Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin β, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.

The equal segregation of chromosomes during cell division requires the co-ordination of many cellular processes, not least the formation of the microtubule spindle. Spindle assembly in itself requires the co-ordination of many processes including microtubule dynamics, the attachment and alignment of chromosomes and the organization of the spindle poles (Compton, 2000). These processes need to be regulated in both a temporal and a spatial manner to ensure the equal segregation of the genetic material to the newly formed daughter cells. While the temporal aspect of spindle assembly is known to be regulated by the cell cycle, the spatial control is less well understood. Recently it has been proposed that the GTPase Ran is a key spatial co-coordinator of spindle assembly (Dasso, 2002). RanGTP, the active form of the protein, was found to stimulate spindle assembly in Xenopus CSF-arrested egg extracts (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999) by affecting several key processes of spindle assembly including microtubule dynamics (Carazo-Salas et al., 2001; Wilde et al., 2001), motor protein activity (Wilde et al., 2001) and microtubule nucleation (Carazo-Salas et al., 2001). In addition, RanGTP was found to be present and restricted around chromatin and the spindle throughout mitosis (Kalab et al., 2002; Trieselmann and Wilde, 2002). RanGTP is generated at this location by the only known nucleotide exchange factor for Ran, RCC1, which remains bound to chromatin throughout mitosis (Moore et al., 2002; Trieselmann and Wilde, 2002). Taken together these data suggest that RanGTP acts as a spatial co-coordinator for spindle assembly by activating specific processes required for spindle assembly only in the vicinity of the chromosomes.

The proposed molecular mechanism utilized by RanGTP to activate spindle assembly is the same as the mechanism it uses in interphase to regulate nuclear transport (Gruss et al., 2001; Nachury et al., 2001; Wiese et al., 2001). RanGTP regulates nuclear transport by regulating the interaction between nuclear transport receptors and their cargo by binding to importin β, part of the nuclear transport receptor complex, which results in the cargo being released from the nuclear transport receptors (Mattaj and Englmeier, 1998). In studies using Xenopus egg extracts a correlation has been made between spindle assembly and the absence of an association of nuclear transport receptors with the spindle assembly factors, TPX2 and NuMA (Gruss et al., 2001; Nachury et al., 2001; Wiese et al., 2001). As nuclear transport receptors bind to nuclear localization sequences (NLSs), and NuMA and TPX2 possess NLSs, it has been proposed that the direct binding of nuclear transport receptors to spindle assembly factors inhibits the activity of spindle assembly factors. Furthermore, the binding of RanGTP to nuclear transport receptors would prevent their inhibitory interaction with spindle assembly factors. Extrapolation of such a model would imply that Ran could modulate the activity of any spindle assembly factor that localizes to the nucleus, such as XCTK2 (Walczak et al., 1997), XMAP310, (Andersen and Karsenti, 1997), MKLP1/CHO1 (Nislow et al., 1992) and Kid (Tokai et al., 1996). However, it has never been shown that the direct binding of nuclear transport receptors to any of these spindle assembly factors directly inhibits their function.

To test this hypothesis we examined the activity of two spindle assembly factors, TPX2 and Kid, in the presence of nuclear transport receptors. Kid is a chromokinesin involved in chromosome orientation during meiosis and mitosis (Antonio et al., 2000; Funabiki and Murray, 2000; Levesque and Compton, 2001). TPX2 was isolated as the factor required for the recruitment of XKLP2, a kinesin of unknown function, to microtubules (Wittmann et al., 2000). However, immunodepletion of TPX2 from Xenopus egg extracts (Wittmann et al., 2000) caused a greater disruption to spindle assembly than the inhibition of XKLP2 activity (Walczak et al., 1998), suggesting that TPX2 had additional activities. Recently TPX2 was found to recruit Aurora A, an essential kinase in spindle assembly (Giet and Prigent, 2000; Glover et al., 1995; Hannak et al., 2001; Roghi et al., 1998; Schumacher et al., 1998), to spindle microtubules (Kufer et al., 2002). In addition a correlation has been made between the inability of CSF-arrested Xenopus egg extract to form spindles and TPX2 being complexed with nuclear transport receptors (Gruss et al., 2001). When TPX2 is not associated with nuclear transport receptors spindle assembly proceeds, suggesting that TPX2 is a spindle assembly factor regulated by the Ran pathway. Therefore, as TPX2 has a central role in spindle assembly it is one of the stronger candidates of all the proposed spindle assembly factors to be regulated by Ran and mediate the effect of RanGTP on spindle assembly.

