A novel big protein TPRBK possessing 25 units of TPR motif is essential for the progress of mitosis and cytokinesis

Gene 511 (2012) 202–217

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A novel big protein TPRBK possessing 25 units of TPR motif is essential for the progress of mitosis and cytokinesis Tomohiro Izumiyama a, Shinsei Minoshima b, Tetsuhiko Yoshida c, Nobuyoshi Shimizu a,⁎ a b c

Advanced Research Center for Genome Super Power, Keio University, Tsukuba, Japan Department of Photomedical Genomics, Basic Medical Photonics Laboratory, Medical Photonics Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Institute for Advanced Sciences, Toagosei Co., Ltd., Tsukuba, Japan

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Article history: Accepted 20 September 2012 Available online 1 October 2012 Keywords: 22q12.1 TetratricoPeptide Repeat (TPR) Centrosome Aurora kinase B Cell division Midbody

a b s t r a c t Through the comprehensive analysis of the genomic DNA sequence of human chromosome 22, we identified a novel gene of 702 kb encoding a big protein of 2481 amino acid residues, and named it as TPRBK (TPR containing big gene cloned at Keio). A novel protein TPRBK possesses 25 units of the TPR motif, which has been known to associate with a diverse range of biological functions. Orthologous genes of human TPRBK were found widely in animal species, from insecta to mammal, but not found in plants, fungi and nematoda. Northern blotting and RT-PCR analyses revealed that TPRBK gene is expressed ubiquitously in the human and mouse fetal tissues and various cell lines of human, monkey and mouse. Immunofluorescent staining of the synchronized monkey COS-7 cells with several relevant antibodies indicated that TPRBK changes its subcellular localization during the cell cycle: at interphase TPRBK locates on the centrosomes, during mitosis it translocates from spindle poles to mitotic spindles then to spindle midzone, and through a period of cytokinesis it stays on the midbody. Co-immunoprecipitation assay and immunofluorescent staining with adequate antibodies revealed that TPRBK binds to Aurora B, and those proteins together translocate throughout mitosis and cytokinesis. Treatments of cells with two drugs (Blebbistatin and Y-27632), that are known to inhibit the contractility of actin–myosin, disturbed the proper intracellular localization of TPRBK. Moreover, the knockdown of TPRBK expression by small interfering RNA (siRNA) suppressed the bundling of spindle midzone microtubules and disrupted the midbody formation, arresting the cells at G2 + M phase. These observations indicated that a novel big protein TPRBK is essential for the formation and integrity of the midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis during mammalian cell cycle. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Mammalian cell cycle is typically divided into five distinct stages (G0, G1, S, G2 and M). G0 is a special stage where a cell is resting until receiving the growth stimuli. G1 and G2 are gaps placed before and after DNA synthesis (S), during which DNA replication or chromosome duplication takes place, and then cell cycle proceeds to mitosis (M). M is a relatively short period but its dynamic events are precisely Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; kb, kilobase(s); mRNA, messenger RNA; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription PCR; UTR, untranslated region; nt, nucleotide(s); NCBI, National Center for Biotechnology Information; aa, amino acid(s); kDa, kilodalton(s); BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ig, immunoglobulin(s); DNase, deoxyribonuclease; RNase, ribonuclease; oligo, oligodeoxyribonucleotide; DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, small interfering RNA. ⁎ Corresponding author at: Advanced Research Center for Genome Super Power, Keio University, 2 Ohkubo, Tsukuba-shi, Ibaraki, 300-2611, Japan. Fax: +81 29 865 0797. E-mail address: [email protected] (N. Shimizu). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.09.061

defined as four sub-phases (prometa-, meta-, ana-, and telo-phases), and then cytokinesis takes place to accomplish cell division. During M, duplicated sets of chromosomes are segregated and pulled toward spindle poles with the aid of spindle microtubules, which are mainly the bundles of α-tubulin and β-tubulin. At the last phase of M (telophase), massive spindle microtubules are assembled to form a central spindle in the spindle midzone (McCollum, 2004; Oegema and Mitchison, 1997). Then, the formation of the central spindle initiates the cytokinesis. The central spindle is a conspicuous network of spindle microtubules and it is formed in cooperation with various proteins, including MKLP1 (mitotic kinesin-like protein 1, a component of the Centralspindlin complex), MKLP2, PRC1 (protein regulator of cytokinesis 1), KIF4 (kinesin family member 4), KIF18 and so on (D'Avino et al., 2005; Gruneberg et al., 2004; Hutterer et al., 2009). Also, multiple kinds of protein kinases (such as Aurora B, PLK1, and CDC14A) accumulate in the spindle midzone to regulate several key components through the proteinphosphorylation. Then, a cleavage site is positioned precisely on the cell membrane, and the contractile ring is organized by assembling a few proteins F-actin, myosin II, and Anillin (actin-binding protein) (Glotzer, 2005). Then, the cleavage furrow is formed and its cellular

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ingression occurs. The contractility of contractile ring is finely tuned by RhoA (Niiya et al., 2006; Piekny et al., 2005) which is activated by ECT2 and inactivated by MgcRacGAP (another component of the Centralspindlin complex) (Li et al., 2010). During cytokinesis, two daughter cells are connected transiently with a projection-like structure called midbody, which is created by dynamic interaction of contractile apparatus with plasma membrane and it acts as a bridge between two newly forming daughter cells, and prior to cell abscission, overlapping microtubules are condensed in the middle of midbody to generate a small electron-dense structure called Flemming body (Matheson et al., 2005; Otegui et al., 2005; Paweletz, 1967; Straight and Fieid, 2000). The formation of midbody and Flemming body is dependent greatly on the harmonious interaction between contractility of contractile ring and bundling of spindle midzone microtubules. At the final stage of cytokinesis, two connecting daughter cells are precisely split at Flemming body. It is further shown that ECT2 and Annexin11 are associated with midbody formation, whereas Cep55 (centrosomal protein of 55 kDa), ESCRT (endosomal sorting complex required for transport) and ALIX (ALG-2 (apoptosis-linked gene 2)-interacting protein X) are required for Flemming body formation (Morita et al., 2007; Simon et al., 2008; Tomas et al., 2004). Moreover, numerous other proteins are shown to assemble on Flemming body. Those proteins include Cep55, Centralspindlin complex, membrane-fusion-inducing SNARE components (syntaxin2 and endobrevin) which are required for a final step in cell cleavage (Low et al., 2003), Septin, membrane–vesicle–tethering Exocyst complex, FIP3 (family of Rab11-interacting protein 3) and Rab11 which target the recycling endosomes to the midbody of dividing cells (Simon and Prekeris, 2008), Centriolin which anchors SNARE and Exocyst complexes at the midbody and integrates membrane-vesicle fusion with cell abscission (Gromley et al., 2005), ESCRT, ALIX and IQGAP1 (IQ motif containing GTPase activating protein 1), and they are shown to facilitate membrane trafficking and the fusion of endocytic vesicles that are brought by recycling endosomes (Prekeris and Gould, 2008). Consequently, those proteins play cooperative roles in closing the split-ends of Flemming body at the final stage of cell abscission or completion of cytokinesis (Prekeris and Gould, 2008). Despite those detailed observations, there are still many unsolved problems on the dynamic events of cell cycle, for instance the precise molecular mechanism of cell abscission. As briefly summarized above, enormous sorts of proteins have been associated with different aspects of mammalian cell division cycle, yet our understanding on the molecular organization and genetic regulation of the entire cell division process is still limited and remains to be further investigated. In this paper we reported a novel gene, TPRBK (TPR containing big gene cloned at Keio) of 702 kb, encoding a big protein of 2481 amino acid residues with 25 units of TPR (TetratricoPeptide Repeat) motif. We will present various data at cellular and molecular levels, which clearly indicate that a novel big protein TPRBK changes its subcellular localization, from spindle poles (centrosomes) to midbody, in association with other proteins during cell division. Moreover, it was proven that TPRBK is essential for the formation and integrity of midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis.

