3190 Research Article Simian virus 40 large T antigen targets the -stabilizing TACC2

Shuchin Tei1,2, Noriko Saitoh1, Tetsushi Funahara1, Shin-ichi Iida3, Yuko Nakatsu1, Kayo Kinoshita1, Yoshikazu Kinoshita2, Hideyuki Saya3 and Mitsuyoshi Nakao1,* 1Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 2Department of Gastroenterology and Hepatology, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan 3Division of Regulation, Institute for Advanced Medical Research, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan *Author for correspondence ([email protected])

Accepted 15 June 2009 Journal of Cell Science 122, 3190-3198 Published by The Company of Biologists 2009 doi:10.1242/jcs.049627

Summary The large T antigens of polyomaviruses target cellular missegregation. These mitotic defects are caused by N-terminal- that control fundamental processes, including p53 and the RB deleted T antigen, which minimally interacts with TACC2, family of tumor suppressors. Mechanisms that underlie T- whereas T-antigen-induced microtubule destabilization is antigen-induced cell transformation need to be fully addressed, suppressed by overexpressing TACC2. Thus, TACC2 might be because as-yet unidentified target proteins might be involved a key target of T antigen to disrupt microtubule regulation and in the process. In addition, recently identified polyomaviruses chromosomal inheritance in the initiation of cell transformation. are associated with particular human diseases such as aggressive skin . Here, we report that simian virus 40 (SV40) large T antigen interacts with the transforming acidic coiled-coil- Supplementary material available online at containing protein TACC2, which is involved in stabilizing http://jcs.biologists.org/cgi/content/full/122/17/3190/DC1 in mitosis. T antigen directly binds TACC2 and induces microtubule dysfunction, leading to disorganized Key words: Large T antigen, TACC2, , Nucleus, mitotic spindles, slow progression of mitosis and Mitosis

Introduction members, WU polyomavirus and KI polyomavirus, were found from

Journal of Cell Science Cellular transformation is essential for progression toward tumor respiratory tract specimens (Allander et al., 2007; Gaynor et al., development. The proteins involved in this process have been 2007). More recently, Feng et al. described a new virus, Merkel identified as oncogene products and tumor suppressors. Simian virus cell polyomavirus, the genome of which is integrated into cellular 40 (SV40), a polyomavirus of rhesus macaque origin, is a DNA DNA in the aggressive human skin cancers (Feng et al., 2008). These tumor virus that can induce tumors in rodents and transform many lines of evidence raise the essential question of how their types of cultured mammalian cells, including those of human origin oncoproteins function in the cells. In addition to the known targets (Ahuja et al., 2005; Fanning and Knippers, 1992; Poulin and of T antigen, this protein was recently reported to interact with Bub1, DeCaprio, 2006). The SV40-encoded replication protein, designated which has dual roles in spindle assembly checkpoint and large T antigen and referred to as T antigen, is a viral oncoprotein chromosome congression (Cotsiki et al., 2004; Hein et al., 2009). that modulates diverse cellular activities in genome integrity and Thus, SV40 T antigen might target as-yet unidentified cellular cell-cycle regulation, thereby promoting the early steps of oncogenic proteins to cause cell transformation and oncogenesis (Chang et al., transformation. Although many studies have reported that T antigen 1997; Moens et al., 2007; Sachsenmeier and Pipas, 2001; Woods inactivates p53 and the RB family of tumor suppressors, it is et al., 1994). suggested that other regulatory proteins are required for SV40- During our investigations, we found that T antigen interacts with induced cellular transformation (Chang et al., 1997; Moens et al., transforming acidic coiled-coil (TACC) protein 2, designated 2007; Sachsenmeier and Pipas, 2001). TACC2. There are at least three TACC family proteins (TACC1, Polyomaviruses are highly specific to their host species but TACC2 and TACC3), all of which contain the conserved TACC encode a similar T antigen (Ahuja et al., 2005; Chang et al., 1997; domain and have been possibly implicated in tumorigenesis Fanning and Knippers, 1992; Moens et al., 2007; Poulin and (Gergely, 2002; Raff, 2002). TACC1 and TACC3 were found to be DeCaprio, 2006; Sachsenmeier and Pipas, 2001). In humans, JC overexpressed in human cancers, and overexpression of TACC1 polyomavirus and BK polyomavirus were discovered to be induced the transformation of primary mouse cells in culture (Still responsible for progressive multifocal encephalopathy and renal et al., 1999a; Still et al., 1999b). By contrast, TACC2 was nephropathy, respectively (Imperiale, 2000; Moens et al., 2007; downregulated as breast tumors became more malignant (Chen et Poulin and DeCaprio, 2006). Analogous to SV40, both viruses al., 2000). Overexpression of TACC2 in these cells reverted encode T antigens that transform the cells in culture and promote malignant phenotypes to benign phenotypes both in vivo and in tumor formation in animals (Allander et al., 2007; Moens et al., vitro, suggesting that TACC2 might function as a potential tumor 2007; Poulin and DeCaprio, 2006). Additionally, two human suppressor. T antigen interacts with TACC2 3191

