates, we incubated sperm chromatin, interphase egg extract, and membranes in the presence of 8 mM BAPTA for 60 min. Pore-free nuclei containing fused nuclear membranes were formed (Macaulay and Forbes, 1996). Nuclear pore assembly was tested under different conditions by diluting an aliquot of these pore-free nuclei 1:10 into fresh cytosol and incubating for another 60 min. The presence of nuclear pores was detected by staining for FG-nucleoporins (mAb414-Alexa555; red), whereas membranes were stained with DHCC (green) and the DNA with Hoechst dye (blue). (B) Addition of 15 μ M MBP as a control allowed nuclear pore assembly; red. (C) Addition of 15 μM GST-TRN inhibited nuclear pore assembly. (D, E) Addition of 15 μM RanQ69L-GTP or 15 μM MBP-M9M with 15 μM GST-TRN rescued nuclear pore assembly. (F) The presence of 15 μM GST-TLB abolished nuclear pore assembly. (G) Addition of 15 μM RanQ69L-GTP with GST-TLB did not rescue nuclear pore assembly; however, (H) addition of 15 μM MBP-M9M to 15 μM GST-TLB did rescue nuclear pore assembly. (I–K) As controls, we found that the addition of 15 μM MBP-M9M, 15 μM RanQ69L-GTP, or a combination of both did not interfere with nuclear pore assembly. (L) Quantitation of five different experiments; between 70 and 100 nuclei were examined for each condition for each experiment. Little variability was observed between experiments within a condition (±1–6%). Only two types of results were observed: blue bars indicate the percentage of nuclei without FG Nups, and red bars indicate the percentage of nuclei with abundant FG-containing nuclear pores. Representative images in A–K.
	FIGURE 7:. Multiple FG nucleoporins and the Nup107–160 complex are transportin-binding targets that are released by M9M. (A) GST or GST-transportin beads were incubated with Xenopus mitotic cytosol for 1 h at room temperature. The bound proteins were analyzed by immunoblotting. An aliquot of mitotic cytosol is shown in lane 1 (Cyt). FG-nucleoporins (Nup358, Nup214, Nup153, and Nup62), members of the Nup107–160 complex (Nup160, Nup133, and Nup43), and ELYS were seen to interact with transportin (GST-TRN; lane 2; Lau et al., 2009). Neither Nup155 nor the non-Nup cdc6 protein, tested as a negative control, associated with transportin (GST-TRN; lane 2). GST-bound beads did not interact with any of the tested nucleoporins (GST; lane 4). When M9M was added in the reaction in lane 3 (GST-TRN+M9M), only Nup153 remained bound to the beads. (B) To determine whether the interaction of endogenous Xenopus ELYS with endogenous transportin was sensitive to M9M, immunoprecipitation from mitotic Xenopus egg extract was performed using anti-xELYS antibodies. This was followed by immunoblotting with anti-ELYS or anti-transportin. Immunoprecipitation of ELYS (lane 2, top) shows that transportin coimmunoprecipitates (lane 2, bottom). Lane 3 shows that addition of M9M to the extract prevents ELYS from binding to transportin. Neither ELYS nor TRN is present in mock immunoprecipitations using control immunoglobulin G (IgG) plus and minus M9M (lanes 4 and 5). (C) The anti-ELYS antiserum is specific. The ELYS doublet was present in mitotic extract mock depleted with IgG beads (ΔMOCK; lanes 4 and 5) but was absent from mitotic cytosol previously subjected to immunodepletion of ELYS (ΔELYS; lanes 2 and 3).
