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• W1974559008 abstract "The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. We show that a human kinetochore oncoprotein, AF15q14/blinkin, a member of the Spc105/Spc7/KNL-1 family, directly links spindle checkpoint proteins BubR1 and Bub1 to kinetochores and is required for spindle checkpoint and chromosome alignment. Blinkin RNAi causes accelerated mitosis due to a checkpoint failure and chromosome misalignment resulting from the lack of kinetochore and microtubule attachment. Blinkin RNAi phenotypes resemble the double RNAi phenotypes of Bub1 and BubR1 in living cells. While the carboxy domain associates with the c20orf172/hMis13 and DC8/hMis14 subunits of the hMis12 complex in the inner kinetochore, association of the amino and middle domain of blinkin with the TPR domains in the amino termini of BubR1 and Bub1 is essential for BubR1 and Bub1 to execute their distinct mitotic functions. Blinkin may be the center of the network for generating kinetochore-based checkpoint signaling. The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. We show that a human kinetochore oncoprotein, AF15q14/blinkin, a member of the Spc105/Spc7/KNL-1 family, directly links spindle checkpoint proteins BubR1 and Bub1 to kinetochores and is required for spindle checkpoint and chromosome alignment. Blinkin RNAi causes accelerated mitosis due to a checkpoint failure and chromosome misalignment resulting from the lack of kinetochore and microtubule attachment. Blinkin RNAi phenotypes resemble the double RNAi phenotypes of Bub1 and BubR1 in living cells. While the carboxy domain associates with the c20orf172/hMis13 and DC8/hMis14 subunits of the hMis12 complex in the inner kinetochore, association of the amino and middle domain of blinkin with the TPR domains in the amino termini of BubR1 and Bub1 is essential for BubR1 and Bub1 to execute their distinct mitotic functions. Blinkin may be the center of the network for generating kinetochore-based checkpoint signaling. Commitment to mitosis activates a number of key cell cycle regulators to promote the formation of kinetochores on centromeric chromatin. These kinetochores are then able to associate with spindle microtubules and two subsequent proteolytic events ensure equal segregation of the sister chromatids to each spindle pole. The ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C) promotes the proteasome-dependent destruction of securin and mitotic cyclin B in anaphase. Whereas the destruction of mitotic cyclin promotes mitotic exit, degradation of securin promotes the separation of sister chromatids as it relieves the inhibitory chaperone activity of securin on separase. Once securin is removed, separase is free to promote anaphase events, which include cleavage of the cohesin linkage. These proteolytic anaphase events are restrained until all the metaphase chromosomes are correctly aligned on the spindle apparatus by a process called spindle assembly checkpoint, feedback control, or mitotic checkpoint (Hoyt et al., 1991Hoyt M.A. Totis L. Roberts B.T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function.Cell. 1991; 66: 507-517Abstract Full Text PDF PubMed Scopus (898) Google Scholar, Li and Murray, 1991Li R. Murray A.W. Feedback control of mitosis in budding yeast.Cell. 1991; 66: 519-531Abstract Full Text PDF PubMed Scopus (931) Google Scholar, Rieder et al., 1994Rieder C.L. Schultz A. Cole R. Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.J. Cell Biol. 1994; 127: 1301-1310Crossref PubMed Scopus (442) Google Scholar; reviewed in Kops et al., 2005Kops G.J. Weaver B.A. Cleveland D.W. On the road to cancer: aneuploidy and the mitotic checkpoint.Nat. Rev. Cancer. 2005; 5: 773-785Crossref PubMed Scopus (918) Google Scholar, Musacchio and Salmon, 2007Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1721) Google Scholar, Pinsky and Biggins, 2005Pinsky B.A. Biggins S. The spindle checkpoint: tension versus attachment.Trends Cell Biol. 2005; 15: 486-493Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The mitotic checkpoint restrains the anaphase promoting proteolysis until correct chromosome alignment occurs, by inhibiting APC/C activation. This dependency relationship prevents premature anaphase. A key molecular link between APC/C and the checkpoint is the inhibitory association of a spindle checkpoint protein, Mad2, with Cdc20/Fizzy/Slp1, an APC/C activator (Hwang et al., 1998Hwang L.H. Lau L.F. Smith D.L. Mistrot C.A. Hardwick K.G. Hwang E.S. Amon A. Murray A.W. Budding yeast Cdc20: a target of the spindle checkpoint.Science. 