Oncogene (2006) 25, 4867–4879 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown

JA Rodriguez1, SMA Lens2, SW Span1, G Vader2, RH Medema2, FAE Kruyt1 and G Giaccone1

1Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands and 2Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

As mitosis progresses, the chromosomal passenger pro- other during mitosis forming one or more protein teins (CPPs) Survivin, Aurora B, INCENP and Borealin complexes that play important roles in early and late dynamically colocalize to mitotic structures. Chromoso- mitoticevents. Aurora B is a member of a conserved mal passenger proteins are already expressed during G2, family of serine/threonine protein kinases originally whereas the nuclear envelope is only disassembled at the identified as a mammalian homologue of Drosophila and end of prophase. However, the mechanisms that modulate yeast proteins required for chromosome segregation their nucleocytoplasmic localization before nuclear envel- (Bischoff et al., 1998; Terada et al., 1998). Aurora B ope breakdown (NEB) are poorly characterized. Using phosphorylates a variety of substrate proteins and epitope-tagged proteins, we show that Aurora B, like regulates many aspects of cell division, from chromo- Survivin, undergoes CRM1-mediated nucleocytoplasmic some condensation to cytokinesis (reviewed by Andrews shuttling, although both proteins lack identifiable ‘classi- et al., 2003). INCENP is a large protein that has been cal’ nuclear transport signals. On the other hand, proposed to act as a scaffold for the binding of the other INCENP resides more stably in the nucleus and contains passenger proteins (Bolton et al., 2002). Aurora B multiple nuclear localization signals. Finally, Borealin was phosphorylates INCENP, which, in turn, activates the detected in the nucleolus and the cytoplasm, but its kinase activity of Aurora B in a positive feedback loop cytoplasmic localization is not directly regulated by (Bishop and Schumacher, 2002; Honda et al., 2003). CRM1. Coexpression experiments indicate that the Survivin is a member of the inhibitor of apoptosis nuclear localization of Aurora B, but not of Survivin, is protein family that plays a dual role as regulator of both modulated by INCENP and that Survivin prevents the apoptosis and cell division (reviewed by Altieri, 2003a). nucleolar accumulation of Borealin. Our data reveal that, Like INCENP, Survivin has been reported to constitute in contrast to their closely related localization during both a substrate and an activator for Aurora B (Chen mitosis, the nucleocytoplasmic localization of the CPPs et al., 2003; Wheatley et al., 2004). Borealin, which was before NEB is largely unrelated. Furthermore, the specific independently isolated as DasraB, is the most recently effect of INCENP and Survivin on the localization of identified and the least characterized subunit of the Aurora B and Borealin, respectively, suggests that chromosomal passenger complex (Gassman et al., 2004; different complexes of CPPs may exist not only during Sampath et al., 2004). Borealin is phosphorylated by mitosis, as recently reported, but also before NEB. Aurora B in vitro but, unlike Survivin and INCENP, Oncogene (2006) 25, 4867–4879. doi:10.1038/sj.onc.1209499; does not appear to increase the activity of the kinase published online 20 March 2006 (Gassman et al., 2004). Several lines of evidence implicate at least two of the CPPs, Survivin and Aurora Keywords: Survivin; Borealin; INCENP; Aurora B; B, in the development and progression of human nuclear envelope breakdown; nuclear transport tumors, and these proteins hold potential as targets for novel anticancer therapies (reviewed by Altieri, 2003b; Giet et al., 2005). The hallmark of CPPs is their dynamicpattern of subcellular localization during mitosis: they associate Introduction with the centromeric region of chromosomes from prophase to metaphase, relocate to the spindle midzone The chromosomal passenger proteins (CPPs) Survivin, and equatorial cortex during anaphase and finally Aurora B, INCENP and Borealin interact with each accumulate in the midbodies during cytokinesis (Adams et al., 2001a). Although their levels are maximal during Correspondence: Dr JA Rodriguez, Department of Medical Oncology, mitosis, the CPPs are already expressed before the VU University Medical Center, Academic Hospital Vrije Universiteit, disassembly of the nuclear envelope, or nuclear envelope KRIGO BR232, Postbus 7057, 1007 MB Amsterdam, The Nether- breakdown (NEB), takes place at the transition between lands. E-mail: [email protected] prophase and prometaphase (reviewed by Burke and Received 8 September 2005; revised 13 January 2006; accepted 9 Ellenberg, 2002). Before NEB, the transport of proteins February 2006; published online 20 March 2006 between the nucleus and cytoplasm occurs exclusively Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4868 through the nuclear pore complex, and is usually an tion, we generated mammalian expression plasmids active process, mediated by soluble import/export encoding full-length human Aurora B, INCENP receptors that bind to nuclear localization signals and Borealin proteins with small VSV-epitope tags (NLSs) and nuclear export signals (NESs) in the cargo fused at their amino-terminal end, and their localization protein, respectively (Mattaj and Englmeier, 1998). in transiently transfected MCF-7 cells was analysed Dynamic nucleocytoplasmic transport constitutes an by immunofluorescence using antitag antibodies important mechanism that regulates the subcellular (Figure 1a). As a marker of nuclear envelope integrity, localization and function of several cancer-associated green fluorescent protein (GFP)-lamin A was co- proteins (Fabbro and Henderson, 2003). transfected with the CPPs. Consistent with our previous In contrast to the detailed knowledge on the findings, Flag-Survivin was predominantly detected in mitotic localization of CPPs, the mechanisms that the cytoplasm in cells where the nuclear envelope contribute to regulate their subcellular distribution in remained intact. VSV-Aurora B was detected in the the presence of an intact nuclear envelope are poorly nucleus and cytoplasm. Variable levels of the protein in characterized. In this respect, we have previously each compartment were observed in different cells, shown that Survivin behaves as a nuclear shuttling ranging from predominantly nuclear (N>C) to mostly protein before NEB, and is actively excluded from the cytoplasmic (C>N). Similar results were obtained using nucleus by a mechanism mediated by the nuclear Aurora B-HA, which bears a carboxy-terminal HA tag export receptor CRM1 (Rodriguez et al., 2002). In the (not shown). On the other hand, VSV-INCENP showed present study, we show that Aurora B also accesses, a clear nuclear localization in most transfected cells. directly or indirectly, the CRM1-dependent pathway Finally, VSV-Borealin was detected in the nucleus, with of nuclear export. In contrast, CRM1 does not appear a prominent nucleolar accumulation, and in the to play a direct role in the nucleocytoplasmic distribu- cytoplasm. Importantly, the three VSV-tagged CPPs tion of INCENP or Borealin. Furthermore, we readily localized to the centromeres during metaphase have functionally tested several potential nuclear and the midbody during telophase/cytokinesis as ex- transport signals in Aurora B and INCENP, and pected (Figure 1a), thus confirming that the small tags identified three independent NLSs in INCENP. Inter- do not grossly interfere with the subcellular targeting of estingly, our results suggest that, despite their closely these proteins. We determined the localization of each of related localization during mitosis, the subcellular the CPPs in at least 200 transfected cells. As illustrated localization of the CPPs before NEB is partially in Figure 1b, Survivin and INCENP were consistently unrelated. In this respect, coexpression experiments localized in the cytoplasm and nucleus, respectively, showed that INCENP induces the nuclear accumulation whereas Aurora B and Borealin showed a more of Aurora B, but not of Survivin. On the other hand, heterogeneous distribution in the population of trans- Survivin, through its carboxy-terminal domain, prevents fected cells. the targeting of Borealin to the nucleolus. Such specific These results indicate that despite their close related effect of INCENP and Survivin on the localization of targeting during mitosis, the CPPs show different Aurora B and Borealin, respectively, suggests that patterns of subcellular localization before NEB, suggest- different complexes of these CPPs may exist before ing that the distribution of these proteins in the presence NEB, in line with recent evidence indicating that of an intact nuclear envelope may be, at least partially, chromosomal passenger complexes of different compo- regulated in an independent manner. sition may exist later during mitosis (Gassman et al., 2004). Our findings, in summary, begin to elucidate the Aurora B undergoes CRM1-mediated nuclear export molecular mechanisms that modulate the subcellular The nucleocytoplasmic distribution of Aurora B fused distribution of the CPPs in the presence of an intact to a MYC tag, similar in size to the VSV and HA nuclear envelope, during periods of the cell cycle at epitopes used here, has been previously shown to be which our knowledge of the biology of these proteins is predominantly nuclear in U2OS cells (Honda et al., more limited. 2003), and both nuclear and cytoplasmic in COS-7 cells (Kawajiri et al., 2003; Yasui et al., 2004). These previous observations, and the heterogeneous distribution we Results found in MCF-7 cells, suggested that the nucleocyto- plasmicdistribution of Aurora B before NEB could be a Subcellular localization of epitope-tagged chromosomal dynamic process. Given that the nucleocytoplasmic passenger proteins before nuclear envelope breakdown shuttling of Survivin is mediated by the CRM1 nuclear We have previously shown that Survivin undergoes export receptor (Rodriguez et al., 2002), we tested the continuous shuttling between nucleus and cytoplasm in possibility that a similar mechanism may operate in the the presence of an intact nuclear envelope (Rodriguez case of Aurora B, by evaluating the effect of the specific et al., 2002). The information regarding the subcellular CRM1 inhibitor leptomycin B (LMB) on the nucleocy- localization of the other CPPs before NEB is very toplasmicdistribution of epitope-tagged Aurora B. limited, which contrasts with the large number of studies Incubation of transfected MCF-7 cells with LMB led addressing the subcellular trafficking of these proteins to a relocation of VSV-Aurora B (Figure 2a) and later during mitosis. To investigate the mechanisms that Aurora B-HA (not shown) from the cytoplasm to the contribute to regulate their nucleocytoplasmic distribu- nucleus. These results indicate that, as in the case of