Here we show that the binding of nuclear transport receptors to TPX2 and Kid inhibit a subset of their known functions, thereby confirming the proposed molecular mechanism that Ran modulates spindle assembly by preventing the inhibitory interaction of nuclear transport receptors with spindle assembly factors.

Plasmid construction, cell transfection and microscopy

Fragments of TPX2 and Kid were generated by polymerase chain reaction (PCR) using a human TPX2 cDNA (accession number BC004136) and a human Kid cDNA (acc. no. R56446) as templates. PCR fragments were first cloned into the pGEM T vector (Promega), and then subcloned into pEGFP N-1 (Invitrogen) or directly into pcDNA3.1/NT-GFP-TOPO (Invitrogen). Positive clones where transfected into HeLa cells growing on glass coverslips using the Polyfect reagent (Qiagen) and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) 24 hours after transfection. Cells were viewed using a Nikon TE800 microscope with a 60× oil immersion lens and micrographs collected using an Orca ER (Hamamatsu) CCD camera driven by Metamorph software (Universal Imaging Inc.).

Protein purification

Protein fragments of TPX2 and Kid were expressed in BL21 E. coli cells, fused to thioredoxin, S and 6 histidine tags using the pET32a vector (Novagen) or GST using the vector pGEX 6P2 (Pharmacia). Bacterial cultures were grown to an A600 of 0.6 and then induced with 0.8 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) for either 3 hours at 37°C or overnight at 15°C. Proteins were purified from the bacteria following the manufacturer's instructions. Purified proteins were dialyzed into 10 mM Hepes pH 7.7, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 50 mM sucrose, 5 mM EGTA overnight, aliquoted, flash frozen in liquid nitrogen and stored at –80°C.

Extract preparation

Mitotic HeLa extract was prepared following the protocol described previously (Stucke et al., 2002). HeLa cells at 80% confluency were treated with 10 μM Taxol for 16 hours, washed with PBS and then harvested in lysis buffer (50 mM Tris-HCl pH 8.0, 1% NP40, 150 mM NaCl). Lysates were centrifuged at 13000 g for 15 minutes at 4°C and the resulting supernatants aliquoted, flash frozen in liquid nitrogen and stored at –80°C.

Binding assays

Purified proteins were immobilized on either Affiprep 10 beads (BioRad), S-protein agarose beads (Novagen) or glutathione agarose beads (Sigma) and incubated with 200 μl of mitotic HeLa extract. Re-isolated beads and associated protein complexes were washed 4 times in 50 mM Hepes pH 7.4, 100 mM NaCl then boiled in SDS-PAGE sample buffer and analyzed by SDS-PAGE. For western blotting, the nitrocellulose membranes were immunoblotted with the relevant antibody.

Antibodies

The anti-TPX2 antibody was generated against the TNT fragment of TPX2 fused to thioredoxin and affinity purified using TNT fused to GST. The anti-Kid antibody was raised against the KCT fragment fused to GST and subsequently affinity purified against the antigen after the serum was pre-cleared with GST (Zhang et al., 2000). GST was detected using a rabbit polyclonal antibody Ab-1 (Oncogene Research), Tubulin was detected using YOL1/34 (Serotec), Aurora A was detected using a mouse monoclonal antibody (Transduction Laboratories) and the activated form of Aurora A, phosphorylated on residue threonine 288, was detected using a rabbit polyclonal antibody (Cell Signaling Technology).

Microtubule sedimentation assays

Tubulin and Taxol-stabilized microtubules were prepared as previously described (Wilde et al., 2001). Microtubules were harvested by centrifugation and resuspended in 80 mM Pipes, 1 mM MgCl2, 1 mM EGTA, pH 6.8 (BRB80) plus 10 μM Taxol. Taxol-stabilized microtubules were added, to a final concentration of 0.5 mg/ml, to 100 μl of mitotic HeLa cell extract or BRB80 containing 10 μM Taxol in the presence or absence of different recombinant proteins. Microtubules were recovered from the solution by centrifugation at 100000 g for 15 minutes and the resulting pellet and supernatant fractions analyzed by SDS-PAGE and immunoblotting.