	FIGURE 8:. Transportin regulates kinetochore binding of ELYS and the Nup107–160 complex. Mitotic Xenopus egg extract was mixed with sperm chromatin, and the chromatin was allowed to remodel and condense for 60 min. Where indicated, GST-transportin (+TRN) or GST-transportin plus MBP-M9M (TRN+M9M) was added at 20 μM at t = 0 min. The presence of nucleoporins on kinetochores was detected by immunofluorescence with Nup-specific antibodies (red dots). In control conditions (+GST), nucleoporins of the Nup107–160 complex and ELYS each showed a dot-like staining on kinetochores (αNup133, αNup160, αELYS, αNup43), as expected (Belgareh et al., 2001; Orjalo et al., 2006; Rasala et al., 2006). Addition of GST-transportin (+TRN; 20 μM) caused almost-complete disappearance of the nucleoporin staining on kinetochores (αNup133, αNup160, αNup43, αELYS). However, inclusion of 20 μM MBP-M9M with 20 μM GST-transportin (+TRN+M9M) restored the kinetochore localization of these particular Nups. Antibodies to the nucleoporins Nup155 and Nup62 did not show any staining on kinetochores under any condition (as previously shown; Orjalo et al., 2006), serving as a negative control. This experiment was performed three times; ∼70 mitotic chromosome packages were observed per condition. Representative images are shown for each condition and antibody probe.
	FIGURE 9:. Mechanistically parallel transportin and importin β GPS pathways. As depicted in the model shown, transportin (TRN) acts by a mechanism parallel to that of importin β, that is, by direct factor inhibition. Spindle assembly at metaphase requires spindle assembly factors or SAFs (blue spheres; top half). As the cell cycle proceeds, a subsequent mitotic assembly event that occurs is the initiation of nuclear pore assembly in telophase. This is characterized by the recruitment and binding of an early set of Nups, the Nup107–160 complex and ELYS, to the surface of the decondensing chromatin (Nups; gray-blue spheres; bottom half). In metaphase, transportin mechanistically acts by binding to and inhibiting spindle assembly factors in regions far from the chromatin; in contrast, SAFs near chromatin are released from transportin by the RanGTP cloud (yellow), produced by the chromatin-bound RanGEF RCC1 (small red spheres), and release promotes spindle assembly. Later, after the spindle has disassembled and telophase begins, nucleoporins such as the Nup107–160 complex and ELYS are inhibited by transportin in regions far from chromatin, but those near chromatin are released from transportin by the RanGTP cloud and initiate nuclear pore assembly on the surface of chromatin. Importin β was shown previously to pursue a similar mechanism of inhibition counteracted by localized RanGTP production, which acts as a GPS system to tell where the mitotic assembly events should take place (see text for references). From the data in Figures 3–8, we conclude that transportin and importin β have mechanistically parallel regulatory mechanisms for the major mitotic assembly events. Of importance, not only is the Nup107–160 complex vital for nuclear pore initiation, but it is in fact also a SAF essential to metaphase kinetochore function. Thus, by regulating this one protein complex, transportin can regulate both spindle and nuclear pore assembly.