Here, we report that human TACC2 has a crucial role in Results microtubule stabilization in cultured cells, and that T antigen binds T antigen interacts with TACC2 TACC2 for inducing mitotic defects and chromosomal instability. To test the effect of T antigen on cellular function independently In addition to changes in chromosomes, T antigen, or the loss of of p53 and RB, we introduced GFP-fused T antigen in HeLa cells TACC2 function, causes abnormalities in nuclear structural after that lacked these functional proteins but stably transmitted cell division. Our findings suggest that TACC2 is a key target of chromosomes during cell division (Fig. 1A). Abnormally nucleated T antigen for promoting mitotic defects leading to abnormal cells, which had multiple nuclei and/or micronuclei, were increased chromosomal and nuclear inheritance. These findings provide the by about fourfold at 72-96 hours after transfection, compared with first evidence that a viral oncoprotein can directly disrupt control cells. This result suggests that T antigen might rapidly target microtubule regulation, and shed light on the molecular basis of other cellular factors rather than p53 and RB. In addition, we found the initiation of cellular transformation. the similar effects of T antigen in rodent CHO cells (supplementary Journal of Cell Science

Fig. 1. T antigen interacts with TACC2. (A) T antigen induces abnormally nucleated cells. HeLa cells were examined 96 hours after transfection with GFP-fused T antigen. Cells expressing GFP alone (control) or GFP-fused T antigen (GFP-TAg) were stained with DAPI. Micronuclei and multiple nuclei are indicated by the arrow and arrowhead, respectively (lower left). Abnormally nucleated cells were counted (right). Values are the means and standard deviations from five independent experiments (n=200). ***Statistical significant difference: P<0.001. (B) Schematic representation of SV40 large T antigen showing the LxCxE motif and bipartite region that bind RB family proteins and p53 for inactivation, respectively (Ahuja et al., 2005; Fanning and Knippers, 1992; Moens et al., 2007). The mitotic checkpoint protein Bub1 associates with the region containing amino acids 89-97 (Cotsiki et al., 2004). Yeast two-hybrid screening using the T antigen region containing amino acids 250-708 as bait identifies the TACC domain of TACC2 (GenBank accession no. NP996742; amino acids 788-996). (C) Human TACC family proteins. C-terminal TACC domains are conserved among the TACC family proteins. Deletion mutants of TACC2 are shown. (D) T antigen binds TACC2 in vitro. GST-fused T antigen(250-708) on glutathione-agarose beads was incubated with His-tagged TACC domains of TACC proteins and p53. Proteins bound on the beads were detected by western blot analysis with anti-His antibodies. The input shows 10% of each protein. (E) Partial colocalization of T antigen with TACC2 at the mitotic spindles. HeLa cells expressing GFP-fused T antigen (green) were stained with anti-TACC2 antibodies (red). 3192 Journal of Cell Science 122 (17)

material Fig. S1A, left). Abnormally nucleated CHO cells promptly TACC protein family (TACC1, TACC2 and TACC3) have been augmented at 48 hours after transfection of T antigen, suggesting identified in human, and are differently implicated in naturally that these alterations induced by T antigen are unlikely to depend occurring cancers (Gergely, 2002; Raff, 2002). To determine a on cell type. direct interaction between T antigen and TACC2, we prepared To identify novel factors that interact with T antigen, we glutathione S-transferase (GST)-fused T antigen(250-708) and performed yeast two-hybrid screening using the region containing His-tagged TACC domains of human TACC proteins, and amino acids 250 to 708 of T antigen as bait (Fig. 1B). From a subjected them to an in vitro pull-down analysis (Fig. 1D). The screening of approximately 7ϫ106 independent transformants of TACC domain of TACC2, but not those of TACC1 or TACC3, 17-day-old mouse embryo cDNA libraries, we isolated four bound T antigen(250-708). The interaction of TACC2 with T cDNA clones encoding the C-terminal TACC domain of TACC2 antigen(250-708) was comparable to that of p53, which is known (amino acids 788-996; Fig. 1C). At least three members of the to bind the bipartite region of T antigen. To further visualize the Journal of Cell Science

Fig. 2. Effect of TACC2 and T antigen on mitotic spindle formation. (A) Small interfering RNA-mediated knockdown (K.D.) of TACC2. Western blot and immunofluorescence analyses show specific depletion of TACC2 in HeLa cells. β-tubulin and γ-tubulin were used as controls. (B) Induction of abnormally nucleated cells by TACC2 depletion. (Upper) Nuclear morphologies were visualized by DAPI staining. (Lower) Quantitative analysis. Values are the means and standard deviations from three independent experiments of over 300 cells. ***P<0.001. (C) Short inter-centrosome distances in TACC2 knockdown and T-antigen- expressing (TAg) cells. HeLa cells were stained with anti-γ-tubulin antibodies and DAPI. Typical short and monopolar spindles are shown. (D) Quantitative analysis of inter- centrosomal distances. The distances between two in prometaphase and metaphase cells were measured using the Lumina Vision program. Short or monopolar spindles are indicated with asterisks (<7 μm). Values are the means and standard deviations from three independent experiments using 50 cells of each type. *P<0.05; **P<0.01. T antigen interacts with TACC2 3193

subcellular locations of T antigen and TACC2, we carried out an Effect of TACC2 and T antigen on microtubule stabilization immunofluorescence analysis in HeLa cells. As previously There are at least two possible mechanisms for incompletely reported (Gergely, 2002; Gergely et al., 2003), TACC2 was stretched bipolar and monopolar spindle formation. One is failure diffusely distributed in the interphase nuclei and at the centrosomes in the formation of stable microtubules, and the other is defects in (supplementary material Fig. S1B). During cell division, TACC2 the microtubule-associated motor proteins that drive the repulsive was concentrated at the centrosomes and mitotic spindles. force between the poles (Sharp et al., 2000). To test the mitotic Simultaneous visualization showed that a portion of T antigen coexists with TACC2 in mitotic spindles (Fig. 1E) and possibly in interphase nuclei (supplementary material Fig. S1C). These data suggest that T antigen directly interacts with TACC2.