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RP11-329J7 (accession no. AL118497) and RP11-436C9 (accession no. AL121825) (Asakawa et al., 1997; Dunham et al., 1999). The repetitive elements in the genomic sequences were masked by RepeatMasker2 program (http://ftp.genome.washington.edu/RM/ RepeatMasker.html). The GC and CpG contents were calculated using our own program “base contents” with 200-bp intervals for both window and slide. The exon prediction was performed using GENSCAN (http://genes.mit.edu/GENSCAN.html) (Burset and Guigo, 1996), MZEF (Zhang, 1997) and X-grail version 1.3c (Edward and Richard, 1991). Homology search of nucleotide or amino acid sequences was carried out on the web server of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Ensembl Genome Browser (http://uswest.ensembl.org/index.html) (Altschul et al., 1990). The prediction of protein motifs was carried out using the program SMART (http://smart.embl-heidelberg.de/) (Letunic et al., 2009; Schultz et al., 1998) and Pfam (http://pfam.sanger.ac.uk/ search) (Finn et al., 2008). The multiple alignment of nucleotide or amino acid sequence was performed with CLUSTAL W version 1.83 (Thompson et al., 1994). Drawing of phylogenetic tree and calculating of bootstrap value were performed using the neighbor-joining (N-J) method. Amino acid sequence homology of each TPR motif was calculated by DNASIS version 3.5. 2.2. cDNA cloning Polymerase chain reaction (PCR) was performed using KOD Plus PCR system (TOYOBO) with primer sets shown in Supplementary Table 1. The cDNA cloning was performed using PCR-amplified products from the human or mouse fetal brain cDNA library of Multiple Tissue cDNA Panels (Takara). To determine 5′ ends of the novel genes, the RACE (rapid amplification of cDNA ends) reaction was performed using Marathon Ready human fetal brain cDNA Amplification Kit (Takara). PCR was performed according to the manufacturer's protocol (94 °C for 15 s, 60 °C for 10 s, and 72 °C for 30 s) for 35 cycles in an automated thermal cycler, TRIO-Thermoblock (Biometra). The amplified products were inserted into the HincII site of pUC118 plasmid vector (Takara), and used as the templates for nucleotide sequencing. 2.3. Northern blot hybridization The hybridization probes for human or mouse TPRBK transcripts were amplified using primers described in Supplementary Table 1. Human Multiple Tissue Northern (MTN) Blot membrane and Human Fetal Normal mRNA Northern Blot membrane were purchased from BioChain (USA). Mouse MTN Blot membrane and Mouse Embryo Northern Blot membrane were purchased from Takara (Japan). The probes were labeled with [α- 32P] dCTP using Random Primer Extension Labeling System (NEN life Science). The membranes were hybridized with the radio-labeled probes overnight at 42 °C. Then, they were washed once at 60 °C with 2.0× standard saline citrate (SSC)/0.5% sodium dodecyl sulfate (SDS) and then with 0.5× SSC/0.5% SDS. Autoradiogram was taken with IP plate (Fujifilm) for 48 h, and then hybridization signal was detected by FLA-3000G system (Fujifilm). 2.4. RNA extraction, RT reaction and RT-PCR

2. Materials and methods 2.1. Computer analysis of DNA nucleotide and protein amino acid sequences The genomic DNA sequences re-analyzed in this work were RP3-477H23 (accession no. AL033538), SC22CB-42E1 (accession no. AL035453), CTA-754D9 (accession no. AL050313), RP6-45P1 (accession no. AL035397), CTA-544A11 (accession no. AL023281), CTA-732E4 (accession no. AL008722), RP11-541J16 (accession no. AL080241),

Cultured cells were washed three times with phosphate buffered saline (PBS), lysed with ISOGEN (WAKO) and total RNA was extracted with Ultra Pure DNase/RNase Free Distilled Water (GIBCO/Invitrogen). For RT reaction, 100 ng of total RNA and 100 units of SuperScript III Reverse Transcriptase (Invitrogen) were used. RT-PCR was performed using Expand High Fidelity PCR system (Roche) with primer sets shown in Supplementary Table 1. The PCR condition was 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min, which was repeated for 35 cycles in TRIO-Thermoblock (Biometra).

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2.5. Generation of antisera to TPRBK Antisera were raised against three synthetic peptides of human TPRBK, including N-terminal CVGMVEAAMKSPMRD, middle CGHHDEALAVAERGR and C-terminal CRHNKKEEGVDKLEL. These peptides were conjugated with keyhole limpet hemocyanin (KLH) and used for rabbit immunization. Specificity of the resultant antibodies was checked by Western blotting using cell lysates from HeLa-S3 that overexpress GFP-tagged TPRBK (Fig. S2). Moreover, the specificity was also checked by Western blotting after pre-treatment with synthetic peptide of TPRBK (Fig. S2). Consequently, rabbit antibody raised against the middle peptide of TPRBK was confirmed to be specific to human TPRBK. This antibody was affinity-purified using NHS-activated HP (Amersham Biosciences) and used for all the study. 2.6. Cell culture, synchronization, drug treatments and flow cytometric analysis All the cell lines (HeLa-S3, A431, HEK293, SH-SY5Y, COS-7, NIH3T3) were maintained in Dulbecco's modified Eagle's Medium (DMEM) containing 4500 mg/l D-glucose (WAKO), 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (GIBCO). Cells were incubated at 37 °C with 5% CO2 in a humidified incubator. Synchronization of HeLa-S3 and COS-7 cells was performed by “double thymidine block method” (Hosono et al., 2004). Briefly, cells at exponential growth phase were treated with 2.5 mM thymidine for 16 h, rinsed and kept in thymidine-free medium for 8 h, and then treated again with 2.5 mM thymidine for 16 h. After the second thymidine block, the cells were rinsed and kept in the thymidine-free medium for 2 h. For drug treatments, those double-thymidine-blocked cells were placed in the medium containing 100 μM Blebbistatin or 50 μM Y-27632. After 6 h, cells were fixed and stained with various antibodies (Straight et al., 2003). Flow cytometric cell cycle analysis was performed using Guava easyCyte 8HT (Millipore). Cells were fixed with ice-cold 70% ethanol, stained with Guava Cell Cycle Reagent, and subjected to the flow cytometer. The ratio of each cell cycle phase was calculated using software CytoSoft Guava Cell Cycle (Millipore). 2.7. Western blot analysis with anti-TPRBK antibody After washing three times with PBS, the cultured cells were lysed on ice using the SDS sample buffer (100 mM Tris–HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol) containing 100 U/ml Benzonase (Merck) and clarified by centrifugation at 13,000 ×g for 20 min at 4 °C. The supernatants were processed for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein bands separated on the gel were transferred to a sheet of PVDF (polyvinylidenefluoride) membrane. Then, the membranes were incubated with 3% (w/v) bovine serum albumin (BSA) (SIGMA life science) in PBS containing 0.1% (v/v) Tween 20 (PBST) for 1 h at room temperature. Then, the BSA-blocked membranes were incubated with rabbit anti-TPRBK antibody at 1:500 dilution, followed by goat anti-rabbit IgG antibody conjugated with HRP (Dako Cytomation) at 1:1000 dilution. The immune-reaction was detected by LAS-1000plus enhanced chemiluminescence system with ECL plus (Fujifilm).