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4869

Figure 1 Subcellular localization of epitope-tagged chromosomal passenger proteins (CPPs) before nuclear envelope breakdown. (a) Examples of the subcellular localization of epitope-tagged CPPs in transfected MCF-7 cells. Flag-Survivin is predominantly located in the cytoplasm, as previously reported. Three different patterns of VSV-Aurora B nucleocytoplasmic localization can be observed: predominantly nuclear (N>C), nuclear and cytoplasmic (N ¼ C) and predominanlty cytoplasmic (C>N). VSV-INCEP efficiently accumulates in the nucleus. Finally, VSV-Borealin localizes into the nucleus, where it accumulates prominently in nucleoli and in the cytoplasm. Co-transfected green fluorescent protein-Lamin A was used as a marker to confirm the integrity of the nuclear envelope. VSV-tagged Aurora B, INCENP and Borealin localize to the centromeres during metaphase and to the midbody during telophase/ cytokinesis. Cells were counterstained with Hoechst 33285 to visualize the DNA. (b) Quantification of the subcellular distribution of CPPs in transfected MCF-7 cells. The localization of each protein was determined using fluorescence microscopy in a single-cell basis, and classified as predominantly cytoplasmic (C>N), cytoplasmic and nuclear (C ¼ N) or predominantly nuclear (N>C). In the case of Borealin, the localization was scored as cytoplasmic (C), cytoplasmic and nucleolar (C ¼ Nol) or predominantly nucleolar (Nol). Bars represent the mean of two independent experiments with less than 10% variation. At least 200 cells were counted per sample.

Survivin, the nucleocytoplasmic localization of Aurora indicating that nucleocytoplasmic transport of Aurora B B before NEB is the result of a dynamicequilibrium does not require its kinase activity. between nuclear import and CRM1-dependent export. Next, we used a time-course analysis of cells treated The steady-state localization and the LMB-induced with LMB to compare the nuclear transport kinetics of nuclear relocation of a kinase dead (KD) Aurora B Aurora B and Survivin. As illustrated in Figure 2b, (the catalytically inactive mutant K106R) was compar- Flag-Survivin in untreated cells was more cytoplasmic able to that of the wild-type protein (not shown), than VSV-Aurora B and, upon LMB treatment, the