Aitchison, Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins. 1996, Pubmed

Aitchison, Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins. 1996, Pubmed
Allen, The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. 2000, Pubmed
Ambrus, Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport. 2010, Pubmed
Andrade, HEAT repeats in the Huntington's disease protein. 1995, Pubmed
Arnaoutov, The Ran GTPase regulates kinetochore function. 2003, Pubmed , Xenbase
Askjaer, Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. 2002, Pubmed
Bayliss, Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. 2000, Pubmed
Bayliss, The molecular mechanism of transport of macromolecules through nuclear pore complexes. 2000, Pubmed
Bayliss, Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors. 2000, Pubmed
Bednenko, Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport. 2003, Pubmed
Belgareh, An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. 2001, Pubmed
Ben-Efraim, Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import. 2001, Pubmed
Ben-Efraim, Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr. 2009, Pubmed , Xenbase
Bernis, Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCF(betatrcp). 2007, Pubmed , Xenbase
Bilokapic, Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex. 2012, Pubmed
Bilokapic, Structural and functional studies of the 252 kDa nucleoporin ELYS reveal distinct roles for its three tethered domains. 2013, Pubmed
Bird, RanGTP and CLASP1 cooperate to position the mitotic spindle. 2013, Pubmed
Bischoff, RanBP1 is crucial for the release of RanGTP from importin beta-related nuclear transport factors. 1997, Pubmed
Bischoff, Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. 1991, Pubmed , Xenbase
Bischoff, RanGAP1 induces GTPase activity of nuclear Ras-related Ran. 1994, Pubmed
Bischoff, Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. 1991, Pubmed
Blevins, Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP. 2003, Pubmed , Xenbase
Boehmer, Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex. 2008, Pubmed
Cansizoglu, Conformational heterogeneity of karyopherin beta2 is segmental. 2007, Pubmed
Cansizoglu, Structure-based design of a pathway-specific nuclear import inhibitor. 2007, Pubmed
Carazo-Salas, Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. 1999, Pubmed , Xenbase
Chan, In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts. 2006, Pubmed , Xenbase
Chook, Karyopherins and nuclear import. 2001, Pubmed
Chook, Uncoupling Kapbeta2 substrate dissociation and ran binding. 2002, Pubmed
Chook, Nuclear import by karyopherin-βs: recognition and inhibition. 2011, Pubmed
Chook, Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp. 1999, Pubmed
Ciciarello, Spatial control of mitosis by the GTPase Ran. 2007, Pubmed
Clarke, Spatial and temporal coordination of mitosis by Ran GTPase. 2008, Pubmed
Clarke, Spatial and temporal control of nuclear envelope assembly by Ran GTPase. 2004, Pubmed , Xenbase
Conti, Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. 1998, Pubmed
Conti, Karyopherin flexibility in nucleocytoplasmic transport. 2006, Pubmed
Cronshaw, Proteomic analysis of the mammalian nuclear pore complex. 2002, Pubmed
Cross, Learning about cancer from frogs: analysis of mitotic spindles in Xenopus egg extracts. 2009, Pubmed , Xenbase
Cross, Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK. 2011, Pubmed , Xenbase
Cross, In vitro nuclear assembly using fractionated Xenopus egg extracts. 2008, Pubmed , Xenbase
Dasso, Running on Ran: nuclear transport and the mitotic spindle. 2001, Pubmed
Delmar, Xenopus importin beta validates human importin beta as a cell cycle negative regulator. 2008, Pubmed , Xenbase
Di Fiore, Mitotic functions of the Ran GTPase network: the importance of being in the right place at the right time. 2004, Pubmed
Dormann, ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. 2010, Pubmed
Ems-McClung, Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal. 2004, Pubmed , Xenbase
Finlay, Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. 1990, Pubmed , Xenbase
Forbes, Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs. 1983, Pubmed , Xenbase
Fornerod, CRM1 is an export receptor for leucine-rich nuclear export signals. 1997, Pubmed , Xenbase
Franz, MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly. 2007, Pubmed , Xenbase
Frey, FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. 2006, Pubmed
Fried, Nucleocytoplasmic transport: taking an inventory. 2003, Pubmed
Galy, MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans. 2006, Pubmed