antibody (N356; Amersham) at 1:500 dilution, mouse anti-Pericentrin antibody (ab28144; Abcam) at 1:200 dilution, mouse anti-Aurora B antibody (ab10735; Abcam) at 1:200 dilution, mouse anti-PLK1 antibody (627802; BioLegend) at 1:200 dilution, and mouse anti-Eg5 antibody (627701; BioLegend) at 1:200 dilution. Secondary antibodies used were: Alexa Fluor 488-labeled goat anti-mouse antibody (Invitrogen) at 1:400 or 1:1000 dilution and Alexa Fluor 555-labeled goat antirabbit antibody (Invitrogen) at 1:1000 dilution. Triple immunofluorescence assay was done using Alexa Fluor 488-labeled goat anti-mouse antibody (red), Alexa Fluor 555-labeled goat anti-rabbit antibody (green), and Zenon Alexa Fluor 647 mouse labeling kit (pink) (Invitrogen/Molecular Probes). 2.9. Duolink in situ PLA (Proximity Ligation Assay) system Interaction between Aurora B and INCENP or TPRBK and Aurora B was examined by Duolink in situ PLA system (Olink Bioscience). HeLa-S3 cells were synchronized by double thymidine block method and fixed with MeOH/acetone (1:1). These fixed cells were first treated with two distinct rabbit and mouse antibodies specific to target proteins. Then, Duolink in situ PLA probe MINUS and Duolink in situ PLA probe PLUS, which are the secondary antibodies conjugated with oligonucleotides, were added and incubated at 37 °C for 2 h. Then, Duolink Ligation solution which contains two kinds of oligonucleotides was added and incubated at 37 °C for 15 min to facilitate a circular DNA formation. Duolink Amplification solution was added and incubated at 37 °C for 90 min to elongate a single strand DNA by the aid of RCA (rolling circle amplification). Duolink Amplification solution contains fluorescent (or HRP)-labeled oligonucleotides and they can hybridize to the elongated single strand DNA. Finally, the cells were treated with Duolink Detection solution at 37 °C for 60 min and incubated with DAPI (4′,6-diamidino-2-phenylindole) solution at room temperature for 5 min. Fluorescent image was obtained by laser microscope LSM 510 META (Zeiss). 2.10. Immunoprecipitation and Western blot analysis After washing three times with PBS, HeLa-S3 cells were lysed on ice for 15 min with the lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl and 1% NP-40) containing protease inhibitor cocktail tablet (Roche Diagnostics). To the clarified cell lysates, anti-TPRBK or anti-Aurora B (ab2254; Abcam) antibodies coupled to the Protein-G Sepharose 4 Fast Flow (Amersham Biosciences) were added and gently shaken at 4 °C overnight. The Sepharose beads were washed three times with the buffer containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl and 0.05% Tween20 (TBST), and treated with SDS sample buffer at 96 °C for 5 min. The resultant eluate was analyzed on SDS-PAGE, followed by Western blotting. For Western blotting, the following primary antibodies were used: affinity-purified rabbit anti-TPRBK antibody, mouse anti-AuroraB antibody (ab10735; Abcam), mouse anti-PLK1 antibody (627802; BioLegend), mouse anti-PRC1 antibody (629001; BioLegend), and mouse anti-α-tubulin antibody (N356; Amersham). Secondary antibodies used are: goat anti-mouse IgG antibody conjugated to HRP (Dako Cytomation) and goat anti-rabbit IgG antibody conjugated to HRP (Dako Cytomation). 2.11. Small interfering RNA (siRNA) treatments

2.8. Indirect immunofluorescent staining HeLa-S3 or COS-7 cells grown on glass coverslips were fixed and permeabilized with methanol:acetone (1:1) mixture on ice for 15 min and blocked with PBS containing 3% BSA at room temperature for 1 h. The BSA-blocked cells were incubated with various primary antibodies at 4 °C overnight, and then treated with secondary antibodies at room temperature for 2 h. Primary antibodies used were: affinity-purified rabbit anti-TPRBK antibody at 1:500 dilution, mouse anti-α-tubulin

The sequences of several siRNAs (TPRBK siRNA, TPRBK scrambled siRNA, positive control GAPDH siRNA, and negative control eGFP siRNA: Sigma-Aldrich) are listed in Supplementary Table 1. The siRNA treatment was combined with the double thymidine block method. After the first thymidine block for 16 h, the cells were incubated for 2 h in the thymidine-free medium and treated with the previously prepared Oligomer(siRNA)-Lipofectamine ™ 2000 complexes at 120 nM for 6 h. Then, cells were synchronized again by the second

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thymidine block for 16 h and released in the thymidine-free medium for 2 h. Again, the cells were treated with Oligomer-Lipofectamine ™ 2000 complexes for 6 h and released from thymidine block for 16 h (Hosono et al., 2010). Those cells were fixed for immunofluorescent staining or harvested for immunoprecipitation and Western blot analysis. 3. Results 3.1. cDNA cloning, genomic structure, and motif prediction of TPRBK Initially, we focused on the previously predicted gene “KIAA1043” that is located within the band q12.1 of human chromosome 22. This gene was actually mapped between two genes (PITPNB and HS747E24) in the 1.1-Mb genomic DNA region. However, our detailed sequence data analysis using 3 different exon prediction programs (GENSCAN, MZEF, Grail) revealed that an open reading frame (ORF) of the KIAA1043 gene passes over its 5′-end and extends to the neighboring exon, suggesting that this gene might be much larger than the previous prediction. Hence, we performed cloning of the full-length cDNA by repetitive use of RT-PCR and RACE (Rapid amplification of cDNA ends), and eventually we succeeded to obtain a large cDNA clone of 11,743 base pairs (bp). The full-length cDNA contained a 7.5-kb ORF, encoding a protein of 2481 amino acid (aa) residues (Figs. 1A-b and A-c). The genome size of this gene was estimated to be 702 kb, consisting of 23 exons and 22 relatively large introns (Fig. 1A-a). This gene turned out to be much larger than LARGE (583 kb mapped on 22q12.3) that was previously claimed to be the largest gene on human chromosome 22. The motif analysis by computer software SMART revealed that the newly found large protein possesses 25 units of TPR (TetratricoPeptide Repeat) motif in the N-terminal portion (Fig. 1A-c). No obvious motifs or domains were found in the C-terminal portion. The TPR motif is composed of a unique amino acid sequence of 34 aa residues, and it is often found as multiple repeats in various proteins that are associated with a diverse range of biological functions, such as protein folding, transcriptional control, protein transport via mitochondria and peroxisome, protein glycosylation and cell cycle regulation (Blatch and Lassels, 1999; Brinker et al., 2002; Das et al., 1998; Gatto et al., 2000; Kimple et al., 2002; Lubas and Hanover, 2000). We named the newly discovered big gene/protein as TPRBK (TPR containing big gene cloned at Keio) (accession no. AB665284) and attempted to perform its further characterization at molecular and cellular levels. 3.2. Orthologous genes of human TPRBK among various species We carried out a homology search in the NCBI and Ensembl databases using BLAST and Dotter analysis programs, and produced a molecular phylogenetic tree of TPRBK genes (Fig. 1B). The orthologs of human TPRBK were found in various species, including mammal (Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus), Aves (Gallus gallus), amphibian (Xenopus tropicalis), Pisces (Takifugu rubripes, Oryzias latipes), Ascidiacea (Ciona intestinalis), insecta (Drosophila melanogaster, Pyretophorus gambiae, Apis mellifera), but no orthologs were found in nematoda (Caenorhabditis elegans), fungi, plants, and lower organisms (Fig. 1B-a). Comparison of the deduced aa sequence of TPRBK proteins among 13 different species by CLUSTAL W program showed high homologies, e.g. 92% identity between human and mouse, and 75% identity between human and fish (Takifugu) (data not shown). All of these TPRBK proteins possessed many (> 22, mostly 25) units of TPR motif in the N-terminal portion. The molecular phylogenetic tree with a total of 175 TPR units among 7 different species (human, mouse, rat, fugu, fly, mosquito and bee) drawn by N-J plot revealed that those orthologous TPR units form 25 branches, which apparently diverged at an early stage of