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4870

Figure 2 Nucleocytoplasmic distribution of Aurora B is regulated by CRM1-dependent nuclear export. (a) Images show an example of the re-localization of VSV-Aurora B to the nucleus of MCF-7 cells after treatment with the specific CRM1 inhibitor leptomycin B (LMB; 6 ng/ml for 6 h). Graphs show a quantification of the effect Figure 3 CRM1 is not a direct determinant of the nucleocyto- of LMB on the localization of VSV-Aurora. Bars represent the plasmic localization of INCENP and Borealin before nuclear proportion of transfected MCF-7 cells showing each pattern of envelope breakdown. (a) Coexpression with the export receptor VSV-Aurora B nucleocytoplasmic distribution and represent the CRM1 does not induce cytoplasmic relocation of INCENP. As a 7 results (mean s.d.) of four independent experiments. (b) Time- positive control (left panels), a nuclear version of Survivin, tagged course analysis of LMB-induced nuclear relocation of Flag- with the SV-40 NLS (Flag-Surv þ NLS), efficiently relocalizes to Survivin and VSV-Aurora B in MCF-7 cells. Graph shows the the cytoplasm when coexpressed with YFP-CRM1. In contrast, the proportion of cells showing predominantly cytoplasmic (C>N) nuclear localization of VSV-INCENP (right panels) does not localization of Flag-Survivin or VSV-Aurora B after treatment change when coexpressed with the export receptor. (b) Leptomycin with 6 ng/ml LMB for the indicated time. B (LMB) treatment does not significantly reduce the partial cytoplasmic localization of VSV-Borealin. The localization of VSV-Borealin was determined by immunofluorescence in trans- fected MCF-7 cells treated with 6 ng/ml LMB for 6 h. In the same nuclear accumulation of Survivin was noticeably faster cells, nuclear relocation of endogenous NF-kB (p65 subunit) was than that of Aurora B. These results indicate that evaluated as an internal control for inhibition of the CRM1- Survivin is more dynamicthan Aurora B in terms of mediated nuclear export pathway. (c) Coexpression with CRM1 does not induce cytoplasmic relocation of Borealin. When nuclear shuttling before NEB. expressed alone or coexpressed with YFP-CRM1, VSV-Borealin is similarly localized to the nucleoli or both the nucleoli and the CRM1is not a direct determinant of the cytoplasm in untreated cells (left panels). Upon incubation with nucleocytoplasmic localization of INCENP and 5 mg/ml actinomycin D for 3 h (right panels), the nucleolar localization of Borealin is disrupted, and the protein disperses Borealin before nuclear envelope breakdown through the nucleus, but does not relocate to the cytoplasm when As CRM1-mediated export contributes, directly or coexpressed with CRM1. indirectly, to determine the localization of Aurora B and Survivin before NEB, we tested the possibility that the same pathway may also regulate the nucleocyto- plasmicdistribution of INCENP and Borealin. comes endogenous CRM1-dependent export of Survivin In the case of INCENP, its predominantly nuclear and enforces its steady-state localization in the nucleus localization hampered the use of LMB to assess the role when expressed alone. However, coexpression with of CRM1-dependent nuclear export. As an alternative YFP-CRM1 efficiently relocates Flag-Survivin þ NLS experimental approach, we coexpressed VSV-INCENP to the cytoplasm, revealing its nuclear shuttling ability. with an yellow fluorescent protein (YFP)-tagged version In contrast to Survivin þ NLS, VSV-INCENP remained of the export receptor CRM1. We have used this in the nucleus when coexpressed with YFP-CRM1 approach previously to demonstrate the role of CRM1 (Figure 3a, right panels). in the export of the predominantly nuclear protein On the other hand, given the partial cytoplasmic BARD1 (Rodriguez et al., 2004). As a positive control, localization of Borealin, we first evaluated the effect of we used Flag-Survivin þ NLS, a modified version of LMB on its nucleocytoplasmic distribution. As illu- Survivin fused to the strong NLS of SV-40 large T strated in Figure 3b, the levels of cytoplasmic Borealin antigen (Kalderon et al., 1984). As shown in Figure 3a did not significantly decrease in cells exposed to LMB (left panels), the SV-40 NLS-mediated import over- for 6 h. As an internal control, nuclear accumulation of

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4871 endogenous NF-kB, whose cytoplasmic localization immunofluorescence. In those interphase cells where requires active CRM1-dependent export from the endogenous Survivin and Aurora B were expressed at nucleus (Johnson et al., 1999), confirmed the effective detectable levels, their localization was predominantly blockage of CRM1 in these cells. In line with the lack of nuclear both in the absence and presence of LMB (not LMB responsiveness, the fraction of Borealin localized shown). Consistent with the LMB-induced mitotic in the nucleus did not undergo relocation to the defects reported by Arnaoutov et al. in HeLa and cytoplasm when coexpressed with YFP-CRM1 U2OS cells, very few MCF-7 cells in anaphase could be (Figure 3c). As nuclear Borealin was largely accumu- detected after the treatment. Interestingly, the levels of lated in the nucleoli, it remained possible that trapping endogenous Survivin (Figure 4a, upper set of panels) in this organelle could prevent its export. To test this and Aurora B (Figure 4a, lower set of panels) in the possibility, cells were incubated in the presence of centromeric region of chromosomes during prophase actinomycin D to disrupt nucleoli. Actinomycin D and (pro)metaphase were significantly reduced in many treatment resulted in a diffuse nuclear localization of VSV-Borealin. However, VSV-Borealin did not relocate to the cytoplasm in CRM1-transfected, actinomycin D- treated cells (Figure 3c). These observations strongly suggest that CRM1- mediated export does not play a major role as a direct determinant of the nucleocytoplasmic localization of these proteins, although our results do not exclude the possibility that INCENP and Borealin may be actively exported from the nucleus under some circumstances.

Sustained treatment with leptomycin B disrupts the localization of Survivin and Aurora B in mitotic cells It has been recently reported that, besides its role as a nuclear export receptor, CRM1 is a critical effector of Ran-GTP during mitosis (Arnaoutov et al., 2005). Thus, inhibition of CRM1 by LMB treatment was shown to disrupt mitoticprogression and chromosome segrega- tion. Given the role of CRM1 as a determinant of the nucleocytoplasmic distribution of Survivin and Aurora B before NEB, we tested the possibility that LMB could also affect the localization of these CPPs during mitosis. To this end, MCF-7 cells were incubated overnight in the presence of 6 ng/ml LMB and the localization of endogenous Survivin and Aurora B was evaluated by