DNA binding assay

DNA binding assays were carried out following the protocol described previously (Tokai et al., 1996). DNA cellulose (Sigma) was hydrated and washed 3 times in binding buffer (50 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 1 mM EDTA and 1 mM DTT). The DNA cellulose was then incubated in the presence or absence of different recombinant proteins for 45 minutes at 4°C. The reaction was then passed over a microcolumn (a pipette tip containing glass beads). The column was then washed with 200 μl of 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA and 1 mM DTT, then 200 μl of 100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA and 1 mM DTT, and finally, 200 μl of 100 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA and 1 mM DTT. The eluates were collected and analyzed by SDS-PAGE.

Identification of NLS-containing domains of TPX2

To test the prediction that TPX2 function can be attenuated through the binding of nuclear transport receptors we first sought to localize the domain of TPX2 required for association with nuclear transport receptors. As nuclear transport receptors bind to NLS motifs that target a protein to the nucleus, we sought to identify domains within TPX2 that are targeted to the nucleus then determine if the function of these domains could be modulated by nuclear transport receptor binding. A series of TPX2 fragments fused to enhanced green fluorescent protein (EGFP) were generated and expressed in HeLa cells (Fig. 1). Whereas EGFP is expressed throughout the cell, fragments of TPX2 encompassing amino acid residues (aa) 236-352 (TNT, 1T 2T and 4T) localized to the nucleus, suggesting that this domain of TPX2 possesses a NLS (Fig. 1A,C). The amino-terminal domain of TPX2 has previously been shown to bind to the kinase Aurora A (Kufer et al., 2002). However, in cells where EGFP-TPX2 fragments of aa 236-352 were overexpressed, and presumably saturated the nuclear import machinery, a sub-population of the TPX2 fragments co-localized with microtubules (Fig. 1B,C). These data suggest aa 236-352 also possess microtubule-binding activity.

Fig. 1.

The amino-terminal domain of TPX2 contains a nuclear localization signal and a microtubule-binding site. Fragments of TPX2 were fused to EGFP, expressed in HeLa cells and then fixed 24 hours after transfection. Microtubules were visualized using an anti-tubulin antibody (YOL1/34), DNA was visualized with DAPI, and the whole cells were viewed by differential interference contrast (DIC) microscopy. (A) TPX2 fragments fused to EGFP that localize to the nucleus (TPX2, TNT and 1T) or the cytoplasm (3T and 5T). (B) EGFP-TPX2 fragments that co-localize with microtubules (MT). (C) Summary outlining the sub-cellular localizations of the different fragments of TPX2 (amino acid residues in parenthesis), N, nuclear localization; C, cytosolic localization; box depicts the NLS-containing domain. Scale bar: 10 μm.

Fig. 1.

The amino-terminal domain of TPX2 contains a nuclear localization signal and a microtubule-binding site. Fragments of TPX2 were fused to EGFP, expressed in HeLa cells and then fixed 24 hours after transfection. Microtubules were visualized using an anti-tubulin antibody (YOL1/34), DNA was visualized with DAPI, and the whole cells were viewed by differential interference contrast (DIC) microscopy. (A) TPX2 fragments fused to EGFP that localize to the nucleus (TPX2, TNT and 1T) or the cytoplasm (3T and 5T). (B) EGFP-TPX2 fragments that co-localize with microtubules (MT). (C) Summary outlining the sub-cellular localizations of the different fragments of TPX2 (amino acid residues in parenthesis), N, nuclear localization; C, cytosolic localization; box depicts the NLS-containing domain. Scale bar: 10 μm.

Analysis of the localization of other fragments suggested that there was an additional microtubule-binding site in the carboxy-terminal domain of TPX2. The TPX2 fragment TCT (aa 345-776), which localized to the cytosol also partially co-localized with microtubules. However, the fragments 3T (aa 345-579) and 5T (aa 547-776), which are also cytosolic, did not co-localize with microtubules. These results suggest that either the microtubule-binding domain in TCT is incorrectly folded in 3T and 5T or it is centered between residues 547 and 579 but extends beyond those residues such that neither 3T nor 5T bind to microtubules. Together our analyses suggest that TPX2 possesses two domains that can mediate its localization to microtubules.