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evolution into two groups: a group of 3 motifs (TPR1 to TPR3) and the other group of 22 motifs (TPR4 to TPR25) (Fig. 1B-b). Furthermore, homology analysis of the multiple TPR motifs among nine different species (the above seven plus chicken and medaka) revealed the intriguing molecular features (Figs. 1B-c, d). The pair-wise comparison of every TPR unit (TPR1 through TPR25) between human and other species showed high homology (40–100%), whereas the pair-wise comparison of TPR units within a given species showed much lower homology (10–55%) (Figs. 1B-c, d, note: the homologies in comparison with TPR19 are shown). These features suggest that an ancestral TPR motif increased its copy number up to 25 by segmental duplication. They apparently diverged into two groups at an early stage of molecular evolution of TPRBK gene, then they evolved independently to increase sequence diversity. 3.3. Expression of TPRBK in human and mouse tissues We performed Northern blot analysis of total RNAs that were prepared from various human and mouse tissues (Fig. 2). In human, when probe 1 specific for the C-terminal region was used, mRNA of TPRBK was detected in most of the eight different fetal tissues and some of the adult tissues (Fig. 2A). A relatively stronger expression is seen in the brain, lung, skeletal muscle, heart, skin and small intestine of fetus, whereas an obvious expression is seen in the testis and ovary. In those cases, three types of mRNA transcripts (11 kb, 9 kb, and 7 kb) were detected. However, when probe 2 specific for the TPR-domain was used, only two larger transcripts (11 kb and 9 kb) were detected (Fig. S1). In mouse, a major transcript of 11 kb and a minor 9 kb transcript were detected in the embryos at different developmental stages (7-day through 17-day) (Fig. 2B). These results suggest the presence of at least three transcript variants in human and two variants in mouse, which must be generated by the alternative splicing depending upon tissue types and developmental stages. Next, we performed RT-PCR and Western blot analyses on six different cell lines established from various origins: human HeLa-S3, A431, HEK293 and SH-SY5Y, monkey COS-7, and mouse NIH3T3 (Fig. 2C and D). The RT-PCR analysis using the specific primers to detect distinct TPR units (see blue arrowheads in a framed diagram of Fig. 2C) revealed that TPRBK gene is expressed in all cell lines. We raised antibody in rabbits by immunizing the synthetic peptide (GHHDEALAVAERGR) corresponding to a middle part of human TPRBK protein (see a blue horizontal bar in a framed diagram of Fig. 2D). Western blotting with the affinity-purified rabbit antibody detected a protein band of 300 kDa which corresponds to the transfected GFP-tagged human TPRBK (lane 2 of Fig. S2A). Similarly, Western blotting using anti-GFP antibody detected a protein band of 300 kDa (lane 1 of Fig. S2A). On the other hand, Western blotting using anti-TPRBK antibody detected a protein band of 270 kDa which corresponds to the size of endogenous human TPRBK (lane 1 of Fig. S2B), and this band disappeared after pre-treatment with synthetic peptide of TPRBK (lane 2 of Fig. S2B). These data confirmed the TPRBK-specificity of antibody. A weak minor band of 200 kDa was detected but this was also eliminated after absorption, suggesting this smaller protein to be a truncated product from a transcript variant of TPRBK that retains the antigenic amino acid sequence. The endogenous TPRBK of 270 kDa was detected for all six cell lines derived from 3 different mammalian species (human, monkey and mouse) (Fig. 2D). 3.4. Subcellular localization of TPRBK protein in COS-7 cells To examine subcellular localization of TPRBK protein, we chose monkey COS-7 cells, which are most suitable for synchronizing at various stages of cell cycle (interphase through cytokinesis). COS-7 cell line was derived from African green monkey (C. aethiops), however we found that the aa sequence (GHHDEALAVAERGR) of the

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human TPRBK peptide used for antibody production is identical to the monkey counterpart (data not shown). Also, the affinity-purified anti-human TPRBK antibody was proven to be immune-reactive to

TPRBKs from human, monkey and mouse (Fig. 2D). Based on these results, we decided to utilize the anti-human TPRBK antibody for immunofluorescent staining of monkey COS-7 cells.

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Fig. 2. Expression profiles of TPRBK in human/mouse tissues and various cell lines. (A) Northern blot analysis of total RNAs from human adult and fetal tissues: probe 1 (human) was designed to detect a part of C-terminal region (see an inserted diagram). Arrowheads indicate three kinds of mRNA transcripts (11-kb, 9-kb and 7-kb). (B) Northern blot analysis of total RNAs from mouse adult and embryo tissues: probe (mouse) was designed to detect a terminal portion of N-terminal half (see an inserted diagram). Arrowheads indicate two kinds of mRNA transcripts (11-kb and 9-kb). (C) RT-PCR analysis of TPRBK expression in six different mammalian cell lines: the PCR primers were designed to detect a small region (550-bp) of the N-terminal TPR-domains (see blue arrowheads in a diagram). (D) Western blotting with anti-human TPRBK antibody: antibody was raised in rabbit using a synthetic peptide around the center (see a blue horizontal bar in a diagram). Arrowheads indicate TPRBK protein as a band of 270 kDa.

Fig. 1. A. Molecular features of human TPRBK gene and protein.(a) Genomic structure: TPRBK is a big gene of 702 kb in size. Twenty three vertical bars indicate the position of exons and the contiguous lines connecting each exon represent the mRNA transcripts. A long arrow indicates the direction of transcription.(b) cDNA structure: the full length cDNA for TPRBK is 11,743 bp in size. White and gray boxes represent exons of different sizes and the gray boxes (nt91–7536) correspond to the open reading frame (ORF).(c) Domain structure: TPRBK protein is composed of 2481 aa residues. White boxes at the N-terminal half of the protein represent 25 units of TPR domain.B. Phylogenetic tree of TPRBK proteins and homology comparison of TPR motifs.(a) Phylogenetic tree of TPRBK proteins: the deduced amino acid sequence of TPRBK was analyzed by CLUSTAL W for 13 different species (from insecta to mammal). Phylogenetic tree was drawn by N-J plot. Horizontal lines are proportional to the extent of sequence divergence from the common ancestor. Bootstrap values are shown at every node.(b) Phylogenetic tree of TPR domains: the molecular tree indicates the repeated duplication events of TPR motifs during the evolution of TPRBK gene. Total 175 orthologous TPR units among seven different species (human, mouse, rat, fugu, fly, mosquito and bee) form 25 branches, which are apparently diverged into two groups at an early stage of evolution; one group of 3 motifs (TPR1 through TPR3) and the other group of 22 motifs (TPR4 through TPR25).(c, d) Homology comparison of 25 different TPR motifs: (c) Pair-wise comparison of every TPR unit between human and other species (mouse, rat, chicken, frog, Takifugu, medaka, Drosophila and mosquito). (d) Pair-wise comparison of TPR units within a given species. The homologies in comparison with TPR19 are shown for each species, which showed the highest homology (10–55%) among others.

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Fig. 3. Subcellular localization of TPRBK at different stages of cell cycle. COS-7 cells were fixed at different stages of cell cycle (interphase to cytokinesis) and the immunofluorescent staining was carried out using anti-TPRBK, anti-α-tubulin and anti-Pericentrin antibodies using Zenon Alexa Fluor 647 mouse labeling kit. Nuclear DNA was stained with DAPI. Independent fluorescent images (red, green, pink and blue) were merged (merge). White arrows indicate the duplicated centrosomes at interphase and TPRBK on the midzone at anaphase. Scale bar: 10 μm.