Effect of TACC2 and T antigen on mitotic spindle formation To address the function of TACC2 in human cells, we performed knockdown of endogenous TACC2 using specific short interfering RNAs (siRNAs; Fig. 2A). Western blot analysis revealed the effectiveness of TACC2 knockdown at 72 hours after transfection. In addition, immunofluorescence signals for this protein were mostly lost in both interphase and mitosis (Fig. 2A). TACC2 mRNA was undetectable at 24 hours after siRNA transfection, whereas TACC1 and TACC3 were not affected in the TACC2-depleted cells (data not shown). Similar to the observations in T-antigen-expressing cells (Fig. 1A), abnormally nucleated cells were significantly increased at 72 hours after TACC2 knockdown in HeLa (Fig. 2B) and CHO cells (supplementary material Fig. S1A, right), suggesting that loss of TACC2 induces mitotic defects. To test whether the occurrence of abnormally nucleated cells is due to mitotic spindle defects, we then detected centrosomes and mitotic chromosomes during cell division. TACC2 knockdown was frequently found to cause incompletely stretched bipolar and monopolar spindles (Fig. 2C, upper and lower left panels, respectively). Very similar results were obtained in T-antigen-expressing cells (Fig. 2C, right). To quantify these data, we measured the distances between two centrosomes in mitosis (Fig. 2D). Compared with control cells, the frequencies of short inter-centrosomal distances (<7 μm) were increased by more

Journal of Cell Science than twofold in TACC2 knockdown and T-antigen-expressing cells. These results suggest that the functional consequences of T antigen introduction or TACC2 depletion are correlated with defects of proper spindle formation.

Fig. 3. Effect of TACC2 and T antigen on microtubule stabilization. (A) Suppression of microtubule formation by TACC2 knockdown (K.D.). Fluorescence image of monopolar HeLa cells (left). TACC2 knockdown and control cells were treated with 100 μM Eg5 inhibitor (monastrol) for 6 hours and then stained with anti-β-tubulin antibodies. The largest diameters of mono-asters were quantitatively assessed (right). Values are the means and standard deviations from three independent experiments of 50 cells of each type. ***P<0.001. (B) Microtubule regrowth assays. The microtubule recoveries after cold-treatment were examined in TACC2 knockdown and control cells. The cells were stained with anti-β-tubulin antibodies. (C) Disorganization of mitotic spindles in TACC2 knockdown cells. HeLa cells were stained with anti-β-tubulin antibodies (green), anti-γ-tubulin antibodies (red) and DAPI (blue). Representative images of normal spindles (upper), short spindles (middle) and monopolar spindles (lower) are shown. (D) Slow progression from prometaphase to metaphase in TACC2 knockdown and T-antigen-expressing cells (TAg). The cells were presynchronized with double thymidine blocks, treated with nocodazole (0.1 μg/ml) for 6 hours, released and further incubated for 1.5 hours in the presence of the proteasome inhibitor MG132 (10 μM) to arrest the cells in metaphase. Chromosomes and centrosomes were visualized by staining with DAPI (blue) and anti-γ-tubulin antibodies (green), respectively. Prometaphase and metaphase cells were counted. Values are the means and standard deviations from three independent experiments of over 100 mitotic cells of each type. **P<0.01; ***P<0.001. 3194 Journal of Cell Science 122 (17)

activity of TACC2, we used an inhibitor of kinesin motor protein in the perinuclear cytoplasmic region (Gergely et al; 2000a; Lee et Eg5, named monastrol, which blocks separation in al., 2001). In agreement with these data, overexpressed TACC2(788- mitosis without affecting microtubule formation (Kapoor et al., 996) was aggregated in the perinuclear region (supplementary 2000). We then examined specific microtubule lengths based on material Fig. S4, right). During our analysis of exogenously the diameters of β-tubulin fluorescent zones around mono-asters. expressed TACC2, we found that FLAG-tagged TACC2(864-989), Measurement of the largest diameters of the monopolar spindles lacking a short stretch of the coiled-coil sequences, showed the same revealed that TACC2-depleted HeLa cells had shorter asters than localization as endogenous TACC2 (supplementary material Fig. control cells (Fig. 3A), suggesting that TACC2 is involved in stable S3A). Rabbit polyclonal antibodies against TACC2, designated anti- microtubule formation. To further examine whether TACC2 TACC antibodies, recognized endogenous TACC2 and exogenously enhances microtubule elongation, we performed a microtubule expressed TACC2(788-996), but not TACC2(864-989) (Gergely et regrowth assay (Fig. 3B). Briefly, cells were chilled on ice to al., 2000a) (supplementary material Fig. S2A,B). Interestingly, depolymerize the mitotic microtubules, returned to 37°C for 1 to 3 overexpression of FLAG-TACC2(864-989) disrupted the minutes to allow microtubule recovery, and then fixed for centrosomal localization of endogenous TACC2 (supplementary immunofluorescence analysis to assess the microtubule material Fig. S3B), suggesting that TACC2(864-989) has a organization. Control cells produced robust microtubule asters in dominant-negative effect on cellular TACC2. The localizations of 1 minute, and nascent spindles were detected after 3 minutes, under TACC1 and TACC3 were not affected by TACC2(864-989) (data the regrowth conditions (Fig. 3B, upper). By contrast, there was not shown). little aster formation in TACC2 knockdown cells, since only short To confirm the specificity of the mitotic defects induced by loss and disorganized microtubules were detected even after 3 minutes of TACC2 function, we expressed TACC2(864-989) and examined under the regrowth conditions (Fig. 3B, lower). These results suggest its effects on mitotic function in HeLa cells. Expression of that microtubule stabilization depends on TACC2 function. TACC2(864-989) induced mitotic abnormalities (supplementary To clarify the consequences of TACC2 knockdown, we visualized material Fig. S3C,D), similar to T-antigen-expressing or TACC2- microtubules relative to centrosomes and mitotic chromosomes (Fig. knockdown cells (Fig. 1A, Fig. 2B). TACC2(864-989)-expressing 3C). Compared with control cells, the mitotic spindles (detected by cells exhibited a significantly higher incidence of abnormal nuclei staining for β-tubulin) and centrosome position relative to (supplementary material Fig. S3C). Furthermore, we observed chromosomes were disorganized in TACC2 knockdown cells. We accumulation of prometaphase cells expressing TACC2(864-989) then checked whether TACC2 knockdown affects progression in (supplementary material Fig. S3D). The proportion of prometaphase mitosis (Fig. 3D). The cells were presynchronized, treated with cells among mitotic cells increased by twofold, following functional nocodazole and then released into metaphase in the presence of a inhibition of TACC2. Collectively, these results suggest that proteasome inhibitor, MG132, which inhibits the metaphase- inactivation of TACC2 by T antigen or the dominant-negative form anaphase transition. Both T-antigen-expressing cells and TACC2- of TACC2 induces mitotic abnormalities. knockdown cells progressed slowly and were delayed at TACC domain-containing proteins in other eukaryotic systems prometaphase, while most control cells entered metaphase. have been shown to recruit the microtubule-associated proteins Furthermore, expression of TACC2(864-989), which has an chTOG (colonic and hepatic tumor-overexpressing gene)