Binding of the amino-terminal domain of TPX2 to microtubules is not inhibited by nuclear transport receptors

Having established that the amino-terminal domain of TPX2 possessed a NLS, we next asked if the amino-terminal domain of TPX2 could bind to importin β, a component of the nuclear transport receptor complex. The amino-terminal domain of TPX2 (TNT) was expressed in E. coli, immobilized onto beads and incubated with mitotic HeLa cell extract. TNT bound to importin β whereas BSA, immobilized onto the same beads, did not (Fig. 2A). The binding of TNT to importin β was prevented in the presence of GST-RanL43E, an allele of Ran locked in the GTP bound form (Lounsbury et al., 1996). To determine if the binding of nuclear transport receptors to TNT could inhibit its ability to bind to microtubules, a microtubule pelletting assay was performed in the presence or absence of nuclear transport receptors or RanL43E (Fig. 2B). The presence of nuclear transport receptors in the reaction did not inhibit the microtubule-binding capacity of TNT suggesting that although the NLS and the microtubule-binding domain are close to each other the binding of nuclear transport receptors to the NLS does not prevent the recruitment of TPX2 to microtubules. Similar results were obtained with GST-4T (data not shown). In addition, in microtubule pelletting experiments in mitotic HeLa cell extracts, nuclear transport receptors did not inhibit TPX2 binding to microtubules (Fig. 3A). These data suggest that within the cell RanGTP does not modulate the recruitment of TPX2 to spindle microtubules.

Fig. 2.

TPX2 can bind to the importin α/β complex, an interaction that prevents TPX2 binding to Aurora A, but not microtubules. (A) The amino terminal domain of TPX2, TNT or BSA was coupled to Affiprep 10 beads, incubated with mitotic HeLa cell extract in the presence or absence of the importin α/β complex and GST-RanL43E, then re-isolated and analyzed by SDS PAGE and immunoblotting to detect importin β. (B) GST-TNT was incubated with Taxol-stabilized microtubules in the presence or absence of the recombinant GST-RanL43E and or the importin α/β complex. Microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immuoblotting to detect GSTTNT. (C) S-tagged TNT was incubated with mitotic HeLa cell extract in the presence of absence of GST-RanL43E and the importin α/β complex. TNT was recovered from the extract with S-protein agarose beads and analyzed by SDS PAGE and immunoblotting to detect Aurora A.

Fig. 2.

TPX2 can bind to the importin α/β complex, an interaction that prevents TPX2 binding to Aurora A, but not microtubules. (A) The amino terminal domain of TPX2, TNT or BSA was coupled to Affiprep 10 beads, incubated with mitotic HeLa cell extract in the presence or absence of the importin α/β complex and GST-RanL43E, then re-isolated and analyzed by SDS PAGE and immunoblotting to detect importin β. (B) GST-TNT was incubated with Taxol-stabilized microtubules in the presence or absence of the recombinant GST-RanL43E and or the importin α/β complex. Microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immuoblotting to detect GSTTNT. (C) S-tagged TNT was incubated with mitotic HeLa cell extract in the presence of absence of GST-RanL43E and the importin α/β complex. TNT was recovered from the extract with S-protein agarose beads and analyzed by SDS PAGE and immunoblotting to detect Aurora A.

Fig. 3.

The importin α/β complex prevents the recruitment of Aurora A to microtubules and its activation. (A) Taxol stabilized microtubules (MT) were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E and the importin α/β complex then re-isolated by centrifugation and the pellet fractions analyzed by SDS-PAGE and immunoblotting to detect TPX2 and Aurora A. (B) Taxol stabilized microtubules were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E or the importin α/β complex. After 30 minutes the extracts were analyzed by SDS-PAGE and immunoblotting to detect Aurora A phosphorylated on amino acid residue threonine 288 (P-Aurora A), then stripped and re-probed to detect all classes of Aurora A.

Fig. 3.

The importin α/β complex prevents the recruitment of Aurora A to microtubules and its activation. (A) Taxol stabilized microtubules (MT) were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E and the importin α/β complex then re-isolated by centrifugation and the pellet fractions analyzed by SDS-PAGE and immunoblotting to detect TPX2 and Aurora A. (B) Taxol stabilized microtubules were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E or the importin α/β complex. After 30 minutes the extracts were analyzed by SDS-PAGE and immunoblotting to detect Aurora A phosphorylated on amino acid residue threonine 288 (P-Aurora A), then stripped and re-probed to detect all classes of Aurora A.