Figure 4 Sustained leptomycin B (LMB) treatment disrupts the localization of endogenous Survivin and Aurora B in mitotic cells. (a) Immunofluorescence microscopy analysis of the localization of endogenous Survivin (upper set of panels) and Aurora B (lower set of panels) in mitoticMCF-7 cellsuntreated or treated with 6 ng/ml LMB for 14 h. A clear colocalization of Survivin and Aurora B with the centromere marker CREST is consistently observed in untreated samples during prophase and metaphase. In LMB- treated samples, the colocalization of the chromosomal passenger proteins (CPPs) with CREST is severely disrupted in many prophase and metaphase cells. The larger panels in the ‘ þ 14 h LMB’ samples show microscopy fields containing cells with correctly localized CPPs (green arrowheads) and cells with disrupted localization of Survivin or Aurora B (red arrowheads). A higher magnification of the cells inside the square is shown on the right. (b) Mitoticlocalizationof Survivin is not altered by a short- term LMB treatment that efficiently disrupts RanBP2 targeting. Cytospins were made from nocodazole-arrested MCF-7 cells that were collected by shake-off, washed and incubated for 1 h in the absence or presence of LMB (6 ng/ml). Localization of RanBP2 to kinetochores is disrupted under these conditions (upper set of panels). A higher magnification of the cells inside the square is shown on the right. In contrast to RanBP2, Survivin remains correctly localized after short-term LMB treatment (lower set of panels).

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4872 cells after LMB treatment. Next, the effect of shorter none of the NES-like sequences in Aurora B was active LMB incubation on the localization of Survivin and in a Rev-GFP-based in vivo nuclear export assay Aurora B was determined in synchronized mitotic cells (Henderson and Eleftheriou, 2000), and the putative that were released from a nocozadole arrest in the NLS was not able to mediate the nuclear accumulation absence or presence of LMB. As a positive control, the of YFP. Thus, none of the candidate nuclear transport localization of RanBP2, whose mitotic targeting is signals identified in Aurora B are functional, raising the rapidly disrupted upon CRM1 inactivation (Arnaoutov possibility that Aurora B may contain non-classical et al., 2005), was determined. In contrast to that of transport signals or access the pathways of nucleocyto- RanBP2 (Figure 4b, upper set of panels), the localiza- plasmictransport in an indirectway, through binding to tion of Survivin (Figure 4b, lower set of panels) and other molecule(s) that may bridge its interaction with Aurora B (not shown) was not disrupted after 1 h in the transport receptors. presence of LMB. Similar results (not shown) were In the case of INCENP, two amino-acid segments obtained using time-lapse microscopy analysis of HeLa resembling bipartite NLSs have been noted previously in cells stably transfected with GFP-Survivin. Thus, in our the murine protein (Saffery et al., 1999). These setting, sustained blockage of CRM1 is required to sequences are conserved in the human homologue disrupt CPP targeting to mitoticstructures, suggesting (Adams et al., 2001b), but their functionality has not that the mechanism by which CRM1 activity affects the been experimentally tested. INCENP segments encom- localization of these CPPs is different from the direct passing these amino-acid sequences and several other mechanism by which the export receptor mediates the candidate NLSs identified by PSORT II analysis were targeting of RanBP2. We are presently carrying out fused to YFP to evaluate their ability to mediate nuclear experiments to further investigate the relationship import in vivo. We identified three short amino-acid between the CRM1 pathway and the localization and sequences that showed nuclear import activity activity of CPPs during mitosis. (Figure 5a). As illustrated in Figure 5b, each of these amino-acid segments was able to promote the nuclear accumulation of YFP, which per se is evenly distributed Functional analysis of putative ‘classical’ nuclear between nucleus and cytoplasm. We termed these transport signals in Aurora B and INCENP sequences NLS 1, NLS 2 (both located in the amino- To further characterize the mechanisms that control the terminal end) and NLS 3 (in the middle region of the nucleocytoplasmic distribution of the CPPs, we next protein). In addition, we noted that INCENP NLS 1 aimed to identify amino-acid sequences in these proteins and NLS 2 led to efficient accumulation of the fused that may function as nuclear transport signals. In our YFP in the nucleoli. Next, using YFP-tagged proteins, previous work, we mapped the region responsible for the we observed that an N-terminal fragment of INCENP nuclear shuttling of Survivin to its carboxy-terminal containing NLS 1, 2 and 3 localized exclusively in the coiled-coil domain, although we could not identify nucleus (Figure 5c), whereas a C-terminal fragment active ‘classical’ nuclear transport signals in this protein distributed throughout the cell. Importantly, small (Rodriguez et al., 2002). On the other hand, two interstitial deletions that eliminate the NLSs readily evolutionarily conserved functional NLSs have been disrupted the nuclear and nucleolar localization of the reported in Borealin sequence (Gassman et al., 2004). amino-terminal fragment of INCENP. Altogether, these Thus, we focused our analysis on Aurora B and results indicate that human INCENP contains at least INCENP. By visual inspection of Aurora B primary three independent functional NLSs, two of which may sequence, we identified two segments of amino acids that also function as nucleolar targeting motifs. resemble the leucine-rich NESs recognized by CRM1 (Table 1). The presence of an NES in the second of these segments was also predicted by using the NetNES INCENP and Survivin modulate the subcellular software (La Cour et al., 2004). On the other hand, localization of Aurora B and Borealin, respectively, before analysis of Aurora B sequence using the PSORT II nuclear envelope breakdown program (Nakai and Horton, 1999) identified a stretch The closely related and interdependent trafficking of the of basicresidues as a putative ‘pat7’ NLS. We tested the CPPs during mitosis (Speliotes et al., 2000; Wheatley ability of each of these putative transport sequences to et al., 2001; Bolton et al., 2002; Lens et al., 2003; act as autonomous NLSs or NESs. As shown in Table 1, Gassman et al., 2004; Sampath et al., 2004) raised the

Table 1 Functional analysis of putative nuclear transport signals in Aurora B Nuclear transport signal Amino-acid sequence Assay Activity

CrmA NES (control) 242-GSYNLVDALVKLGLTEVF IVEA Yes Aurora B putative NES1 115-KEGVEHQLRREIEIQAHL IVEA No Aurora B putative NES2 202-KPENLLLGLKGELKIADF IVEA No SV-40 large T Ag NLS (control) 126-PKKKRKV F-YFP Yes Aurora B putative NLS 226-PSLRRKT F-YFP No

Abbreviations: NES, nuclear export signal; NLS, nuclear localization signal; IVEA, in vivo GFP-based nuclear export assay; F-YFP, fusion to YFP. The hydrophobic residues that conform to the ‘leucine-rich’ NES consensus sequence are underlined.