Goldfarb, Importin alpha: a multipurpose nuclear-transport receptor. 2004, Pubmed
Görlich, Transport between the cell nucleus and the cytoplasm. 1999, Pubmed
Görlich, Distinct functions for the two importin subunits in nuclear protein import. 1995, Pubmed , Xenbase
Görlich, Identification of different roles for RanGDP and RanGTP in nuclear protein import. 1996, Pubmed , Xenbase
Görlich, Isolation of a protein that is essential for the first step of nuclear protein import. 1994, Pubmed , Xenbase
Görlich, Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. 1993, Pubmed
Groves, The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. 1999, Pubmed
Gruss, Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. 2001, Pubmed , Xenbase
Hachet, Importin alpha associates with membranes and participates in nuclear envelope assembly in vitro. 2004, Pubmed , Xenbase
Hahn, Classical NLS proteins from Saccharomyces cerevisiae. 2008, Pubmed
Harel, Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores. 2003, Pubmed , Xenbase
Harel, Importin beta negatively regulates nuclear membrane fusion and nuclear pore complex assembly. 2003, Pubmed , Xenbase
Harel, Importin beta: conducting a much larger cellular symphony. 2004, Pubmed
Hetzer, The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. 2002, Pubmed
Hinshaw, Architecture and design of the nuclear pore complex. 1992, Pubmed , Xenbase
Hintersteiner, Identification of a small molecule inhibitor of importin β mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries. 2010, Pubmed
Hu, Midbody assembly and its regulation during cytokinesis. 2012, Pubmed
Hua, Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2. 1998, Pubmed , Xenbase
Iijima, Two motifs essential for nuclear import of the hnRNP A1 nucleocytoplasmic shuttling sequence M9 core. 2006, Pubmed
Izaurralde, The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. 1997, Pubmed
Kalab, Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. 2002, Pubmed , Xenbase
Kalab, The ran GTPase regulates mitotic spindle assembly. 1999, Pubmed , Xenbase
Kalab, The RanGTP gradient - a GPS for the mitotic spindle. 2008, Pubmed
Kaláb, Analysis of a RanGTP-regulated gradient in mitotic somatic cells. 2006, Pubmed , Xenbase
Kehlenbach, Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1. 2001, Pubmed
Kehlenbach, A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. 1999, Pubmed
Kimura, Identification of cargo proteins specific for importin-β with importin-α applying a stable isotope labeling by amino acids in cell culture (SILAC)-based in vitro transport system. 2013, Pubmed
Kutay, Reorganization of the nuclear envelope during open mitosis. 2008, Pubmed
Lachish-Zalait, Transportin mediates nuclear entry of DNA in vertebrate systems. 2009, Pubmed , Xenbase
Lau, Transportin regulates major mitotic assembly events: from spindle to nuclear pore assembly. 2009, Pubmed , Xenbase
Lee, Kap104p-mediated nuclear import. Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna. 1999, Pubmed
Lee, Rules for nuclear localization sequence recognition by karyopherin beta 2. 2006, Pubmed
Li, A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo. 2003, Pubmed , Xenbase
Lim, Biology and biophysics of the nuclear pore complex and its components. 2008, Pubmed
Lohka, Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. 1983, Pubmed , Xenbase
Lohka, Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. 1985, Pubmed , Xenbase
Loïodice, The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. 2004, Pubmed
Macaulay, Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA. 1996, Pubmed , Xenbase
Mackay, Coordinating postmitotic nuclear pore complex assembly with abscission timing. 2011, Pubmed
Mackay, Defects in nuclear pore assembly lead to activation of an Aurora B-mediated abscission checkpoint. 2010, Pubmed
Marelli, The dynamics of karyopherin-mediated nuclear transport. 2001, Pubmed
Marelli, A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope. 2001, Pubmed
Maresca, Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts. 2006, Pubmed , Xenbase
Marshall, Nuclear envelope assembly after mitosis. 1997, Pubmed
Melchior, Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. 1993, Pubmed
Melchior, Two-way trafficking with Ran. 1998, Pubmed
Mishra, The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores. 2010, Pubmed , Xenbase
Moore, Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells. 2002, Pubmed , Xenbase
Mosammaparast, Karyopherins: from nuclear-transport mediators to nuclear-function regulators. 2004, Pubmed