Journal of Cell Science inhibitory effect on cellular TACC2, induced similar mitotic homologues to centrosomes and mitotic spindles (Kinoshita et al., abnormalities (supplementary material Figs S2 and S3). 2005; Lee et al., 2001). In HeLa cell, endogenous chTOG was Exogenously expressed full-length TACC2 or the TACC domain detected in the centrosomes and mitotic spindles (supplementary of this protein was previously reported to show polymer formation material Fig. S4A, left), and was recruited to exogenously expressed

Fig. 4. T antigen targets TACC2 to induce mitotic defects. (A) Localization of GFP-fused TACC2Δ(788- 863). HeLa cells expressing GFP-TACC2⌬(788-863) (green) were stained with anti-γ-tubulin antibodies (red) and DAPI (blue). Similar to endogenous TACC2 (supplementary material Fig. S1C), TACC2Δ(788-863) was localized to centrosomes and mitotic spindles. (B) Overexpression of TACC2Δ(788-863) suppresses microtubule defects induced by T antigen. As described in Fig. 3A, transfected HeLa cells were treated with 100 μM monastrol for 6 hours. The diameter of the mono-asters were measured. Values are the means and standard deviations from three independent experiments of more than 100 cells of each type. **P<0.01. T antigen interacts with TACC2 3195

TACC domain of TACC2 protein (supplementary material Fig. S4A, TACC2, we performed a microtubule elongation assay using right). However, the localization of chTOG was maintained even monastrol-treated HeLa cells. As shown in Fig. 3A, the ability of in TACC2-depleted unstable mitotic spindles (supplementary TACC2 to promote microtubule assembly can be quantitatively material Fig. S4B), suggesting that TACC2 stabilizes microtubule assessed by measuring the diameters of the monopolar spindles. T- independently of chTOG (Cassimeris and Morabito, 2004; Gergely antigen-expressing cells had shorter asters than control cells (Fig. et al., 2003; Holmfeldt et al., 2004). In addition, TACC2 knockdown 4B), as did the TACC2-depleted cells (Fig. 3A), providing the affected microtubule organization to some degree in interphase cells evidence that T antigen abrogates microtubule assembly. (supplementary material Fig. S2C), suggesting that TACC2 might Importantly, the effect of T antigen on microtubule formation was stabilize microtubules throughout the . suppressed by co-expressing TACC2Δ(788-863) to the control levels (Fig. 4B). The expression of TACC2Δ(788-863) alone did not T antigen targets TACC2 to induce mitotic defects increase the diameter of the spindles (data not shown). We further To prove that the interaction between T antigen and TACC2 is confirmed that TACC2Δ(788-863) interacted with T antigen in the biologically important, we tested whether the effect of T antigen cells, using an immunoprecipitation analysis (supplementary on microtubule formation is suppressed by overexpressing TACC2. material Fig. S4C). Therefore, based on the interaction between T As previously reported (Gergely et al., 2000; Lee et al., 2001), the antigen and TACC2, these data suggest that TACC2Δ(788-863) exogenous expression of full-length TACC2 or TACC2(788-996) rescues T antigen-induced short or unstable microtubules. induced cytoplasmic aggregate formation (supplementary material Fig. S4A). Since FLAG-TACC2(864-989) had the proper N-terminal-deleted T antigen that interacts with TACC2 causes localization and did not cause cytoplasmic aggregates mitotic defects (supplementary material Fig. S3A), the use of TACC2Δ(788-863), We then checked the direct binding of TACC2 to various T antigen full-length TACC2 lacking this small region, enabled us to perform mutants (supplementary material Fig. S5A). Maltose-binding protein the rescue experiment. GFP-fused TACC2Δ(788-863) was localized (MBP) pull-down experiments revealed that ΔC1 (amino acids 1- to mitotic spindle, similar to endogenous TACC2 (Fig. 4A). To 487) and ΔC2 (amino acids 1-250) as well as full-length T antigen demonstrate a functional relationship between T antigen and bound the TACC domain of TACC2. For this reason, we had Journal of Cell Science Fig. 5. N-terminal-deleted T antigen that interacts with TACC2 causes mitotic defects. (A) Prometaphase delay induced by a T antigen(250-708) that minimally interacts with TACC2. HeLa cells were analyzed by staining with anti-γ-tubulin antibodies and DAPI to determine prometaphase cells in mitosis. T antigen(250-708) can interact with TACC2 but not Bub1. Values are the means and standard deviations from three independent experiments of over 200 mitotic cells. **P<0.01; ***P<0.001. (B) Time-lapse microscopy analysis of HeLa cells expressing GFP-fused histone H2B. Control cells show normal mitosis (upper panels), whereas TACC2 knockdown or T antigen(250-708)- expressing cells exhibit prolonged mitosis with unusual chromosome movements (middle and bottom panels, respectively). Lagging chromosomes and bi-nucleated cells are indicated with white and red arrows, respectively. (C) Complex formation by T antigen and endogenous TACC2. Immunoprecipitation in HeLa cells expressing FLAG-tagged T antigen was performed with the indicated antibodies. The input shows 10% of each lysate. K.D., knockdown; TAg, T antigen. 3196 Journal of Cell Science 122 (17)