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The culture of COS-7 cells was processed for synchronization by the double thymidine block method, and the cells at a distinct cell cycle stage were prepared for the analysis by immunofluorescence staining with adequate antibodies. At interphase, the immunofluorescence staining with anti-TPRBK antibody detected two distinct spots (red) adjacent to the nucleus (see a framed enlarged photo in Fig. 3). The immune staining with a second antibody which reacts with α-tubulin showed a wide spread network (green) of microtubules. Then, we carried out the dual immunofluorescence staining using a third antibody against Pericentrin, which is known as a marker for centrosome. The dual fluorescent image clearly showed that the red spots of TPRBK overlap with two distinct pink spots of Pericentrin, indicating that TPRBK is located on the centrosomes at interphase. This situation is clearly seen in the merged image (Fig. 3). At prometaphase, TPRBK spread over the nucleus in close association with a network of the spindle microtubules (green) and spindle poles (pink), and the location of TPRBK was not much changed at metaphase (Fig. 3). At anaphase, substantial portion of TPRBK was accumulated on a middle region, namely the midzone, of the overlapping microtubules (Fig. 3). White arrow indicates that TPRBK also localized on the midzone. At telophase, TPRBK was condensed on the central spindles, which are known to stimulate the formation of cleavage furrow (Fig. 3). During cytokinesis, most of the TPRBK was condensed on the distinct structure “midbody” (Fig. 3). Throughout the entire cell cycle, Pericentrin stayed at the centrosomes and its subcellular location relative to TPRBK and α-tubulin is clearly seen in the merged images (Fig. 3). We also carried out the dual immunofluorescence staining with anti-TPRBK antibody and anti-Eg5 antibody. Eg5 is a motor protein in the kinesin-like family and known to play a role in the assembly and organization of mitotic spindles. At metaphase, TPRBK and Eg5 overlapped each other on the mitotic spindle, which plays a critical role for the progress of chromosome segregation (Fig. S3). At telophase, TPRBK and Eg5 were together localized in the spindle poles and central spindle (Fig. S3). This particular photo was taken at the beginning of telophase, but at a later time, both TPRBK and Eg5

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were found on the midzone (data not shown). During cytokinesis, TPRBK and Eg5 together stayed on the midbody (Fig. S3). However, the distribution of both signals (red and green) in the merged image indicates that some of TPRBK locate independently of Eg5 in the center of midbody. The coordinated movement of TPRBK and Eg5 during metaphase and anaphase can be monitored by the 3D-image video (see a Supplementary Video A). These results again indicated that in association with Eg5, TPRBK must play a significant role in the progress of cell division toward cytokinesis. 3.5. TPRBK interacts with Aurora B, which binds to other proteins, such as PLK1 and PRC1 To examine if any other proteins interact with TPRBK, we carried out the immunoprecipitation with anti-TPRBK antibody for the lysates of human HeLa-S3 cells, and the resulting immunoprecipitates were processed for Western blot analysis (Fig. 4). As seen, in the immunoprecipitates, Aurora B was found together with TPRBK, whereas 3 other tested proteins (PLK1, PRC1 and α-tubulin) were not found (Fig. 4A). Conversely, in the immunoprecipitates that were separately generated with anti-Aurora B antibody, TPRBK as well as two relevant proteins PLK1 and PRC1 were found (Fig. 4B). These results strongly indicate the tight interaction of TPRBK with Aurora B and further confirm the previous finding that Aurora B interacts with PLK1 and PRC1. Aurora B is a serine/threonine protein kinase and it is known to act as a chromosome passenger protein (CPC), which corrects inappropriate attachment of the mitotic spindle (microtubules) to the kinetochores and facilitate the proper chromosome segregation. Thus, these results indicate that TPRBK must function in tight association with a key protein Aurora B, which interacts independently with PLK1 and PRC1. 3.6. TPRBK associates with Aurora B throughout mitosis and cytokinesis Next, we performed the dual immunofluorescence staining to determine the precise localization of TPRBK and Aurora B during cell cycle using the synchronized COS-7 cells. At prometaphase, TPRBK

Fig. 4. Immunoprecipitation assay using anti-TPRBK and anti-Aurora B antibodies. (A) Western blotting of the immunoprecipitates with anti-TPRBK antibody: HeLa-S3 cell lysates were processed for immunoprecipitation (IP) with anti-TPRBK antibody, and the immune-complexes were analyzed by Western blotting (WB) with various antibodies (anti-TPRBK, anti-Aurora B, anti-PLK1, anti-PRC1 and anti-α-tubulin). Lane 1, HeLa-S3 cell lysates (control); lane 2, eluates after IP with anti-TPRBK antibody; lane 3, eluates after IP with preimmune rabbit serum (control). Arrows indicate the relevant bands of immune reaction in lane 2 as compared to lane 1. (B) Western blotting of the immunoprecipitates with anti-Aurora B antibody: HeLa-S3 cell lysates were processed for immunoprecipitation (IP) with anti-Aurora B antibody, and the immune-complexes were analyzed by Western blotting (WB) with various antibodies in the same way as (A). Lane 1, HeLa-S3 cell lysates (control); lane 2, eluates after IP with anti-TPRBK antibody; lane 3, eluates after IP with preimmune rabbit serum (control). Arrows indicate the relevant bands of immune reaction in lane 2 as compared to lane 1.

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was spread broadly on the spindle microtubules and more obviously Aurora B was spread on the condensing chromosomes (Fig. 5A). At metaphase, TPRBK was assembled on the spindle microtubules,

ranging from spindle poles to equatorial plane, whereas Aurora B was condensed on the kinetochores, which are assembled on an equatorial region (Fig. 5A). At anaphase, TPRBK was located on the spindle

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Fig. 5. Subcellular localization of TPRBK, Aurora B and PLK1 during cell cycle. (A) Dual immunofluorescent staining of TPRBK and Aurora B in COS-7 cells throughout the cell cycle: COS-7 cells were fixed at different stages of cell cycle (prometaphase through cytokinesis) and immunofluorescent staining was carried out using two 1st antibodies (rabbit-anti-TPRBK antibody or mouse-anti-Aurora B antibody), followed by the 2nd fluorescent antibodies (Alexa Fluor 555-labeled goat-anti-rabbit IgG antibody or Alexa Fluor 488-labeled goat-anti-mouse IgG antibody). Nuclear DNA was stained with DAPI. Independent fluorescent images (red, green and blue) were merged (merge). At anaphase, the areas indicated with white arrows are magnified (see also at telophase). Bar: 10 μm. (B) Triple immunofluorescent staining of TPRBK, Aurora B and PLK1 during cytokinesis: COS-7 cells were fixed at early/late/final stages of cytokinesis and immune-staining was performed for TPRBK and Aurora B as described in (A). For anti-PLK1 antibody, Zenon Alexa Fluor 647 mouse labeling kit was used. Nuclear DNA was stained with DAPI. Independent fluorescent images (red, green, pink and blue) were merged (merge). See enlarged views at bottom-left corners. Bar: 10 μm.