TPX2 binding to Aurora A is inhibited by nuclear transport receptors

As the amino-terminal domain of TPX2 also binds to the kinase Aurora A (Kufer et al., 2002), we determined if nuclear transport receptor binding to the amino-terminal domain affected the interaction of TPX2 with Aurora A. The amino-terminal domain of TPX2 (TNT) fused to an S-Tag was expressed in E. coli, purified and incubated with mitotic HeLa cell extract. TNT was recovered from the extract using S-protein beads and analyzed by SDS-PAGE and immunoblotting for the co-recovery of Aurora A. Aurora A bound to TNT in the presence or absence of RanL43E but not in the presence of exogenously added recombinant nuclear transport receptors (Fig. 2C). The inhibition of Aurora A binding to TNT in the presence of nuclear transport receptors was reversed in the presence of RanL43E. These data suggest that nuclear transport receptors inhibit the interaction of TPX2 with Aurora A but not microtubules.

Nuclear transport receptors prevent Aurora A activation

As previous studies have suggested that TPX2 is required for the recruitment of Aurora A to spindle microtubules (Kufer et al., 2002), we determined if Aurora A recruitment to microtubules was inhibited by the nuclear transport receptors. Taxol-stabilized microtubules were added to mitotic HeLa cell extract in the presence or absence of either the nuclear transport receptors or RanL43E and then isolated by centrifugation. Whilst TPX2 co-sedimented with microtubules independently of exogenously added nuclear transport receptors or RanL43E, co-sedimentation of Aurora A with microtubules was inhibited by the addition of nuclear transport receptors, an inhibition reversed by the presence of RanL43E (Fig. 3A). We next sought to determine if the recruitment of Aurora A to microtubules was required for its activation as judged by the phosphorylation of amino acid residue threonine 288 (Walter et al., 2000). The phosphorylation of Aurora A on threonine 288 did not occur in the absence of microtubules or in the presence of nuclear transport receptors (Fig. 3B), suggesting that the recruitment of Aurora A to microtubules by TPX2 is required for its activation and that this process will not occur in the presence of nuclear transport receptors.

Kid has multiple NLSs

TPX2 is just one of the potential spindle assembly factors that could be regulated by the Ran pathway. To determine if Ran has a broader role in spindle assembly, we examined the potential of another spindle assembly factor, the chromokinesin Kid, to be regulated by the Ran pathway. Kid has a role in moving chromosome arms to the metaphase plate in both meiosis and mitosis (Antonio et al., 2000; Funabiki and Murray, 2000; Levesque and Compton, 2001). Previous studies of Kid have described a putative NLS in the DNA binding domain (Tokai et al., 1996). In addition, when expressed in tissue culture cells the amino-terminal domain localized to the nucleus suggesting the presence of a NLS in this domain of Kid. Expression of fragments of Kid fused to EGFP revealed at least 3 different NLSs in Kid (Fig. 4). One NLS resides between aa 255 and 413 in the amino-terminal microtubule-binding domain of Kid, another between aa 547 and 566 encompassing the previously described putative NLS in the DNA binding domain of Kid and a third NLS exists between aa 566 and 665 in a domain with, as yet, no characterized function. Recombinant fragments of Kid, KNT encoding the amino terminus (aa 1-413) and KCT encoding the carboxy terminus (aa 407-665), were fused to GST and tested for their ability to bind importin β. KNT, KCT and BSA were immobilized onto beads and incubated with mitotic HeLa cell extract. Upon re-isolation the beads were analyzed by SDS-PAGE and immunoblotting to detect importin β. Importin β bound to KNT and KCT but not in the presence of GST-RanL43E (Fig. 5A).

Fig. 4.

Kid has 3 NLS-containing domains. Fragments of Kid were fused to EGFP and expressed in HeLa cells and then fixed 24 hours after transfection. DNA was visualized with DAPI and the cells viewed by differential interference contrast (DIC) microscopy. The sub-cellular localization of the different Kid fragments is summarized in the table. N, nuclear; C, cytosolic; open box, kinesin homology domain; hatched box, DNA binding domain. Scale bar: 10 μm.