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4873

Figure 5 INCENP contains multiple functional nuclear localization signals (NLSs). (a) Amino-acid sequence of the INCENP fragments containing putative NLSs that were tested for import activity. Underlined residues indicate the sequences in human INCENP homologous to the previously proposed putative NLS in the mouse protein (Saffery et al., 1999). Each of these INCENP fragments was fused to YFP and the localization of the fusion proteins was compared to that of YFP alone (negative control) and YFP-NLS (positive control) using fluorescence microscopy. This analysis identified three small INCENP segments that induce nuclear accumulation of YFP and thus, behave as autonomous functional NLSs. (b) Representative examples of transfected MCF-7 cells showing an even distribution of YFP through the nucleus and the cytoplasm, and the nuclear accumulation of YFP induced by the fusion of INCENP NLS 1, NLS 2 and NLS 3. Note that both INCENP NLS 1 and NLS 2 efficiently targeted YFP to the nucleoli. (c) Schematic representation and subcellular localization of INCENP fragments containing or lacking NLS 1, 2 and 3. YFP-INCENP(1– 454) is exclusively nuclear, whereas YFP-INCENP(455–919) is more diffusely distributed throughout the cell. Small interstitial deletions eliminating the NLSs efficiently abrogate the nuclear accumulation of YFP-INCENP(1–454).

possibility that these proteins might also modulate the Borealin when coexpressed with each of the other CPPs. localization of each other before NEB. We addressed As a control for specificity, we used YFP-NLS, a fusion this issue using co-transfection experiments. We rea- protein of YFP and the SV-40 NLS, whose coexpression soned that concomitantly raising the levels of two CPPs should not alter the localization of the CPPs. would reveal any influence on each other’s localization, The nucleocytoplasmic distribution of Aurora B although it must be noted that endogenous levels of the coexpressed with Survivin or Borealin was similar to four CPPs are present in all cases. Figure 6A illustrates its localization when expressed alone or with the YFP- the localization of Aurora B, Survivin, INCENP and NLS control. In contrast, coexpression with INCENP

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4874 led to a prominent relocation of Aurora B to the bears a carboxy-terminal domain completely unrelated nucleus. As shown in Figure 6B, localization of Aurora to that of Survivin, was unable to prevent the nucleolar B was predominantly nuclear in more than 60% of localization of Borealin (Figure 7b). Second, we have INCENP-co-transfected cells. We confirmed that the previously reported that the carboxy-terminal region of effect of INCENP on the localization of Aurora B was Survivin mediates its nucleocytoplasmic transport (Ro- specific using control experiments (data not shown) in driguez et al., 2002). Thus, binding of Borealin could which coexpression with INCENP did not induce modulate the access of Survivin to the nuclear transport nuclear accumulation of the shuttling protein Snurpor- machinery and vice versa. As illustrated in Figure 7c, tin I (Paraskeva et al., 1999), or the cytoplasmic protein LMB treatment of co-transfected cells led to the efficient TRAF2 (Birbach et al., 2002). nuclear accumulation of YFP-Survivin, demonstrating The predominantly cytoplasmic localization of Survi- that the nucleocytoplasmic shuttling ability of Survivin vin, on the other hand, did not significantly change is not impaired in cells coexpressing VSV-Borealin. In when coexpressed with any of the other CPPs, although some cells, we observed simultaneous nuclear relocation a faint nucleolar signal was noted in some cells of Survivin and Borealin, but, in most cases, only coexpressing Borealin. It is remarkable that, in contrast Survivin relocated to the nucleus after LMB treatment to Aurora B, Survivin remained in the cytoplasm when (Figure 7c). Next, we coexpressed VSV-Borealin with coexpressed with INCENP. YFP-Survivin þ NLS, a version of Survivin targeted to The localization of INCENP, like that of Survivin, the nucleus by fusion of the SV40 NLS. As shown in was not significantly altered by coexpression of any of Figure 7d, coexpression of YFP-Survivin þ NLS led to a the other CPPs, and this protein remained nuclear in diffuse nuclear localization of Borealin. In contrast to virtually all transfected cells. the LMB-induced nuclear accumulation of Survivin, Finally, a striking change in the localization of SV40 NLS-mediated nuclear targeting of Survivin Borealin was observed in cells coexpressing Survivin. invariably led to the concomitant nuclear relocation of In these cells, the nucleolar localization of Borealin was coexpressed Borealin, suggesting that the interaction of completely abrogated. Survivin and Borealin colocalized Borealin and Survivin is more efficient when the nuclear in the cytoplasm in approximately 90% of the co- import of Survivin is mediated by a heterologous transfected cells (Figure 6C). In contrast to Survivin, sequence outside its carboxy-terminal domain. neither Aurora B nor INCENP disrupted the nucleolar accumulation of Borealin. We must point out, however, that the coexpression efficiency of VSV-INCENP and Discussion YFP-Borealin was consistently low in the several experiments we attempted and only a limited number The CPPs Survivin, Aurora B, INCENP and Borealin of co-transfected cells could be examined. sequentially colocalize to the chromosome centromeres, In summary, the results of these experiments uncover the spindle midzone, the cleavage furrow and finally to a novel role for INCENP and Survivin as specific the midbody as mitosis progresses (Adams et al., modulators of the subcellular distribution of Aurora B 2001a, b; Gassman et al., 2004; Sampath et al., 2004). and Borealin, respectively, before NEB. The CPPs are already expressed at the G2/M transition and, at least in the case of Survivin, expression at earlier Modulation of the subcellular localization of Borealin by cell cycle stages has been reported in different types of Survivin both normal and tumor cells (Krysan et al., 2004; Song As Borealin is the less well characterized of the CPPs, we et al., 2005). Before NEB, which takes place at the explored in more detail how Survivin modulates its transition between prophase and prometaphase, the localization before NEB. First, we carried out a deletion nuclear envelope poses a physical barrier between the analysis (Figure 7a) to determine what region of nucleus and the cytoplasm. During most of the cell Survivin mediates its effect on Borealin localization. cycle, dynamic nucleocytoplasmic transport across the As shown in Figure 7b, the results of this analysis nuclear envelope determines the localization of many indicated that the carboxy-terminal domain of Survivin cancer-related proteins, including Survivin (Rodriguez is both necessary and sufficient to prevent the nucleolar et al., 2002). Here, we have used epitope-tagged versions accumulation of Borealin. This observation is consistent of the other CPPs to investigate the molecular mechan- with our finding that this region of Survivin is sufficient isms that determine the nucleocytoplasmic distribution to bind Borealin in co-immunoprecipitation experiments of Aurora B, INCENP and Borealin in the presence of (data not shown), and raised two interesting possibi- an intact nuclear envelope. lities. First, it is known that alternative splicing of Our results reveal that the localization of the CPPs Survivin mRNA gives origin to several Survivin protein before NEB is not as closely related as it is during isoforms (Mahotka et al., 1999) that contain different mitosis and that their nucleocytoplasmic transport is, at carboxy-terminal amino-acid sequences (depicted in least in part, independently regulated. Our findings are Figure 7a) and thus might differ in their ability to summarized in the model depicted in Figure 8. We show modulate Borealin localization. Indeed, Survivin-2B, that Aurora B, like Survivin, accesses the CRM1- which contains an in-frame novel sequence of 23 amino dependent nuclear export pathway and undergoes acids, prevented nucleolar localization of Borealin as LMB-sensitive shuttling between the nucleus and the efficiently as Survivin, whereas Survivin-DEx3, which cytoplasm, whereas CRM1-mediated export does not