microtubules, while Aurora B was located around the microtubules near the spindle poles. Furthermore, TPRBK and Aurora B were also observed as dotted signals on the midzone (Fig. 5A). At telophase, they were more condensed closely to each other at the central spindle (Fig. 5A). At an early stage of cytokinesis, both TPRBK and Aurora B were found on the midbody, which is connecting two daughter cells (Fig. 5A). At a later stage of cytokinesis, almost all of them were aligned on the elongated midbody. The merged image indicates that most of the red and green signals are overlapped, generating yellow signals, but significant amounts of red signal (TPRBK) remain independently of green (Aurora B) in the stems of midbody (Fig. 5A). Thus, a novel protein TPRBK drastically changes its location during cell cycle, and its translocation is closely associated with a protein kinase, Aurora B. Their association on the midbody is remarkable during cytokinesis, especially at the late stage of cytokinesis prior to cell abscission. As was confirmed by immunoprecipitation assay (Fig. 4B), Aurora B binds directly to PLK1, another serine/threonine protein kinase, that functions throughout M-phase (Goto et al., 2006). Triple immunofluorescence staining using three antibodies against TPRBK, Aurora B and PLK1 revealed that at early and late stages of cytokinesis, those three proteins are located together on the midbody (Fig. 5B). The merged image indicates that their relative position on the midbody

is in the order of “TPRBK–AuroraB–PLK1”. TPRBK apparently forms a broad structural foundation on which the other two proteins reside. Interestingly, at the final stage of cytokinesis, PLK1 was no longer found on the midbody, whereas both TPRBK and Aurora B remained on the midbody throughout the cytokinesis (Fig. 5B). To examine if TPRBK binds directly to Aurora B, we employed a novel method “Duolink in situ PLA system” (Fig. S4). To justify the Duolink method, we tested a typical case for Aurora B and INCENP using the synchronized HeLa-S3 cells, since those proteins are known as the interacting components on the chromosome passenger complex (Terada, 2001). As expected, a distinct red spot of hybridization signal was detected at an early stage of cytokinesis (Fig. S4). Similarly, a distinct red hybridization signal was seen for TPRBK and Aurora B at the early stage of cytokinesis (data not shown). However, at the later stage of cytokinesis, two relatively large condensed red spots (white arrows) and several small spots are seen along the elongated midbody, confirming the close interaction of TPRBK with Aurora B (Fig. S4). Moreover, the differential interference contrast (DIC) microscopy clearly showed no signals in the center of the midbody, where Flemming body is formed (see +DIC in Fig. S4). These results suggest that TPRBK may bind directly to Aurora B during anaphase and both together translocate toward the midbody, where TPRBK and Aurora B play a

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coordinated role in accomplishing the cytokinesis. On the other hand, we found that PRC1 was not co-immunoprecipitated with TPRBK (Fig. 4A), so that no cross-linking signals were seen for TPRBK and PRC1 on the cells at a late stage of cytokinesis (Fig. S4). 3.7. Treatment with inhibitors of actin-myosin contractility, Blebbistatin and Y-27632, confine TPRBK and Aurora B at a disrupted structure named “postmitotic spindle” We examined if the subcellular localization of TPRBK is affected by two inhibitors, Blebbistatin and Y-27632, which have been shown to interfere with the contractility of actin and myosin during mitosis and cytokinesis. Blebbistatin inhibits non-muscle Myosin II and is known to block the contraction of cleavage furrow, but it does not disrupt the mitosis and allows formation of the contractile ring. On the other hand, Y-27632 is an inhibitor of the Rho-associated coiled-coil forming protein kinase, ROCK, which phosphorylates Myosin II to disrupt assembly of the contractile ring. In the untreated control COS-7 cells, at telophase, TPRBK and Aurora B were found on the numerous bundles of antiparallel

midzone microtubules, where Aurora B was localized in the center as compared to TPRBK (Fig. 6—column a). At cytokinesis, they were aligned on the midbody (Fig. 6—column b). In the cells treated with Blebbistatin or Y-27632, the midzone microtubules were not aligned, and both TPRBK and Aurora B were detected on an unusually spindle-shaped structure, which resides between two nuclei (Fig. 6— columns c, d and e). These disrupted structures have been named as “postmitotic spindles” since they are the remnants of mitotic spindles but the ends of spindle microtubules are independent to the centrosomes (see white arrows) (Martineau et al., 1995). It is noteworthy that the treatment with inhibitors often caused bi-nucleated cells, in which TPRBK and Aurora B were not well assembled (see Fig. 6— column-f for the inhibitor Y27632). The merged images, especially under the DIC microscopy, clearly show that the midbody organization was disrupted, but yet TPRBK and Aurora B were tightly associated in the inhibitor-treated cells (Fig. 6—4th and 5th rows). These results indicate that the adequate processing of microtubule organization, such as contraction of the cleavage furrow and assembly of the contractile ring, is an essential prerequisite for TPRBK and Aurora B to properly contribute to the midbody formation.

Fig. 6. Effects of Blebbistatin and Y-27632 on subcellular localization of TPRBK and Aurora B during telophase and cytokinesis. COS-7 cells were fixed at telophase and cytokinesis, and immunofluorescent staining was carried out using anti-TPRBK and anti-Aurora B antibodies as described in Fig. 5. Nuclear DNA was stained with DAPI. Independent fluorescent images (red, green and blue) were merged (Merge) and the merged images were photographed under a differential interference contrast (DIC) microscope (Merge + DIC). White arrows indicate the central spindle (a), midbody (b) and postmitotic spindles (c, d and e), respectively (see text for details). Bar: 10 μm.

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Fig. 7. Knockdown of TPRBK expression and its inhibitory effects on cell cycle progression. (A) RT-PCR and Western blot analysis: HeLa-S3 cells were treated with four types of TPRBK-siRNAs (targets 1 through 4), and the expression of TPRBK was analyzed by RT-PCR and Western blotting. GAPDH was used as a control. (B) Growth suppression of HeLa-S3 cells by TPRBK-siRNAs: cells were synchronized by 1st thymidine block for 16 h from −40 h to −24 h, released from thymidine block for 2 h, and treated with either TPRBK-siRNA (mix of targets 3 and 4) or scrambled (scr)-siRNA for 6 h. Then, cells were synchronized again by 2nd thymidine block for 16 h, kept for 2 h without thymidine, and treated with TPRBK siRNA and/or scr-siRNA for 6 h. After that, cells were cultured with the medium containing 10% FBS without thymidine for 16 h. (C) Cell cycle profiles of HeLa-S3 cells by FACS analysis: cells treated with TPRBK-siRNA or scr-siRNA were fixed and stained with Guava Cell Cycle Reagent (Millipore), then analyzed by a flow cytometer Guava easyCyte 8HT (Millipore). The ratio of each cell cycle phase was calculated using software CytoSoft Guava Cell Cycle (Millipore). (a) Cells were synchronized and treated with TPRBK scr-siRNA. Three peaks (fractions 1 through 3) correspond to G1, S and G2 + M-phases. (b) Cells were synchronized and treated with TPRBK-siRNA. The inserted table shows the changes in the cell cycle distribution. (D) Morphology change of HeLa-S3 cells after TPRBK-knockdown. Immunofluorescent staining was performed on the cells treated with TPRBK-siRNA or TPRBK-scr-siRNA using anti-TPRBK antibody as described in Fig. 5. Upper two photos indicate the presence of TPRBK on the midbody. The lower two photos indicate the loss of TPRBK and midbody (arrows indicate the expected positions). Bar: 20 μm.

3.8. Knockdown of TPRBK by small interfering RNA (siRNA) arrests cell cycle at G2 + M phase We further examined the functional aspects of TPRBK on the cell cycle. For this, we designed small interfering RNAs (siRNAs) against different target regions (target 1 through 4) of TPRBK gene, and HeLa-S3 cells were treated with each of those siRNAs (Fig. 7 and Table S1). By RT-PCR and immunoblotting analyses, we found that all four siRNAs substantially reduced the expression of TPRBK gene and two of them (siRNAs for target 3 and 4) exhibited the knockdown of TPRBK protein synthesis (Fig. 7A). If TPRBK plays an important role for cell division, the rate of cell proliferation would be reduced in the TPRBK-knockdown cells. So, we analyzed the possible growth suppression of HeLa-S3 cells by treating with TPRBK-siRNAs (mix of two siRNAs for targets 3 and 4) and scrambled (scr)-siRNA as a control. We found that proliferation of the TPRBK-knockdown cells by TPRBK-siRNAs was greatly reduced as compared to the scrambled (scr)-siRNA-treated cells (Fig. 7B).