Fig. 4.

Kid has 3 NLS-containing domains. Fragments of Kid were fused to EGFP and expressed in HeLa cells and then fixed 24 hours after transfection. DNA was visualized with DAPI and the cells viewed by differential interference contrast (DIC) microscopy. The sub-cellular localization of the different Kid fragments is summarized in the table. N, nuclear; C, cytosolic; open box, kinesin homology domain; hatched box, DNA binding domain. Scale bar: 10 μm.

Fig. 5.

The microtubule-binding activity, but not the DNA binding activity of Kid is inhibited by the importin α/β complex. (A) The amino-terminal domain (KNT) and the carboxy-terminal domain (KCT) of Kid and BSA were immobilized onto Affiprep 10 beads, incubated with mitotic HeLa cell extract then re-isolated and analyzed by SDS-PAGE and immunoblotting to detect importin β. (B) GST-KCT was incubated with DNA cellulose at 4°C for 1 hour and then successively washed with buffers containing 50 mM NaCl (50), 300 mM NaCl (300) and 500 mM NaCl (500). The eluates were analyzed by SDS-PAGE and Coomassie Blue staining. L, load. (C) GST-KCT was incubated with DNA cellulose in the presence or absence of GST-RanL43E and the importin α/β complex. The DNA cellulose was washed with 50 mM NaCl followed by 500 mM NaCl. The initial amount of KCT (L) incubated with the DNA cellulose was compared to that eluted with 500 mM NaCl (E). (D) GST-KNT was incubated with Taxol-stabilized microtubules in the presence or absence of GST-RanL43E and the importin α/β complex. The microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immunoblotting to detect GST.

Fig. 5.

The microtubule-binding activity, but not the DNA binding activity of Kid is inhibited by the importin α/β complex. (A) The amino-terminal domain (KNT) and the carboxy-terminal domain (KCT) of Kid and BSA were immobilized onto Affiprep 10 beads, incubated with mitotic HeLa cell extract then re-isolated and analyzed by SDS-PAGE and immunoblotting to detect importin β. (B) GST-KCT was incubated with DNA cellulose at 4°C for 1 hour and then successively washed with buffers containing 50 mM NaCl (50), 300 mM NaCl (300) and 500 mM NaCl (500). The eluates were analyzed by SDS-PAGE and Coomassie Blue staining. L, load. (C) GST-KCT was incubated with DNA cellulose in the presence or absence of GST-RanL43E and the importin α/β complex. The DNA cellulose was washed with 50 mM NaCl followed by 500 mM NaCl. The initial amount of KCT (L) incubated with the DNA cellulose was compared to that eluted with 500 mM NaCl (E). (D) GST-KNT was incubated with Taxol-stabilized microtubules in the presence or absence of GST-RanL43E and the importin α/β complex. The microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immunoblotting to detect GST.

Nuclear transport receptors inhibit Kid binding to microtubules but not DNA

To determine if the DNA binding capacity of Kid could be affected by the nuclear transport receptor complex, a DNA binding assay was performed using KCT (Tokai et al., 1996). KCT bound to DNA cellulose in low salt concentration and was eluted from the DNA cellulose in 500 mM NaCl (Fig. 5B). However, the DNA binding ability of KCT was not affected by nuclear transport receptors (Fig. 5C), suggesting that the Ran pathway does not regulate the DNA binding activity of Kid.

Next we determined if RanGTP and the nuclear transport receptor complex could modulate the microtubule-binding activity of Kid. Using the microtubule pelleting assay described above, the microtubule-binding activity of KNT in the presence of nuclear transport receptors was determined. Nuclear transport receptors inhibited the microtubule-binding activity of KNT but not in the presence of RanL43E (Fig. 5D). We conclude that nuclear transport receptors can inhibit the interaction of Kid with microtubules but not DNA.