Murray, Cell cycle extracts. 1991, Pubmed
Nachury, Importin beta is a mitotic target of the small GTPase Ran in spindle assembly. 2001, Pubmed , Xenbase
Nakielny, Transportin: nuclear transport receptor of a novel nuclear protein import pathway. 1996, Pubmed
Nelson, Nuclear import strategies of high risk HPV16 L1 major capsid protein. 2002, Pubmed
Newmeyer, Egg extracts for nuclear import and nuclear assembly reactions. 1991, Pubmed , Xenbase
Newport, Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. 1987, Pubmed , Xenbase
Newport, Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. 1992, Pubmed , Xenbase
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Ohba, Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. 1999, Pubmed , Xenbase
Ohtsubo, The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. 1989, Pubmed
Orjalo, The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly. 2006, Pubmed , Xenbase
Pfaller, Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro. 1991, Pubmed , Xenbase
Pollard, A novel receptor-mediated nuclear protein import pathway. 1996, Pubmed
Powers, The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. 1997, Pubmed , Xenbase
Prunuske, The nuclear envelope: form and reformation. 2006, Pubmed
Quimby, The small GTPase Ran: interpreting the signs. 2003, Pubmed
Rasala, Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly. 2008, Pubmed , Xenbase
Rasala, ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. 2006, Pubmed , Xenbase
Rebane, Transportins 1 and 2 are redundant nuclear import factors for hnRNP A1 and HuR. 2004, Pubmed
Reichelt, Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. 1990, Pubmed , Xenbase
Ren, Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis. 1993, Pubmed
Ribbeck, NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules. 2006, Pubmed , Xenbase
Rotem, Importin beta regulates the seeding of chromatin with initiation sites for nuclear pore assembly. 2009, Pubmed , Xenbase
Rout, The yeast nuclear pore complex: composition, architecture, and transport mechanism. 2000, Pubmed
Ryan, The Ran GTPase cycle is required for yeast nuclear pore complex assembly. 2003, Pubmed
Ryan, The karyopherin Kap95 regulates nuclear pore complex assembly into intact nuclear envelopes in vivo. 2007, Pubmed
Sagata, The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. 1989, Pubmed , Xenbase
Sauer, Proteome analysis of the human mitotic spindle. 2005, Pubmed
Siomi, A nuclear localization domain in the hnRNP A1 protein. 1995, Pubmed
Soderholm, Importazole, a small molecule inhibitor of the transport receptor importin-β. 2011, Pubmed , Xenbase
Strawn, Minimal nuclear pore complexes define FG repeat domains essential for transport. 2004, Pubmed
Süel, Modular organization and combinatorial energetics of proline-tyrosine nuclear localization signals. 2008, Pubmed
Tsai, A mitotic lamin B matrix induced by RanGTP required for spindle assembly. 2006, Pubmed , Xenbase
Wälde, The Part and the Whole: functions of nucleoporins in nucleocytoplasmic transport. 2010, Pubmed
Walther, The conserved Nup107-160 complex is critical for nuclear pore complex assembly. 2003, Pubmed , Xenbase
Walther, RanGTP mediates nuclear pore complex assembly. 2003, Pubmed , Xenbase
Wandke, Enclosing chromatin: reassembly of the nucleus after open mitosis. 2013, Pubmed
Wente, The nuclear pore complex and nuclear transport. 2010, Pubmed
Wiese, Role of importin-beta in coupling Ran to downstream targets in microtubule assembly. 2001, Pubmed , Xenbase
Wiese, Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event. 1997, Pubmed , Xenbase
Wilde, Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. 1999, Pubmed , Xenbase
Wilde, Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. 2001, Pubmed , Xenbase
Wilson, A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. 1988, Pubmed , Xenbase
Wood, CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. 1997, Pubmed , Xenbase
Xu, Recognition of nuclear targeting signals by Karyopherin-β proteins. 2010, Pubmed
Yokoyama, Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate. 2008, Pubmed , Xenbase
Zhang, Role of importin-beta in the control of nuclear envelope assembly by Ran. 2002, Pubmed , Xenbase
Zhang, Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS). 2012, Pubmed
Zhang, Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts. 1999, Pubmed , Xenbase
Zhang, Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly. 2002, Pubmed , Xenbase
Zuccolo, The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions. 2007, Pubmed