Then, using flow cytometry, we examined which phase of the cell cycle (G1, S and G2 + M phases) was most affected. The cell cycle distribution of the control scr-siRNA-treated cells was used as a reference (Fig. 7C-a). This profile was significantly changed after treating with TPRBK-siRNA, showing that substantially more cells were arrested at G2 + M phase (57.6% vs 72.4%) (Fig. 7C-b). Interestingly, the TPRBKsiRNA-treated cells often showed abnormal morphology, having two nuclei and no midbody although they seem to have finished nuclear division (see white arrows in Fig. 7D). These results suggest that the knockdown of TPRBK causes the failure of cytokinesis, leaving cells at a two-nucleated state. 3.9. Knockdown of TPRBK disrupts midbody structure and proper localization of proteins relevant for making midbody We further examined if the knockdown of TPRBK affects the intracellular localization of three relevant proteins (α-tubulin, Aurora B and Eg5), which are involved in the formation of midbody. For this,

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we first confirmed the knockdown of TPRBK (Fig. 8A). In the cells treated with scr-siRNA (control), TPRBK and Eg5 were seen on the midbody at cytokinesis, whereas no signals for TPRBK are seen in the cells treated with TPRBK-siRNA, indicating the knockdown of TPRBK. Also, we observed the intracellular localization of α-tubulin, Aurora B and INCENP (Figs. 8B and S5). In the control cells treated with scr-siRNA, Aurora B is seen as numerous spots on midzone at anaphase, and it is aligned in the middle of spindle midzone microtubules at telophase (Fig. 8B). At cytokinesis, Aurora B is assembled as two parallel broad bands in a central part of midbody, which is surrounded by massive α-tubulin bundles (Fig. 8B). Contrary to these normal situations, in the TPRBK-knockdown cells, the bundling of microtubules is disturbed and formation of midbody is not seen, although the acto-myosin ring (contractile ring) seems assembled. Moreover, a scatter of spindle midzone microtubules are only observed on an equatorial plane and Aurora B is not correctly localized across the central spindle (Fig. 8B). The merged images with DIC microscopy clearly showed the locations of midzone, midbody and contractile ring as to the dividing two nuclei (see “Merge + DIC” in Fig. 8B). Analysis on INCENP (inner centromere protein), a factor of proteins which compose the CPC, provided essentially the same results as was seen for Aurora B (Fig. S5). Thus, in the TPRBK-knockdown cells, INCENP was misplaced due to the lack of midbody formation, although the acto-myosin ring seems assembled during cytokinesis. These results strongly suggest that TPRBK is involved in the condensation of spindle midzone microtubules, leading to the formation of midbody, which is an essential structure for completing cytokinesis. 4. Discussion 4.1. Molecular components necessary for formation and integrity of midbody Cytokinesis is an essential process for cell division to form two daughter cells, and the formation of midbody and its central part Flemming body is indispensable to facilitate cell abscission. A great many proteins are involved in the organization of midbody/Flemming body, which is formed in tight association with compaction and bundling of the spindle midzone microtubules. These proteins include Centralspindlin, CPC (chromosome passenger complex, composed of INCENP, Survivin, Borealin and mitotic kinase Aurora B), PLK1 (Polo-like kinase 1), PRC1, Annexin11, Tektin2 (Tek2), MKLP2, Cep55, and so on (Durcan et al., 2008; Fabbro et al., 2005; Hutterer et al., 2009; Kaur et al., 2007; Kurasawa et al., 2004; Richard et al., 2001; Tomas et al., 2004). Disruption of any one of these cell division-related proteins results in improper formation of midbody/Flemming body, leading to incompletion of cytokinesis (Mishima et al., 2002; Mollinari et al., 2002). In this study, we further added a new member “TPRBK” to the above lengthy list as an essential component for formation and integration of midbody/Flemming body during cytokinesis. 4.2. TPRBK is a large gene and produces a big protein with multiple TPR motifs The TPRBK gene was found between two known genes (PITPNB and HS747E24) at the band q12.1 of human chromosome 22. The genome size of this gene was 702 kb, consisting of 23 exons and relatively large 22 introns (Fig. 1A-a), encoding a big protein of 2481 amino acid (aa) residues (Fig. 1A-b). The predicted protein possesses

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25 units of TPR (TetratricoPeptide Repeat) motif in the N-terminal half and no obvious motif in the C-terminal half of the protein (Fig. 1A-c). 4.3. TPRBK orthologous genes are commonly found in the animal kingdom By homology search, we found TPRBK-like genes (orthologs) in many animal species from insecta to the mammal (Fig. 1B-a). However, we could not find any orthologs in nematoda, plant, fungi, and protozoan. All the animal orthologs of TPRBK possessed multiple (>22 units) TPR motifs in the N-terminal half of the proteins, although they showed different aa sequences. The molecular phylogenetic tree indicated the divergence of TPR motifs (TPR1 through 25) into two groups: a group of 3 motifs (TPR1 to TPR3) and the other group of 22 motifs (TPR4 to TPR25), which were duplicated and diverged at an early stage during evolution of TPRBK gene (Fig. 1B-b). The aa sequences of 25 different TPR motifs in the human TPRBK protein are quite different each other. Moreover, the pair-wise comparison of every TPR motif (TPR1 through TPR25) between human and seven other species showed high homology (40–100%), whereas the pair-wise comparison of TPR units within a given species showed much lower homology (10–55%). These molecular features strongly suggest that every TPR motif might play a similar but independent role in reinforcing the functions of the entire molecule. Since TPR motifs are known to act as an interaction site to a protein partner, multiple TPR motifs with sequence diversity might have been favorable to increase partners. This principle may be applicable to all the TPR-containing proteins including human TPRBK although at present only a few partners have been identified. It will be interesting to determine which of 25 TPR motifs is interacting with a particular one of several partners. The C-terminal half of the TPRBK proteins possess a highly homologous region among orthologs (data not shown). This conserved aa sequence would suggest the unknown functional domain in this region. Nonetheless, TPRBK gene is found mostly in the animal kingdom and not in plants. Indeed, TPRBK is ubiquitously expressed and its expression is especially high in human fetal and mouse embryo tissues, and in several cell lines of human, monkey and mouse (Figs. 2 and S1). Hence, TPRBK was considered to play an essential role in animalspecific cell physiological function. Regarding the function of big protein TPRBK, it is noteworthy that the TPR-motif-containing proteins are often involved in the progress of cell division. Those proteins include BBS4 (Bardet–Biedl syndrome 4), OGT (O-GlcNAc transferase), APC/C (anaphase promoting complex/ cyclosome), CDC16 (cell division cycle 16), CDC23, CDC27, APC5, and APC7 (Kim et al., 2004; for reviews see Peters, 2006; Slawson et al., 2008). These observations strongly suggested that a novel big protein TPRBK with multiple TPR motifs must play a critical role in the mechanism of cell division. In fact, it was found that TPRBK is involved in cell division while interacting with many other important proteins, and especially during cytokinesis it provides a basis to form and integrate the midbody. 4.4. TPRBK moves with Aurora B along a network of spindle midzone microtubules TPRBK continuously changes its location from spindle poles to midbody during mitosis and cytokinesis (Fig. 3). At the end of mitosis,

Fig. 8. Knockdown of TPRBK affects cytokinesis. (A) Knockdown of TPRBK protein synthesis by treating with TPRBK siRNA. Upper panel: HeLa-S3 cells were treated with TPRBK-scr-siRNA (control) and fixed at cytokinesis. Immunofluorescent staining was carried out using anti-TPRBK and anti-Eg5 antibodies. Lower panel: HeLa-S3 cells were treated with TPRBK-siRNA and stained with anti-TPRBK and anti-α-tubulin antibodies. Nuclear DNA was stained with DAPI. Independent fluorescent images (red, green and blue) were merged (Merge) and the merged image was observed under a differential interference contrast (DIC) microscope (Merge + DIC). White arrows indicate the location of TPRBK protein. Bar: 20 μm. (B) Knockdown of TPRBK affects subcellular localization of Aurora B and α-tubulin. HeLa-S3 cells were fixed at different stages of cell cycle (anaphase, telophase and cytokinesis) after treating with control TPRBK-scr-siRNA or TPRBK-siRNA, then stained with anti-Aurora B and anti-α-tubulin antibodies. Other conditions were the same as A. White arrows indicate midzone (anaphase), central spindle (telophase), midbody (cytokinesis) and midzone (TPRBK siRNA), respectively. Bar: 20 μm.