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4875

Figure 6 INCENP and Survivin modulate the subcellular localization of Aurora B and Borealin, repectively, before nuclear envelope breakdown. (A) Images show representative examples of the localization of epitope-tagged Aurora B, Survivin, INCENP and Borealin when coexpressed with each of the other chromosomal passanger proteins (CPPs) or YFP-nuclear localization signals (NLS) as a control for specificity. The tag used is indicated inside the panels. Coexpression with INCENP induced nuclear relocation of Aurora B (panel c1), but not of Survivin (panel c2). The largely cytoplasmic localization of Survivin and the nuclear localization of INCENP were not substantially altered by coexpression with any of the other CPPs. Coexpression with Survivin prevented accumulation of Borealin in the nucleoli, and increased its cytoplasmic localization (panel b4). Coexpression with the negative control YFP-NLS (panels e1–e4) did not alter the localization of any of the CPPs. (B) Quantification of the effect of INCENP coexpression on the localization of Aurora B. Graphs show the proportion of cells expressing HA-Aurora B predominantly in the cytoplasm (C>N), at similar levels in the cytoplasm and nucleus (C ¼ N) or predominantly in the nucleus (N>C), when expressed alone or coexpressed with VSV-INCENP. The percentage of cells expressing predominantly nuclear Aurora B drastically increases in the presence of coexpressed INCENP. Bars represent the mean7s.d. of three independent experiments. (C) Quantification of the effect of Survivin coexpression on the localization of Borealin. Graphs show the proportion of cells expressing VSV-Borealin predominantly in the cytoplasm (C), in both the cytoplasm and nucleolus (C þ Nol) or predominantly in the nucleolus (Nol), when expressed alone or coexpressed with YFP- Survivin. Bars represent the mean of two independent experiments with less than 10% variation. More than 200 cells per sample were counted in each experiment. seem to play a major role as a direct determinant of the of the nucleocytoplasmic shuttling of Survivin and nucleocytoplasmic distribution of INCENP and Bor- Aurora B before NEB remain to be established. ealin. Several groups, including ours, have shown that However, it has been recently reported that, besides its the nucleocytoplasmic localization of Survivin in tumor function as a nuclear export receptor, CRM1 has a role cells determined by immunohistochemistry (IHC) is determining the localization of proteins to chromosome related to prognosis in patients with different types of kinetochores during mitosis (Arnaoutov et al., 2005). In cancer (Altura et al., 2003; Vischioni et al., 2004; Tonini this respect, our initial experiments demonstrate that et al., 2005). It is presently unknown if a similar extended LMB treatment disrupts the localization of relationship with clinical parameters exists in the case endogenous Survivin and Aurora B to the centromeric of Aurora B localization, as the number of studies region of prophase and (pro)metaphase cells. We show evaluating the expression Aurora B in human tumors that, unlike that of RanBP2 (Arnaoutov et al., 2005), using IHC is very limited. The functional consequences the localization of the CPPs is only disrupted after

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4876

Figure 7 Modulation of the subcellular localization of Borealin by Survivin before nuclear envelope breakdown. (a) Schematic representation of the YFP-tagged deletion mutants and splice variants of Survivin used in the experiments. The BIR domain of Survivin is depicted in gray. The regions of Survivin-2B and Survivin-DEx3 containing amino-acid sequences different from Survivin are depicted in black. (b) The carboxy-terminal region of Survivin is necessary and sufficient to prevent nucleolar localization of Borealin. Images show representative examples of the effect of coexpressing each of the fragments and isoforms of Survivin on the subcellular localization of VSV-Borealin. Like the full-length protein, the carboxy-terminal region of Survivin (74–142), and Survivin- 2B abrogate nucleolar accumulation of Borealin. In contrast, Borealin remains in the nucleoli when coexpressed with the amino- terminal region of Survivin (1–74) or with Survivin-DEx3. (c) Survivin remains a nuclear shuttling protein when coexpressed with Borealin. MCF-7 cells coexpressing YFP-Survivin and VSV-Borealin were treated with leptomycin B (LMB) (6 ng/ml for 3 h). In untreated samples, YFP-Survivin and VSV-Borealin colocalize in the cytoplasm. After LMB treatment, Survivin efficiently accumulates in the nucleus. In a proportion of co-transfected cells (the mean7s.d. of three independent experiments is indicated inside the panels), simultaneous nuclear relocation of Borealin was observed. (d) Nuclear targeting of Survivin mediated by a heterologous nuclear localization signal (NLS) leads to nuclear localization of coexpressed Borealin. Images show that VSV-Borealin is diffusely localized within the nucleus (not accumulated in the nucleoli) when coexpressed with YFP-Survivin þ NLS, a nuclear version of Survivin bearing an SV40 NLS fused to its carboxy-terminal end.