1998; 279: 1041-1044Crossref PubMed Scopus (463) Google Scholar, Kim et al., 1998Kim S.H. Lin D.P. Matsumoto S. Kitazono A. Matsumoto T. Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint.Science. 1998; 279: 1045-1047Crossref PubMed Scopus (319) Google Scholar). Together with Mad2, BubR1 (Bub1-related kinase), Bub3, and Cdc20 form a mitotic checkpoint complex MCC that inhibits the APC/C (Millband and Hardwick, 2002Millband D.N. Hardwick K.G. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner.Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar, Sudakin et al., 2001Sudakin V. Chan G.K. Yen T.J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (665) Google Scholar, Yu, 2002Yu H. Regulation of APC-Cdc20 by the spindle checkpoint.Curr. Opin. Cell Biol. 2002; 14: 706-714Crossref PubMed Scopus (289) Google Scholar). BubR1 is similar to the fungal spindle checkpoint protein Mad3 (Taylor et al., 1998Taylor S.S. Ha E. McKeon F. The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase.J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar), but its carboxy terminus contains a kinase domain that is not present in Mad3 (Millband and Hardwick, 2002Millband D.N. Hardwick K.G. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner.Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar, Taylor et al., 1998Taylor S.S. Ha E. McKeon F. The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase.J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). Bub1 phosphorylates Cdc20, has a key role in the assembly of checkpoint proteins, and seems to have an additional noncheckpoint function (Johnson et al., 2004Johnson V.L. Scott M.I. Holt S.V. Hussein D. Taylor S.S. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.J. Cell Sci. 2004; 117: 1577-1589Crossref PubMed Scopus (264) Google Scholar, Yu and Tang, 2005Yu H. Tang Z. Bub1 multitasking in mitosis.Cell Cycle. 2005; 4: 262-265Crossref PubMed Scopus (48) Google Scholar). The association of the mitotic checkpoint proteins with the kinetochore is regulated during mitosis. Vertebrate Mad2 is bound to unattached kinetochores (Chen et al., 1996Chen R.H. Waters J.C. Salmon E.D. Murray A.W. Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores.Science. 1996; 274: 242-246Crossref PubMed Scopus (379) Google Scholar, Li and Benezra, 1996Li Y. Benezra R. Identification of a human mitotic checkpoint gene: hsMAD2.Science. 1996; 274: 246-248Crossref PubMed Scopus (520) Google Scholar). Other checkpoint proteins in vertebrates, Bub1 and BubR1 (Taylor et al., 2001Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar) and fungal Mad2, Bub1, and Mad3 (Gillett et al., 2004Gillett E.S. Espelin C.W. Sorger P.K. Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast.J. Cell Biol. 2004; 164: 535-546Crossref PubMed Scopus (153) Google Scholar, Millband and Hardwick, 2002Millband D.N. Hardwick K.G. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner.Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar) also transiently associate with kinetochores. The universality of temporal kinetochore association suggests that the kinetochore binding per se is mechanistically relevant in checkpoint control. The target of the checkpoint proteins, APC/C, is abundant in nonkinetochore regions, however, whereas only a portion is present in the kinetochores (Acquaviva et al., 2004Acquaviva C. Herzog F. Kraft C. Pines J. The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint.Nat. Cell Biol. 2004; 6: 892-898Crossref PubMed Scopus (90) Google Scholar, Kallio et al., 1998Kallio M. Weinstein J. Daum J.R. Burke D.J. Gorbsky G.J. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events.J. Cell Biol. 1998; 141: 1393-1406Crossref PubMed Scopus (221) Google Scholar). It has therefore been proposed that a Mad2-containing protein complex at the kinetochore acts as a template to propagate a conformational change of Mad2 that promotes its affinity for Cdc20 that then amplifies this “wait anaphase” signal throughout the entire cell (De Antoni et al., 2005De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. et al.The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Other models, which include a kinetochore-independent anaphase inhibitor role for the MCC (e.g., Sudakin et al., 2001Sudakin V. Chan G.K. Yen T.J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (665) Google Scholar), have also been proposed. A key step in unlocking the enigma of the spindle checkpoint is the elucidation of the mechanism by which checkpoint proteins are recruited to kinetochores. Establishing that certain kinetochore proteins are required for the kinetochore recruitment of checkpoint proteins was a first step toward this aim (Martin-Lluesma et al., 2002Martin-Lluesma S. Stucke V.M. Nigg E.A. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.Science. 2002; 297: 2267-2270Crossref PubMed Scopus (375) Google Scholar, McAinsh et al., 2006McAinsh A.D. Meraldi P. Draviam V.M. Toso A. Sorger P.K. The human kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome segregation.EMBO J. 2006; 25: 4033-4049Crossref PubMed Scopus (64) Google Scholar). One such kinetochore component is hMis12 (Goshima et al., 2003Goshima G. Kiyomitsu T. Yoda K. Yanagida M. Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.J. Cell Biol. 2003; 160: 25-39Crossref PubMed Scopus (196) Google Scholar). hMis12 is essential for chromosome segregation and forms a stable complex with several proteins, including AF15q14 (Cheeseman et al., 2004Cheeseman I.M. Niessen S. Anderson S. Hyndman F. Yates 3rd, J.R. Oegema K. Desai A. A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension.Genes Dev. 2004; 18: 2255-2268Crossref PubMed Scopus (334) Google Scholar, Obuse et al., 2004Obuse C. Iwasaki O. Kiyomitsu T. Goshima G. Toyoda Y. Yanagida M. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1.Nat. Cell Biol. 2004; 6: 1135-1141Crossref PubMed Scopus (213) Google Scholar). AF15q14 belongs to the Spc105 protein family (the founding member is the budding yeast Spc105; Nekrasov et al., 2003Nekrasov V.S. Smith M.A. Peak-Chew S. Kilmartin J.V. Interactions between centromere complexes in Saccharomyces cerevisiae.Mol. Biol. Cell. 2003; 14: 4931-4946Crossref PubMed Scopus (77) Google Scholar, Wigge et al., 1998Wigge P.A. Jensen O.N. Holmes S. Soues S. Mann M. Kilmartin J.V. Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.J. Cell Biol. 1998; 141: 967-977Crossref PubMed Scopus (270) Google Scholar) and is similar to fission yeast Spc7, nematode KNL-1, and fly dmSpc105R (Desai et al., 2003Desai A. Rybina S. Muller-Reichert T. Shevchenko A. Shevchenko A. Hyman A. Oegema K. KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans.Genes Dev. 2003; 17: 2421-2435Crossref PubMed Scopus (182) Google Scholar, Kerres et al., 2004Kerres A. Vietmeier-Decker C. Ortiz J. Karig I. Beuter C. Hegemann J. Lechner J. Fleig U. The fission yeast kinetochore component Spc7 associates with the EB1 family member Mal3 and is required for kinetochore-spindle association.Mol. Biol. Cell. 2004; 15: 5255-5267Crossref PubMed Scopus (37) Google Scholar, Przewloka et al., 2007Przewloka M.R. Zhang W. Costa P. Archambault V. D'Avino P.P. Lilley K.S. Laue E.D. McAinsh A.D. Glover D.M. Molecular analysis of core kinetochore composition and assembly in Drosophila melanogaster.PLoS ONE. 2007; 2: e478Crossref PubMed Scopus (103) Google Scholar). It was initially identified because an oncogenic chromosome translocation resulted in the fusion of AF15q14 with the MLL gene in acute myeloid leukemia (Hayette et al., 2000Hayette S. Tigaud I. Vanier A. Martel S. Corbo L. Charrin C. Beillard E. Deleage G. Magaud J.P. Rimokh R. AF15q14, a novel partner gene fused to the MLL gene in an acute myeloid leukaemia with a t(11;15)(q23;q14).Oncogene. 2000; 19: 4446-4450Crossref PubMed Scopus (43) Google Scholar). We report that the human protein AF15q14 is the direct target kinetochore protein for both Bub1 and BubR1. AF15q14 interacts with the tetratrichopeptide repeat (TPR) motifs present in both Bub1 and BubR1 and these interactions are required for their mitotic functions. Our data also suggest that this kinetochore protein has a role not only in the association with checkpoint proteins Bub1 and BubR1, but also in the attachment to the spindle microtubules. Mass spectroscopic analysis (Cheeseman et al., 2004Cheeseman I.M. Niessen S. Anderson S. Hyndman F. Yates 3rd, J.R. Oegema K. Desai A. A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension.Genes Dev. 2004; 18: 2255-2268Crossref PubMed Scopus (334) Google Scholar, Obuse et al., 2004Obuse C. Iwasaki O. Kiyomitsu T. Goshima G. Toyoda Y. Yanagida M. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1.Nat. Cell Biol. 2004; 6: 1135-1141Crossref PubMed Scopus (213) Google Scholar) established that human hMis12 coprecipitates with six centromere/kinetochore proteins (PMF1, DC8, c20orf172, HEC1/Ndc80, Zwint-1, and AF15q14). With the exception of Zwint-1, putative homologs of all of these proteins are present in S. cerevisiae and S. pombe (Figure S1, see the Supplemental Data available with this article online). Because kinetochore recruitment of Bub1 and BubR1 (hereafter designated Bubs) requires the presence of the hMis12 complex (Kline et al., 2006Kline S.L. Cheeseman I.M. Hori T. Fukagawa T. Desai A. The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation.J. Cell Biol. 2006; 173: 9-17Crossref PubMed Scopus (160) Google Scholar, McAinsh et al., 2006McAinsh A.D. Meraldi P. Draviam V.M. Toso A. Sorger P.K. The human kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome segregation.EMBO J. 2006; 25: 4033-4049Crossref PubMed Scopus (64) Google Scholar; unpublished data), we examined whether Bubs coprecipitated with hMis12. For this approach, we generated a HeLa cell line that stably expressed FLAG-hMis12, and extracts were immunoprecipitated using anti-FLAG antibodies (Figure 1A). The resulting precipitates contained Bubs. Relatively little Bubs precipitated with FLAG-Mis12 in the asynchronous (AS) or S-phase arrested (DTB, double thymidine block) cultures, but the levels rose sharply when cells were arrested in mitosis using nocodazole (Noc). Control precipitations from nontagged HeLa cells did not coprecipitate with Bubs, indicating that Bubs formed a complex that contained hMis12 specifically during Noc arrest. Consistently, Bub1 coprecipitated with both endogenous hMis12 and FLAG-hMis12 in the reverse immunoprecipitation experiment (Figure S2). To identify the molecules that associate with Bubs, we conducted pairwise 2- and 3-hybrid (Tirode et al., 1997Tirode F. Malaguti C. Romero F. Attar R. Camonis J. Egly J.M. A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.J. Biol. Chem. 1997; 272: 22995-22999Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) screens between Bubs and all components of the hMis12 kinetochore complex. The panel for a set of 2-hybrids using 10 kinetochore proteins (Figure 1B) revealed interactions between AF15q14 and Bubs (the fragments Bub1 N150 and BubR1 N203 were used). We propose to use a human-specific name, blinkin (bub-linking kinetochore protein), for AF15q14. Blinkin was truncated into three fragments, the amino-terminal BLKN (1–728), the middle BLKM (729–1833), and the carboxy-terminal BLKC (1834–2316). Both Bub1 and BubR1 interacted with the amino-terminal BLKN. Blinkin is one of the largest kinetochore proteins and may directly interact with multiple kinetochore proteins. Extending the 2-hybrid analysis to pairwise combinations with all kinetochore components established that BLKC interacted with Zwint-1 (Figure 1C), whereas no 2-hybrid interaction was detected with hMis12. Human hMis12 is thought to form a heterotetrameric complex (Kline et al., 2006Kline S.L. Cheeseman I.M. Hori T. Fukagawa T. Desai A. The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation.J. Cell Biol. 2006; 173: 9-17Crossref PubMed Scopus (160) Google Scholar). Judging from the results of extensive 2- and 3-hybrid analyses, two heterodimers (hMis12-hNnf1/PMF1 and hMis13/c20orf172-hMis14/DC8) formed (Figure S3A) and were connected by further interaction between hMis13 and hMis12-hNnf1 (Figure S3B and Figure S3C). The bifunctional nature of hMis13 might thus enable the formation of a heterotetrameric complex. Among the many constructs examined, a positive 3-hybrid interaction was detected between BLKC and hMis13-hMis14 (Figure 1D and data not shown). BLKN thus interacts with Bubs and BLKC interacts with Zwint-1 and the hMis13-hMis14 complex. No interaction was detected between any domain of blinkin and HEC1 (or HEC1/Ndc80 complex subunits), whereas HEC1 interacted with Zwint-1 (Figure 1C and Figure S3D). A mouse monoclonal antibody was raised against the amino-terminal 22 amino acid peptide of blinkin (Experimental Procedures). A band was detected at the expected MW of ∼300 kD in immunoblots of HeLa extracts (Figure 1E), which disappeared in RNAi cells (described below). The band shifted to a slower migrating form in Noc-arrested cells (Figure 1F, left), in which the Bub1 band is transformed into multiple intense bands that migrate more slowly than Bub1 in untreated extracts (Taylor et al., 2001Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar). Subsequent removal of Noc resulted in a change of the upper band back to the lower