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TPRBK together with Aurora B moves along network of spindle midzone microtubules, and those proteins eventually overlap on the midbody (Fig. 5A). Co-immunoprecipitation assay confirmed that TPRBK binds to Aurora B (Fig. 4), and Duolink in situ PLA system further proved that TPRBK and Aurora B are localized close enough to be linked to each other on the midbody (Fig. S4). It was also proven that Aurora B by itself interacts with PLK1 and PRC1 (Fig. 4B). These results are consistent with previous findings that during anaphase, the CPC moves together with chromosomes toward spindle poles, and it moves to the midzone along a network of spindle midzone microtubules and forms a complex with several other proteins, such as PLK1 and PRC1 during the late stage of cell division (Goto et al., 2006; Zhu and Jiang, 2005). These results further indicated that TPRBK functions in tight association with Aurora B until their separation at the final stage of cytokinesis. Triple immunofluorescence staining revealed that the relative position of three components on the midbody is “TPRBK–AuroraB– PLK1”, where TPRBK provides a foundation, on which the other two proteins reside. Interestingly, at the final stage of cytokinesis, PLK1 was no longer found on the midbody, whereas both TPRBK and Aurora B remained on the midbody throughout cytokinesis (Fig. 5B). The molecular event that TPRBK moves in close association with Aurora B during cytokinesis can be monitored by 3D-image video (see Supplemental Video B). 4.5. Translocation of TPRBK to midbody is associated with cleavage furrow and acto-myosin ring During the late mitosis (anaphase and telophase), spindle midzone microtubules determine a cleavage plane to induce the creation of cleavage furrow. Then, the contractile ring is formed on the plane and furrow ingression is promoted by many other proteins (such as MgcRacGAP, RhoA and Rac), which regulate the “acto-myosin ring dynamics”. During furrow ingression, a few more proteins (FIP3, Rab11, SNAREs, Centriolin and Exocyst) accumulate to couple with recycling endosome-derived membrane traffic toward the final stage of cytokinesis (D'Avino et al., 2005; Montagnac and Chavrier, 2008; Simon and Prekeris, 2008; Wilson et al., 2005). In this regard, we examined how the subcellular localization of TPRBK is affected by two inhibitors (Blebbistatin and Y-27632), which are known to prevent contraction of an acto-myosin ring. In those inhibitor-treated cells, TPRBK was restricted to a “postmitotic spindle” which resides between two nuclei (Figs. 6-c, d and e). Aurora B behaved in the same way as TPRBK. Thus, the process of microtubule organization is necessary for proper destination of TPRBK and Aurora B. In other words, translocation of a big protein TPRBK to the midbody is related to the formation of cleavage furrow and the contraction of the acto-myosin ring. It is clear that TPRBK is an essential structural component of midbody, but it is unclear whether TPRBK plays any regulatory role in the mechanism of cytokinesis. 4.6. Knockdown of TPRBK by siRNAs disrupts the formation of midbody The siRNAs for TPRBK substantially reduced the expression of TPRBK gene and exhibited the knockdown of protein synthesis (Fig. 7). In these TPRBK-knockdown cells, the proliferation was arrested at G2 + M phase. Those TPRBK-knockdown cells clearly showed disruption of midbody. However, the ingression of cleavage furrow and contraction of contractile ring were still observed although at least two proteins, Aurora B and INCENP, were not found at the proper location (Figs. 8B and S5). Thus, the presence of TPRBK is necessary for the condensation of spindle midzone microtubules, the formation of midbody, and the completion of cytokinesis. It is noteworthy that siRNAs for Tek2 generated similar knockdown cells in which midbody structure was disorganized (Durcan et al., 2008). Tek2 is known to bind tubulins through its conserved aa

sequence (RPNVELCRD), stabilizing midzone microtubules (Amos, 2008). Moreover, Tek2 is shown to serve for the proper localization of Aurora B, MKLP1 and PRC1 on the midzone, facilitating the formation of midbody matrix. In this regard, it is interesting that co-immunoprecipitation assay showed no interaction between TPRBK and α-tubulin. In fact, TPRBK does not possess the conserved aa sequence (RPNVELCRD) necessary for binding to tubulins. Hence, TPRBK may indirectly facilitate the bundling of microtubules via tubulin-binding ability of other proteins such as Tek2 and MKLP1. Another protein Cep55 also possesses a microtubule-bundling ability and appears to be involved in establishing the midbody structure, especially at the final stage of cytokinesis. Like Tek2, Cep55 is required for correct localization of Aurora B. In the cells treated with Cep55 siRNA, the components of midbody were either absent or mislocated (Zhao et al., 2006), and this abnormal phenotype is much similar to the TPRBK-knockdown cells. Moreover, it is known that subcellular localization of Cep55 is controlled by a protein complex Centralspindlin, through Aurora B-dependent phosphorylation. Thus, Cep55 is involved in the function of midbody. However, it remains to be investigated whether TPRBK interacts with Cep55. 4.7. TPRBK is essential for the integrity of midbody structure and completion of cytokinesis Cytokinesis proceeds on the midbody, and Flemming body appears in the central part of the expanded midbody at the final stage of cytokinesis (Morita et al., 2007). TPRBK was necessary for providing a foundation for the formation and integrity of midbody, but no direct evidence was seen for the formation of Flemming body (Figs. 3, 5 and S4). A great many proteins are involved in the mammalian cell cycle events. Among those, three proteins are particularly noteworthy since they have been known to participate in formation of midbody and Flemming body. Those proteins are: Annexin11, ESCRT and ALIX (Morita et al., 2007; Tomas et al., 2004). At present, there are no evidences whether these proteins interact with TPRBK. Further study will be necessary to elucidate how those multiple proteins interact with a novel protein TPRBK of 270 kDa, which is apparently the biggest protein component of midbody. Nevertheless, TPRBK seems to play a major role in the formation and maintaining integrity of midbody. In this study, we provided substantial evidences that a novel big protein TPRBK with multiple TPR motifs is essential for the progress of mitosis and cytokinesis. Further analysis on the role of TPRBK in a series of dynamic molecular process of cell division would undoubtedly facilitate better understanding of a fascinating animal cell division. Furthermore, it would be worthy to explore the possible association of the loss of TPRBK function with certain human diseases, such as cancer and some neuropathy (Singhmar and Kumar, 2011). Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.09.061. Acknowledgments This work was supported by the Fund for “Research for the Future” Program from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Priority Areas “Medical Genome Science” and the 21st Century Center of Excellence (COE) Program entitled “Understanding and Control of Life's via Systems Biology (Keio University)” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), a Grant-in-Aid for Young Scientist B from MEXT. The authors also thank a special research fund from Toagosei Co., Ltd, Japan.

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