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.

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