sustained blockage of CRM1 activity, suggesting that pathway that is relevant for events that take place after the presence of LMB during the preceding interphase NEB. may contribute to the observed effect. Therefore, further Despite their dynamicnucleocytoplasmictransport, experimental evidence is needed to clarify the relation- our functional assays failed to identify active NLSs or ship between the CRM1 pathway and the CPPs during NESs of the ‘classical’ type in Aurora B (this report) or mitosis. For example, it remains to be established if the Survivin (Rodriguez et al., 2002). Although it remains altered localization of Survivin and Aurora B is a direct possible that other ‘non-classical’ signals in these or indirect consequence of blocking the CRM1 pathway. proteins mediate their direct interaction with nuclear In the light of these results, however, it is tempting to transport receptors, our findings suggest that the speculate that the CRM1-dependent nuclear shuttling of nucleocytoplasmic traffic of Survivin or Aurora B could Aurora B and Survivin before NEB may reflect a be indirectly modulated through interaction with other functional interaction of the CPPs with the CRM1 NES- or NLS-containing proteins. In this regard, both

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4877 Borealin before NEB was its accumulation in nucleoli. The nucleolar localization of GFP-Borealin was con- sidered a non-physiological feature in the initial report, because the authors did not detect endogenous Borealin in this organelle using immunocytochemistry (ICC) (Gassman et al., 2004). More recently, however, a highly sensitive proteomics approach using mass spec- trometry has revealed the presence of endogenous Borealin in the nucleolus (Andersen et al., 2005, see the Nucleolar Proteome Database available at http:// www.lamondlab.com/NOPdb/), suggesting that Borea- lin is a bona fide nucleolar protein. In fact, not only Borealin but also Aurora B and INCENP were found to copurify with human nucleoli. These findings raise the intriguing possibility that the nucleolus may function as a reservoir for a fraction of Borealin, Aurora B and INCENP before NEB, and it would be interesting to dissect the mechanisms that modulate the trafficking of the CPPs in and out of this nuclear organelle. In this context, we noted that two of the INCENP NLSs strongly targeted the fused YFP to the nucleoli, Figure 8 Nucleocytoplasmic localization of the chromosomal passenger proteins (CPPs) before nuclear envelope breakdown. suggesting that they may contribute to the nucleolar Model summarizing the mechanisms that regulate the nucleocyto- localization of INCENP. plasmic distribution of the CPPs in the presence of an intact More importantly, our data reveal a previously nuclear envelope. Aurora B undergoes CRM1-mediated nuclear unknown role for Survivin as a potent negative export and is, like Survivin, able to shuttle between nucleus and modulator of the nucleolar accumulation of Borealin. cytoplasm. INCENP contains multiple functional nuclear localiza- tion signals (NLSs) and is efficiently imported/retained into the We show that the coexpression of both proteins results nucleus. Borealin also contains functional NLSs (Gassman et al., in Survivin efficiently blocking the nucleolar localization 2004) and, after translocation into the nucleus, it accumulates in of Borealin. Thus, the low levels of endogenous Borealin the nucleolus. Although not depicted in the model, the presence of in nucleoli, undetectable by ICC, could be explained by Aurora B and INCENP (but not of Survivin) in the nucleolus has also been reported (Andersen et al., 2005). INCENP modulates the its balanced expression with endogenous Survivin. This nucleocytoplasmic distribution of Aurora B (probably through novel role of Survivin is mediated by its carboxy- increased nuclear import and/or reduced nuclear export/enhanced terminal end and is retained in the splice variant retention of the kinase in the nucleus), but not of Survivin. Finally, Survivin-2B, but lost in Survivin-DEx3. Survivin iso- Survivin prevents the targeting of Borealin to the nucleolus. forms have been previously reported to differ in their antiapoptoticactivity (Mahotka et al., 1999). Our results identify another functional divergence between INCENP (this report) and Borealin (Gassman et al., these proteins that is most likely non-related to 2004) contain functional ‘classical’ NLSs, and therefore apoptosis regulation, as the isoform Survivin-2B, which they were obvious candidates to modulate the nucleo- has reportedly lost the antiapoptoticability (Mahotka cytoplasmic distribution of Survivin and Aurora B. et al., 1999), is still able to prevent the nucleolar Indeed, coexpression with INCENP dramatically localization of Borealin. increased the nuclear accumulation of Aurora B. It Finally, we show that nuclear shuttling of Survivin is could be argued that coexpression of any protein with not impaired when coexpressed with Borealin, although an NLS-bearing binding partner would lead to the the carboxy-terminal region of Survivin mediates both nuclear localization of both proteins. Remarkably, its nuclear transport and its effect on Borealin. however, coexpression with INCENP did not induce Importantly, only a minor fraction of Borealin relocates nuclear relocation of Survivin, although the ability of to the nucleus along with Survivin in the presence of these two proteins to physically interact is well LMB. In contrast, enforced nuclear accumulation of established (Bolton et al., 2002; Wheatley et al., 2001). Survivin by fusion of a heterologous NLS invariably Thus, these results indicate that INCENP plays a results in the concomitant nuclear relocation of Bor- specific role as modulator of Aurora B nucleocytoplas- ealin. These results are consistent with a model in which mic distribution by increasing its nuclear import and/or the binding of proteins that mediate its nuclear its retention in the nucleus. transport would largely impair the interaction of In contrast to INCENP, Borealin did not significantly Survivin with Borealin. Therefore, we suggest that, alter the nucleocytoplasmic distribution of neither although Survivin modulates the nucleolar localization Aurora B nor Survivin. Rather, our data indicate that of Borealin before NEB, the nucleocytoplasmic trans- it is Survivin that modulates the subcellular localization port of both proteins is mostly unrelated. of Borealin before NEB. As previously described using a In conclusion, we show that INCENP and Survivin GFP-tagged version (Gassman et al., 2004), a prominent are able to specifically modulate the subcellular localiza- characteristic of the subcellular distribution of VSV- tion of Aurora B and Borealin, respectively, before