difficulties in narrowing down the region of T antigen that interacts (supplementary material Fig. S6A). Control cells stably maintained with TACC2 and in generating a mutant of T antigen that is chromosome numbers (2n), suggesting that chromosomal specifically defective for TACC2 binding. Since mutation in the inheritance during mitosis is regulated. By contrast, TACC2 region containing amino acids 89-97 of T antigen caused the loss knockdown cells exhibited not only the normal complement of of binding to mitotic checkpoint protein Bub1 (Cotsiki et al., 2004), chromosomes (2n) but also some cells had 4n, together with loss we used N-terminally deleted T antigen (amino acids 250-708), or gain of a few chromosomes, indicating that TACC2-depleted cells which can interact with TACC2 but not Bub1 (Fig. 5A). have unstable chromosome transmission. In addition, T-antigen- Approximately 20% of control HeLa cells were normally in expressing cells showed similar patterns of chromosomal changes, prometaphase during mitosis, whereas expression of T antigen together with an increase in hypodiploid chromosomes (<2n). The markedly increased the number of delayed prometaphase cells. effect of T antigen might be underestimated probably because of Similar to the case for TACC2 knockdown, T antigen(250-708) various expression levels of this protein in the cells studied. These comparably increased such prometaphase cells. In addition, we data suggest that TACC2 dysfunction induces chromosomal found that a part of T antigen(250-708)-expressing cells or TACC2 instability. knockdown cells tended to undergo apoptosis. To determine whether Abnormally nucleated cells as a consequence of mitotic defects the increase in delayed prometaphase cells is due to short or unstable were characterized by the appearance of multiple nuclei and/or microtubules, we checked the elongation of monastrol-induced micronuclei (Fig. 1A, Fig. 2B and supplementary material Fig. S3C). monopolar spindles in T-antigen(250-708)-expressing cells. Since nuclear organization is modified in many cancers and Expression of T-antigen(250-708) resulted in the formation of transformed cells (Zaidi et al., 2007), we examined whether T shorter spindles (supplementary material Fig. S5B), similar to the antigen affects nuclear organization in interphase (supplementary effect of TACC2 knockdown or T antigen expression (Fig. 3A, Fig. material Fig. S6B). Immunofluorescence analysis was performed 4B). Thus, the T antigen(250-708) that preferentially interacts with using specific antibodies against various nuclear structural markers. TACC2 can induce mitotic abnormalities. Interestingly, in T-antigen-expressing cells the nuclear envelope To further investigate the dynamics in cell-cycle progression, we became irregular and had multiple segments, compared with control carried out a time-lapse imaging analysis of HeLa cells stably cells. We further found that the nuclear envelope was discontinuous expressing GFP-fused histone H2B (Fig. 5B). In control cells, the in asymmetrically divided micronuclei, suggesting that such time between prometaphase onset and anaphase entry was incomplete nuclear envelope formation may cause loss of DNA approximately 31±9 minutes. By contrast, TACC2 knockdown cells content, thereby leading to aneuploid karyotypes. Similar results and T-antigen(250-708)-expressing cells exhibited unusually slow were found in TACC2 knockdown cells (data not shown). Taken progression of prometaphase (53±23 minutes and 50±21 minutes, together, interaction of T antigen with TACC2 may promote respectively). Cells expressing full-length T antigen spent about abnormal mitotic defects as a result of microtubule dysfunction, 39±12 minutes on the transition from prometaphase to anaphase leading to chromosomal instability and nuclear structural entry (data not shown), suggesting that full-length T antigen disorganization. expression does not cause significant mitotic delays, compared with To finally demonstrate complex formation by T antigen and TACC2 knockdown or T antigen(250-708) expression, probably TACC2 in vivo, we performed an immunoprecipitation analysis in