position within 30 to 60 min without a significant reduction in band intensity (Figure 1F, right). This finding contrasts with Bub1 and cyclin B1 bands, which are both lost during recovery from Noc arrest. Thus, blinkin seemed to be modified during Noc arrest and its level did not fluctuate with cell cycle progression. Monoclonal antibodies were used to compare the distribution of blinkin with that of Bub1 using immunofluorescence microscopy. The closely apposed kinetochore signals were intense at both prometaphase and metaphase (Figure 1G), whereas Bub1 kinetochore localization was only faintly observed in metaphase cells as previously described (Taylor et al., 2001Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar). Examination of the kinetochore localization of blinkin alongside GFP-hMis12 in the HeLa cell line revealed that the two proteins had very similar distributions (Figure S4). Weak centromeric signals were observed in interphase nuclei. The mitotic kinetochore signals became strong from prophase to late anaphase, but greatly diminished from the telophase and cytokinesis to early G1. This kinetochore behavior was similar to that of GFP-hMis12. We hypothesized that blinkin may bind to sequences that are conserved between Bub1 and BubR1 (Figure 1H, blue and red). The amino termini of both proteins contain a TPR (tetratrichopeptide repeat) motif (Bolanos-Garcia et al., 2005Bolanos-Garcia V.M. Beaufils S. Renault A. Grossmann J.G. Brewerton S. Lee M. Venkitaraman A. Blundell T.L. The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity.Biophys. J. 2005; 89: 2640-2649Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) (Figure 1H, red box). The amino fragments of Bub1 and BubR1 exhibited 2-hybrid interactions with BLKN, but not with BLKC (Figure 1I). The middle domain, BLKM, had a less intense interaction with the Bub1 fragment. Full-length Bub1 interacted weakly with BLKN and BLKM, but interacted strongly with Bub3. The central domain of Bub1 interacts with Bub3 (Taylor et al., 1998Taylor S.S. Ha E. McKeon F. The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase.J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). We constructed substitution mutants in the two Bub1 residues (indicated by the arrows in Figure 1H) required for the essential conformation of the TPR motif (D'Andrea and Regan, 2003D'Andrea L.D. Regan L. TPR proteins: the versatile helix.Trends Biochem. Sci. 2003; 28: 655-662Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, Hirano et al., 1990Hirano T. Kinoshita N. Morikawa K. Yanagida M. Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+.Cell. 1990; 60: 319-328Abstract Full Text PDF PubMed Scopus (238) Google Scholar), and examined the impact of these changes upon the 2-hybrid interactions. Three amino acids, D, G, and W, were used to alter the A106 residue. The only change that could maintain the 2-hybrid interaction was a change to the similarly-sized G (Gly). This is consistent with the hypothesis that the knob-hole-like conformation predicted for the TPR motif (Hirano et al., 1990Hirano T. Kinoshita N. Morikawa K. Yanagida M. Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+.Cell. 1990; 60: 319-328Abstract Full Text PDF PubMed Scopus (238) Google Scholar) is functionally relevant (Figure 1J). For the L122 residue, which has a bulky hydrophobic side chain, substitution by the smaller side chain of G abolished the interaction, whereas substitution by the bulky F did not diminish the strong positive interaction. Furthermore, the insertion of G between A104 and W105 abolished the 2-hybrid interaction. This is consistent with the predicted structure of the TPR conformation. Similar results were obtained for the BubR1 substitution mutants in TPR (Figure 1K). The substitution of A159 by G, but not by W, maintained a positive 2-hybrid interaction, and the substitution of F175 by L, but not by A, maintained the interaction with blinkin. Thus, the TPR motifs of Bubs specifically interact with BLKN. RNA interference was performed in HeLa cells to determine whether blinkin was required for the recruitment of Bubs to the kinetochores. The protein level was negligible 24 hr after RNAi (Figure 2A, upper panel). Consistently, the kinetochore signals were abolished in mitotic cells (Figure 2A, lower panel). The Bubs signals were diffused in blinkin RNAi cells (Figure 2B, top). The kinetochore signals of blinkin were unaffected, however, when the Bubs themselves were knocked down by