In this study we demonstrate that nuclear transport receptors can directly interfere with specific functions of the spindle assembly factors TPX2 and Kid. The inhibitory action of nuclear transport receptors can be overcome by the presence of RanGTP. Furthermore, nuclear transport receptors do not inhibit all the functions of Kid and TPX2, just a subset. With these precedents we suggest that the other spindle assembly factors proposed to be in the Ran spindle assembly pathway, MKLP1/CHO1, XCTK2, NuMA and XMAP310, will have some aspect of their activity inhibited by binding of nuclear transport receptors. As RanGTP concentrations are highest around the chromosomes (Kalab et al., 2002; Trieselmann and Wilde, 2002) nuclear transport receptors will not be able to inhibit selective functions of spindle assembly factors close to the chromosomes. Therefore, Ran will serve as a spatial regulator of spindle assembly ensuring that spindle assembly factors are only in the active state around the chromatin. Away from the chromatin where RanGTP is low but importin β persists (Trieselmann and Wilde, 2002), spindle assembly factors will be inhibited thereby preventing spindle assembly at sites distal to the chromatin.

The implication for these findings within the cell are that RanGTP mediates its effect on spindle assembly, at least in part by regulating the recruitment and activation of Aurora A to microtubules in the vicinity of chromatin. As Aurora A activity is required for spindle assembly (Tsai et al., 2003), targeting that activity to microtubules surrounding condensed chromatin would ensure the correct orientation of the spindle for viable chromosome segregation. Our findings in a mitotic system are consistent with recent observations using a meiotic system (Tsai et al., 2003) suggesting that Ran can influence both meiotic and mitotic spindle assembly by regulating the activation of Aurora A. Few downstream effectors of Aurora A are known but candidates from in vitro studies include TPX2 (Tsai et al., 2003), dTACC [a centrosomal protein (Giet et al., 2002)] and Eg5 [a tetrameric kinesin essential for correct spindle assembly (Giet et al., 1999)]. Since Eg5 activity is altered in CSF-arrested Xenopus egg extracts in the presence of RanGTP (Wilde et al., 2001), it is tempting to speculate that Aurora A activity mediates this effect. By facilitating the localized activation of Aurora A to the site of spindle assembly, Ran could activate a signal cascade, thereby amplifying the RanGTP signal. The observation that Aurora A phosphorylation in the activation loop is dependent on microtubules (Tsai et al., 2003) (this study) raises the possibility that the Aurora A activating activity is associated with microtubules, as binding to TPX2 alone does not result in phosphorylation of the threonine in the activation loop.

Although Ran's regulation of Aurora A recruitment to microtubules in itself would play a pivotal role in orchestrating spindle assembly, we have shown that Ran has the potential to modulate another spindle assembly process by modulating the activity of the chromokinesin Kid. Kid is involved in generating the polar ejection force that pushes chromosomes arms away from the spindle pole toward the metaphase plate (Antonio et al., 2000; Funabiki and Murray, 2000; Levesque and Compton, 2001). The requirement of this force in chromosome congression appears to be greater in meiotic systems (Antonio et al., 2000; Funabiki and Murray, 2000; Zhang et al., 1990) as opposed to mitotic systems (Levesque and Compton, 2001). By regulating chromosome attachment to microtubules through the modulation of Kid activity, the RanGTP gradient surrounding the chromosomes would ensure the correct alignment of chromosomes upon the spindle, especially in meiotic systems, thereby ensuring the equal segregation of the genetic material into the two newly formed daughter cells. However, it is possible that Ran may regulate an uncharacterized function of Kid in the activation of MPF after germinal vesicle break down during Xenopus oocyte maturation (Perez et al., 2002). If Kid expression was inhibited during oocyte maturation, oocytes would enter an interphase like state after meiosis I, whereas, if a mutant of Kid lacking the DNA binding domain was ectopically expressed in these Kid-depleted oocytes, the oocyte could progress to meiosis II. This function of Kid does not require the carboxy-terminal DNA binding domain and therefore the activity may lie in or require the amino-terminal microtubule-binding domain that we have demonstrated can be regulated by RanGTP and nuclear transport receptors.

Taken together these findings show that the Ran pathway has the potential to regulate various aspects of spindle assembly by modulating the direct binding of the nuclear transport receptor complex to spindle assembly factors.

We would like to thank Drs B. Andrews and B. Lavoie for helpful discussion and reading the manuscript and Dr J. Glover for technical assistance. S.A. and J.R. were supported by University of Toronto Life Sciences summer studentships and A.W. is supported by grants from the National Cancer Institute of Canada, the Terry Fox Foundation, the Canada Foundation for Innovation and a Canada Research Chair.

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