Journal of Cell Science because of the failure of the mitotic checkpoint. Importantly, TACC2 HeLa cells (Fig. 5C). FLAG-tagged T antigen was expressed in the knockdown or T antigen(250-708) expression induced chromosome cells and immunoprecipitated by anti-TACC2 and anti-FLAG missegregation, leading to the appearance of lagging chromosomes antibodies. Western blot analysis revealed that endogenous TACC2 (white arrows in Fig. 5B) and bi-nucleated cells (red arrows in Fig. was present in immunoprecipitates with FLAG-T antigen, but absent 5B); the latter probably arose because of incomplete cytokinesis. from the control. T antigen was also detected in immunoprecipitates Analysis of more than 10 live images for each cell type (control with TACC2, suggesting that T antigen complexes with TACC2. cells, TACC2 knockdown cells, T antigen(250-708)-expressing cells Collectively, our data show that (1) T antigen directly interacts with and T antigen-expressing cells) indicated that T antigen(250-798) TACC2, (2) T-antigen-induced microtubule destabilization is caused similar mitotic defects to those induced by TACC2 suppressed by overexpressing TACC2Δ(788-863), and (3) mitotic knockdown (data not shown). Characteristic movements of mitotic defects are caused by T antigen(250-708), which minimally interacts chromosomes were observed as follows: (1) condensed with TACC2, suggest the possibility that T antigen inhibits TACC2 chromosomes were aligned on the metaphase plate, segregated once function, leading to mitotic errors. to opposite poles, then collapsed to one pole and repeatedly moved as a pendulum between the poles (Fig. 5B, middle panel); and (2) Discussion after alignment of most chromosomes on the metaphase plate, they This study has identified the centrosome and mitotic spindle- were spread and aligned on the metaphase plate again, followed by localizing protein TACC2 as a novel T antigen target. Our data their segregation in the presence of lagging chromosomes (Fig. 5B, suggest that, besides known anti-tumor proteins such as p53 and lower panel). Furthermore, it has been reported that multiple RB, T antigen selectively interacts with TACC2 to disrupt mitotic chromosomal aberrations are observed in T-antigen-transformed spindle formation possibly toward cell transformation. These cells (Chang et al., 1997; Ray et al., 1990; Stewart and Bacchetti, findings represent the first evidence that a viral oncoprotein can 1991; Woods et al., 1994), and that nuclear organization is modified directly target the mitotic spindle apparatus to induce chromosome in many cancers and transformed cells (Zaidi et al., 2007). To instability, and that TACC2 is involved in stabilizing microtubules elucidate the consequence of the mitotic errors, we examined in mitosis. Perturbation of p53 and RB was reported to be required, whether TACC2 knockdown and T antigen affect chromosome but not sufficient, for the transforming activity of T antigen transmission during mitosis and nuclear organization in interphase (Sachsenmeier and Pipas, 2001). In addition, inhibition of RB and (supplementary material Fig. S6). We prepared chromosome spreads Bub1 by T antigen was reported to induce mitotic defects and from HeLa cells and counted the number of chromosomes per cell chromosomal instability (Cotsiki et al., 2004; Quartin et al., 1994; T antigen interacts with TACC2 3197

Sotillo et al., 2007; Woods et al., 1994). Our study has further throughout the cell cycle (supplementary material Fig. S4D). revealed that T antigen(250-708), which does not interact with these Recent studies have reported that TACC2 might be involved in proteins, can induce mitotic defects including delayed progression transcriptional control, via associations with histone through mitosis, suggesting that inhibition of TACC2 by T antigen acetyltransferases and chromatin remodeling factors (Gangisetty et causes mitotic defects independently of RB and Bub1. Thus, al., 2004). Our data do not exclude the possibility that T antigen impairment of both TACC2 and Bub1 by T antigen might affects the function of TACC2 in interphase as well as mitosis. Taken synergistically enhance mitotic defects and avoid mitotic checkpoint together, our findings provide insights into the molecular basis of response. the chromosomal instability and nuclear structural disorganization The chromosomal abnormalities such as aneuploidy and promoted by T antigen. polyploidy are common characteristics of naturally occurring human cancers, which result from incorrect cell division (Draviam Materials and Methods et al., 2004; Kops et al., 2005; Weaver and Cleveland, 2006). Yeast two-hybrid screening Yeast strain AH109 carrying pAS2-1-T antigen (amino acids 250-708) was Recently, several lines of evidence have indicated that chromosomal transformed with mouse E17 whole embryo cDNA libraries constructed in pACT2 instability is a crucial early event during and might (Clontech). Plasmids harboring cDNA were recovered from both histidine- and be a cause, rather than a consequence, of cellular transformation adenine-positive colonies and subjected to DNA sequencing. (Weaver and Cleveland, 2006). Although this idea is still under Plasmid construction debate, T antigens of polyomaviruses are likely to provide a clue cDNAs encoding T antigen and TACC2 were cloned into pcDNA3 to create the in improving our understanding of chromosomal instability. The following plasmids: pcDNA3-FLAG-T antigen, pcDNA3-FLAG-TACC2(788-996), infected oncoviruses might actively disturb the mitotic spindle pcDNA3-FLAG-TACC2(864-989), pcDNA3-EGFP-T antigen, pcDNA3-EGFP-T formation of the host cells, possibly leading to the increase of cell antigen(250-708), pcDNA3-EGFP-TACC2(788-996), pcDNA3-EGFP-TACC2(864- 989) and pcDNA3-EGFP-TACC2Δ(788-863). death. However, chromosomal instability induced by T antigen might produce some transformed cells from the small numbers of Cell culture surviving cells as the benefit to further viral replication. HeLa and CHO cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s minimum essential medium and Ham’s F-12 nutrient medium (Sigma) supplemented Human TACC family proteins have been implicated in with 10% (v/v) heat-inactivated fetal bovine serum. (Gergely, 2002; Raff, 2002). Although knockout of TACC2 in mice showed no obvious phenotypes (Schuendeln et al., 2004), this might Transfection and RNA interference not contradict our data that abrupt loss of TACC2 induced mitotic Cells were transfected with plasmid DNAs using FuGENE 6 and FuGENE HD (Roche Applied Science). For siRNA experiments, cells were transfected with siRNA duplex abnormalities in somatic cells. There could be developmental oligonucleotides using RNAiMAX (Invitrogen) or co-transfected with siRNA duplex complements by other cellular factors in TACC2-deficient mice. oligonucleotides and pcDNA3-EGFP using Lipofectamine 2000 (Invitrogen). Furthermore, TACC2 is likely to be the sole target for T antigen Knockdown cells were analyzed 72 hours after each transfection. The siRNA duplexes among the TACC family members. In addition, we have found used were designed to target the mRNAs encoding human and Chinese hamster TACC2. The sequences of the siRNAs (Japan Bioservice) were as follows: human functional differences between human TACC2 and TACC domain- TACC2, 5Ј-AGGACGCUGGAGCAGAAGA-3Ј and Chinese hamster TACC2, 5Ј- containing proteins in other species. The TACC proteins in AGAACGCUGGAGCAGAAGA-3Ј. The results were confirmed using more than Caenorhabditis, Drosophila and Xenopus were reported to stabilize two siRNA duplex oligonucleotides against different sequences. siRNA against firefly luciferase GL3 was used as a control, as described previously (Saitoh et al., 2006).