Oncogene Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4878 NEB. The existence of at least two different chromoso- NLS were annealed, phosphorylated and cloned into HindIII/ mal passenger complexes in mitotic cells has been BamHI-digested pEYFP-C1 to make the control construct recently proposed (Gassman et al., 2004). One of these YFP-NLS. The high-fidelity DNA polymerase Pfu (Strata- complexes contains only Aurora B and INCENP, gene, La Jolla, CA) was used in all PCR reactions and the whereas the second complex also includes Survivin and sequence of the inserts was verified by DNA sequencing. The sequence of the oligonucleotides used in plasmid construction Borealin. Consistent with this view, our findings suggest is available upon request. that different complexes of CPPs may also exist before NEB and undergo different dynamics of transport between the nucleus and the cytoplasm. Functional analysis of putative transport signals in Aurora B and INCENP DNA fragments encoding two putative NESs identified in Aurora B protein sequence were generated by PCR using Materials and methods Aurora B cDNA as template. PCR products were cloned into the pRev(1.4)-GFP plasmid and the export activity of these Cell culture, transfection and drug treatments sequences was tested in a nuclear export assay, as previously Human breast carcinoma cells MCF-7 were grown in described (Henderson and Eleftheriou, 2000). On the other Dulbecco’s modified Eagle’s medium (DMEM) (BioWhit- hand, Aurora B and INCENP sequences encoding putative taker, Walkersville, MD, USA), supplemented with 10% fetal NLSs were cloned into HindIII/BamHI-digested pEYFP-C1. calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin The import activity of these sequences was tested by (Gibco BRL, Gaithersburg, MD, USA). Cells were seeded comparing the nucleocytoplasmic distribution of the fusion onto sterile glass coverslips in 12-well trays and transfected proteins to that of the YFP protein alone (negative control) with 0.5–2 mg of plasmid DNA using the FuGene6 transfection and YFP-NLS (positive control). reagent (Roche Molecular Biochemicals). Leptomycin B (LMB, a generous gift from Dr Minoru Yoshida, University of Tokyo, Japan) and actinomycin D (Sigma, St. Louis, MO, Immunofluorescence and microscopy analysis USA) were added to the culture medium to a final concentra- Cells were fixed with 3.7% formaldehyde in phosphate-buffered tion of 6 ng/ml and 5 mg/ml, respectively. Mitotic-arrested cells saline (PBS) for 30 min and permeabilized with 0.2% Triton X- were collected by shake-off after overnight incubation with 100 in PBS for 10 min. After 1 h incubation in blocking solution 50 ng/ml nocodazole (Sigma) and released from the arrest in (3% bovine serum albumin (BSA) in PBS), immunocytochem- nocodazole-free DMEM with or without LMB (6 ng/ml). After ical detection of endogenous proteins was carried out using a 1 h incubation, cytospins were prepared using a Cytospin 2 rabbit anti-NF-kB polyclonal antibody (diluted 1:300; Santa (Shandon, Waltham, MA, USA). Cruz, Biotechnology, Santa Cruz, CA, USA), mouse anti- Survivin monoclonal antibody 6E4 (diluted 1:200; Cell Signal- ing Technology, Danvers, MA, USA), mouse anti-AIM-1 Plasmids and cloning procedures (Aurora B) monoclonal antibody (diluted 1:300; BD Bios- The plasmids encoding HA-TRAF2, HA-Snurportin I and ciences, Franklin Lakes, NJ, USA), a human anti-CREST GFP-Lamin A were generously provided by Dr Colin Duckett antibody provided by J Kuijpers (University Medical Center (University of Michigan, USA), Dr Sylvain Meloche (Institute Utrecht, The Netherlands) and a goat anti-RanBP2 antibody de Recherches Cliniques de Montreal, Canada) and Dr David provided by F Melchior (Georg-August Universita¨ t Gottingen, Gilbert (State University of New York, USA). The plasmids Germany). Detection of epitope-tagged proteins was performed encoding YFP-CRM1, Flag-Survivin, YFP-Survivin, YFP- with anti-VSV monoclonal antibody (diluted 1:20 000; Sigma), Survivin-2B and YFP-Survivin-DEx3 have been previously anti-HA rabbit polyclonal antibody Y-11 (diluted 1:300; Santa described (Rodriguez and Henderson, 2000; Rodriguez et al., Cruz) or anti-Flag M2 monoclonal antibody (diluted 1:200; 2002). Survivin deletion mutants YFP-Survivin(1–74) and Stratagene). Primary antibodies were detected using anti- YFP-Survivin(74–142) were generated by PCR using Survivin mouse, anti-rabbit, anti-human or anti-goat secondary anti- complementary DNA (cDNA) as template. Aurora B, bodies conjugated to fluorescein isothiocyanate (Santa Cruz Borealin and INCENP cDNAs were obtained by reverse and Sigma), Alexa Fluor 594 or Alexa Fluor 488 (Molecular 0 transcriptase–PCR and cloned in-frame to a 5 sequence Probes, Carlsbad, CA, USA). The chromosome dye Hoechst encoding the VSV epitope into pCR3 (Aurora B and Borealin) 33285 (Sigma) was used to counterstain the nuclei. Finally, the or pCDNA3 (INCENP) vectors to generate VSV-Aurora B, coverslips were mounted onto microscope slides using Vecta- VSV-Borealin and VSV-INCENP, as described in detail shield (Vector, Burlingame, CA, USA). elsewhere (Vader et al., 2006). Carboxy-terminally tagged Fluorescence microscopy analysis was carried out using an Aurora B-HA, YFP-Borealin, YFP-INCENP(1–454) and inverted Leica DMIRB/E fluorescence microscope. Images YFP-INCENP(455–919) were generated by PCR using Aurora were collected using the Q500MC Quantimet software V01.01 B, Borealin and INCENP cDNA as templates. The PCR (Leica Cambridge Ltd, Cambridge, UK). To quantitatively products were cloned into pCDNA3 (Aurora B) and pEYFP- determine the subcellular distribution of each protein, its C1 (Borealin and INCENP). On the other hand, site-directed localization was assessed in a single-cell basis in at least 200 mutagenesis was used to introduce the K106R point mutation transfected cells per sample. in Aurora B sequence, generating the catalytically inactive Aurora B KD, and to delete INCENP NLSs in YFP- INCENP(1–454). Survivin þ NLS was constructed using Acknowledgements PCR to add a DNA sequence encoding the SV-40 large T- antigen nuclear localization signal (SV-40 NLS) to the 30 end We thank Drs C Duckett, S Meloche, D Gilbert and BR of Survivin. The PCR product was cloned as a HindIII/BamHI Henderson for providing plasmids, Drs J Kuijpers and F fragment into the pCDNA3 and pEYFP-C1 vectors to Melchior for providing antibodies and Dr M Yoshida for generate Flag-Survivin þ NLS and YFP-Survivin þ NLS, re- providing LMB. JA Rodriguez was supported by the Walter spectively. Finally, cDNA oligonucleotides encoding the SV-40 Bruckerhoff Stiftung.

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