RNAi (Figure 2B, middle). Control RNAi also did not affect the kinetochore signals for blinkin, Bub1, or BubR1 (Figure 2B, bottom). Kinetochore localization of blinkin was thus not affected by the absence of Bubs, whereas Bubs' localization at the kinetochore required blinkin. Note that the protein levels of Bubs assayed by immunoblot did not decrease in blinkin RNAi (Figure 2C). Levels of blinkin and hMis12 were not affected in Bubs' RNAi cells. The level of blinkin was considerably diminished in hMis12 RNAi, however, and hMis12 was slightly diminished in blinkin RNAi. Consistently, the kinetochore signals of blinkin were diminished in hMis12 RNAi cells (Figure 2C, right panel). To determine whether spindle checkpoint control was retained in blinkin RNAi cells, a number of movies were taken to determine the timing of anaphase after nuclear envelope breakdown (NEBD) (Figure 2D, table). Control RNAi cells underwent commitment to anaphase with normal timing (duration = 29 min, n = 22; Figure 2E and Movie S1). Chromosome alignment and segregation were also normal in all of the control RNAi cells. In great contrast, in blinkin RNAi cells accelerated mitosis with chromosome misalignment occurred frequently (∼65%, n = 69). The remaining cells had a mitotic delay, probably due to the incomplete knockdown of blinkin protein that activated the spindle checkpoint. The same phenotypes were observed with different siRNA sequences (Figure S5) and were rescued by RNAi-resistant blinkin plasmids (see Figure 6H and data not shown), indicating that the phenotypes were due to a loss of blinkin. Three example cells with accelerated mitosis (timing ≈ 20 min) are shown in the movies represented in Figure 2F (Movies S2–S4). All control BubR1 RNAi cells (n = 25) produced accelerated aberrant mitosis (Figure 2G and Movie S5), mostly accompanied by brief and partial alignment (21/25). If, however, double RNAi of BubR1 and Bub1 was performed, accelerated mitosis with extensive chromosome misalignment was observed (Figure 2H and Movie S6). The phenotypes of chromosome behavior during accelerated mitosis in blinkin RNAi and the double BubR1 and Bub1 RNAi were very similar. Most control Bub1 RNAi cells, however, had delayed mitosis with chromosome alignment defects (Movie S7). Immunofluorescence microscopy revealed that micronuclei frequently formed after blinkin RNAi (Figure 2I, arrows). Other cells with many nuclear-like structures in various sizes were abundant (Figure 2I, arrowhead). These results suggested that chromosome segregation was greatly impaired in accelerated mitosis. Another criterion for the spindle checkpoint was the arrest in mitosis by the addition of the microtubule poisons Noc or taxol. In control RNAi, 70%–75% of cells became round, indicating mitosis, while only 30% cells were round after BubR1 or blinkin RNAi (Figure 2J). These results showed that mitotic stage was not maintained in blinkin RNAi if tubulin poisons were added. Through-focus images of fixed mitotic cells were taken in the presence of MG132, a proteasome inhibitor, using antibodies against tubulin and CENP-C and Hoechst 33342 for DNA. The kinetochore-spindle attachment was defective in blinkin RNAi cells (Figure 3A and Figure S6). Consistent with the lack of proper metaphase configuration, the mean distance between sister kinetochores was 0.77 ± 0.18 μm (n = 77), similar to that in prophase 0.70 ± 0.17 μm (n = 45) in control cells, but significantly shorter than that of 1.38 ± 0.22 μm (n = 37) in control metaphase cells that were under bipolar tension due to the attachment of the metaphase spindle. Microtubules attached to kinetochore are resistant to cold treatment (DeLuca et al., 2005DeLuca J.G. Dong Y. Hergert P. Strauss J. Hickey J.M. Salmon E.D. McEwen B.F. Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites.Mol. Biol. Cell. 2005; 16: 519-531Crossref PubMed Scopus (199) Google Scholar), so blinkin RNAi cells were cold treated in the presence of MG132 to determine if the microtubules were attached to the kinetochores. The kinetochore dots remained within the zone of the kinetochore microtubules in control RNAi cells (Figure 3B, left), while in blinkin RNAi cells, kinetochore signals were broadly distributed in the absence of cold-resistant kinetochore microtubules (Figure 3B, right). These" @default.
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• W1974559008 creator A5004377531 @default.
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