Journal of Cell Science microtubules by recruiting ZYG9, Msps and XMAP215, respectively, which are homologues of human chTOG, to Protein expression centrosomes and mitotic spindles (Gergely et al., 2003; Lee et al., Human cDNAs encoding TACC1 (amino acids 605-805), TACC2 (amino acids 788- 2001; Peset et al., 2005). XMAP215 can bind microtubules and 996) and TACC3 (amino acids 638-838) were cloned into pET28a (Novagen). The cDNA encoding TACC2(788-996) was also cloned into pMAL-c2X (New England enhance microtubule growth at the plus end (Kinoshita et al., 2005). Biolabs). A cDNA encoding T antigen(250-708) was cloned into pGEX4T-1 However, human chTOG was able to localize to centrosomes and (Amersham Bioscience). Bacterial expressions of His-TACC2(788-996), MBP- mitotic spindles in TACC2-depleted cells but failed to stabilize the TACC2(788-996), His-p53 and GST-T antigen(250-708) were similarly carried out. microtubules (supplementary material Fig. S4B). In human cells, Antibodies chTOG is likely to be recruited by redundant functions of TACC1 Anti-human TACC2 polyclonal antibodies were generated by immunizing a rabbit and TACC3. TACC2 might stabilize microtubule formation, with His-tagged TACC2(788-996), and affinity purified using TACC2 coupled to independently of chTOG. Since T antigen forms a complex and HiTrap Protein G HP (GE Healthcare). Rabbit anti-human TACC1, TACC2 and TACC3 antibodies, and rabbit anti-chTOG antibodies were described previously coexists with TACC2 at mitotic spindles (Fig. 1), we speculate that (Gergely et al., 2000; Kinoshita et al., 2005). Other antibodies were obtained from TACC2 stabilizes microtubule formation together with as-yet the following sources: mouse anti-T antigen (BD Biosciences PharMingen), mouse unknown regulatory proteins, and that T antigen inhibits their anti-FLAG (M5; Sigma), mouse anti-His (Qiagen), mouse anti-GST (DAKO), mouse anti-β-tubulin (Sigma), mouse anti-γ-tubulin (Sigma), rabbit anti-γ-tubulin (Santa Cruz cooperation. Our data in Fig. 1A and Fig. 2B showed that either Biotechnology), and mouse anti-lamin A/C and rabbit anti-RanBP2 antibodies (Saitoh the expression of T antigen or the knockdown of TACC2 induced et al., 2006). abnormally nucleated cell phenotypes. In comparison with these conditions, the combination of T antigen expression and TACC2 Immunoprecipitation and pull-down experiments knockdown tended to have a somewhat additive effect on the For immunoprecipitation experiments, cells were lysed with RIPA buffer [50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1.5 mM MgCl2, 2.5 mM ZnCl2, 0.5% Triton X-100, occurrence of the abnormal nucleation (supplementary material Fig. 10% (v/v) glycerol] supplemented with 1 mM Na3VO4 and protease inhibitors for S7). In addition, the data in Fig. 4B showed that overexpression of 30 minutes on ice. After centrifugation for 30 minutes, the supernatant was collected, μ TACC2Δ(788-863) rescued T antigen-induced short or unstable incubated with specific antibodies for 1 hour at 4°C, mixed with 20 l of protein A/G-agarose beads (Amersham Bioscience) and further incubated for 1 hour. The microtubules, although we have not found that T antigen-induced beads were washed, and bound proteins were detected by western blot analysis. For abnormal nucleation was suppressed by TACC2Δ(788-863) (data GST pull-down assays, bacterially expressed GST and GST-fused T antigen (1 μg) not shown), probably because of the involvement of multiple factors were immobilized on glutathione-agarose beads and incubated with His-tagged TACC2 proteins (1 μg) and His-tagged p53 protein in a buffer consisting of 0.1% in the abnormally nucleated cell phenotype. Moreover, we have Triton X-100, 50 mM Hepes pH 7.5, 50 mM NaCl, 5% (v/v) glycerol, 1 mM found that TACC2 contributes to microtubule stabilization dithiothreitol and protease inhibitors for 1 hour at 4°C. The beads were washed, and 3198 Journal of Cell Science 122 (17)

bound proteins were detected by western blot analysis. Maltose-binding protein (MBP) Gaynor, A. M., Nissen, M. D., Whiley, D. M., Mackay, I. M., Lambert, S. B., Wu, G., pull-down assays were carried out in a similar manner, except that the supernatant Brennan, D. C., Storch, G. A., Sloots, T. P. and Wang, D. (2007). Identification of a was incubated with bacterially expressed MBP-fusion proteins (1 μg) immobilized novel polyomavirus from patients with acute respiratory tract infections. PLoS Pathog. on Amylose resin (New England Biolabs). 3, e64. Gergely, F. (2002). Centrosomal TACCtics. BioEssays 24, 915-925. Immunofluorescence analysis Gergely, F., Karlsson, C., Still, I., Cowell, J., Kilmartin, J. and Raff, J. W. (2000). The TACC domain identifies a family of centrosomal proteins that can interact with Cells were fixed with methanol at –20°C for 3 minutes, washed with PBS, blocked microtubules. Proc. Natl. Acad. Sci. USA 97, 14352-14357. with 0.5% bovine serum albumin in PBS, incubated with specific primary antibodies Gergely, F., Draviam, V. M. and Raff, J. W. (2003). The ch-TOG/XMAP215 protein is for 1 hour at room temperature, and then incubated with appropriate secondary essential for spindle pole organization in human somatic cells. Dev. 17, 336-341. antibodies for 1 hour. The following secondary antibodies were used: Alexa-Fluor- Hein, J., Boichuk, S., Wu, J., Cheng, Y., Freire, R., Jat, P. S., Roberts, T. M. and 488-conjugated donkey anti-mouse IgG (Molecular Probes); Alexa-Fluor-488- Gjoerup, O. V. (2009). Simian virus 40 large T antigen disrupts genome integrity and conjugated donkey anti-rabbit IgG (Molecular Probes); Cy3-conjugated donkey anti- activates a DNA damage response via Bub1 binding. J. Virol. 83, 117-127. mouse IgG (Jackson ImmunoResearch); and Cy3-conjugated donkey anti-rabbit IgG Holmfeldt, P., Stenmark, S. and Gullberg, M. (2004). Differential functional interplay (Jackson ImmunoResearch). Cells were counterstained with DAPI (1 mg/ml) before of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis. EMBO mounting. For immunofluorescence experiments analyzing nuclear structure, cells J. 23, 627-637. were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Images Imperiale, M. J. (2000). The human polyomaviruses, BKV and JCV: molecular were acquired with a Model IX71 microscope (Olympus) and analyzed using Lumina pathogenesis of acute disease and potential role in cancer. Virology 267, 1-7. Vision (Mitani Corporation). Kapoor, T. M., Mayer, T. U., Coughlin, M. L. and Mitchison, T. J. (2000). Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic Microtubule regrowth assay and monastrol treatment kinesin, Eg5. J. Cell Biol. 150, 975-988. Kinoshita, K., Noetzel, T. L., Pelletier, L., Mechtler, K., Drechsel, D. N., Schwager, A., For microtubule regrowth assays, cells were synchronized at mitosis by double thymidine Lee, M., Raff, J. W. and Hyman, A. A. (2005). Aurora A phosphorylation of blocks and released, placed on ice for 30 minutes to depolymerize the microtubules TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis. and then incubated in warm medium (37°C) for 1, 2 or 3 minutes to allow microtubule J. Cell Biol. 170, 1047-1055. recovery (Petretti et al., 2006). The cells were fixed with methanol at –20°C and stained Kops, G. J., Weaver, B. A. and Cleveland, D. W. (2005). On the road to cancer: aneuploidy β with anti- -tubulin antibodies. For monastrol (an inhibitor of kinesin motor protein and the mitotic checkpoint. Nat. Rev. Cancer 5, 773-785. Eg5) treatment, TACC2-depleted and T-antigen(250-708)-expressing cells were Lee, M. J., Gergely, F., Jeffers, K., Peak-Chew, S. Y. and Raff, J. W. (2001). synchronized by double thymidine blocks. At 6 hours after release of the blocks, 100 Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule μM monastrol was added and the cells were incubated for a further 6 hours. behaviour. Nat. Cell Biol. 3, 643-649. Marumoto, T., Honda, S., Hara, T., Nitta, M., Hirota, T., Kohmura, E. and Saya, H. Time-lapse imaging (2003). Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa HeLa cells stably expressing GFP-tagged histone H2B were grown on 35-mm glass- cells. J. Biol. Chem. 278, 51786-51795. based dishes (IWAKI) (Marumoto et al., 2003). Fluorescence and DIC time-lapse Moens, U., Van Ghelue, M. and Johannessen, M. (2007). Oncogenic potentials of the analyses were performed using a microscope (Model XI70; Olympus). The camera, human polyomavirus regulatory proteins. Cell Mol. Life Sci. 64, 1656-1678. shutters and filter wheel were controlled by MetaMorph imaging software (Universal Peset, I., Seiler, J., Sardon, T., Bejarano, L. A., Rybina, S. and Vernos, I. (2005). Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis. Imaging), and images were collected every 5 minutes with 50-msecond exposures. J. Cell Biol. 170, 1057-1066. Through-focus z-series stacks consisting of three frames were acquired at each time Petretti, C., Savoian, M., Montembault, E., Glover, D. M., Prigent, C. and Giet, R. point. (2006). The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation. EMBO Rep. 7, 418-424. We are grateful to the members in our laboratory for helpful Poulin, D. L. and DeCaprio, J. A. (2006). Is there a role for SV40 in human cancer? J. discussions. We thank Jordan W. Raff (The Gurdon Institute, Clin. Oncol. 24, 4356-4365. Quartin, R. S., Cole, C. N., Pipas, J. M. and Levine, A. J. (1994). 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