Human NUF2 Interacts with Centromere-associated Protein E and Is Essential for a Stable Spindle Microtubule-Kinetochore Attachment

Abstract

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here, we show that Homo sapiens (Hs) NUF2 is required for stable kinetochore localization of centromere-associated protein E (CENP-E) in HeLa cells. HsNUF2 specifies the kinetochore association of CENP-E by interacting with its C-terminal domain. The region of HsNUF2 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pulldown and yeast two-hybrid assays. Suppression of synthesis of HsNUF2 by small interfering RNA abrogated the localization of CENP-E to the kinetochore, demonstrating the requirement of HsNUF2 for CENP-E kinetochore localization. In addition, depletion of HsNUF2 caused aberrant chromosome segregation. These HsNUF2-suppressed cells displayed reduced tension at kinetochores of bi-orientated chromosomes. Double knockdown of CENP-E and HsNUF2 further abolished the tension at the kinetochores. Our results indicate that HsNUF2 and CENP-E are required for organization of stable microtubule-kinetochore attachment that is essential for faithful chromosome segregation in mitosis. Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here, we show that Homo sapiens (Hs) NUF2 is required for stable kinetochore localization of centromere-associated protein E (CENP-E) in HeLa cells. HsNUF2 specifies the kinetochore association of CENP-E by interacting with its C-terminal domain. The region of HsNUF2 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pulldown and yeast two-hybrid assays. Suppression of synthesis of HsNUF2 by small interfering RNA abrogated the localization of CENP-E to the kinetochore, demonstrating the requirement of HsNUF2 for CENP-E kinetochore localization. In addition, depletion of HsNUF2 caused aberrant chromosome segregation. These HsNUF2-suppressed cells displayed reduced tension at kinetochores of bi-orientated chromosomes. Double knockdown of CENP-E and HsNUF2 further abolished the tension at the kinetochores. Our results indicate that HsNUF2 and CENP-E are required for organization of stable microtubule-kinetochore attachment that is essential for faithful chromosome segregation in mitosis. The kinetochore is a supramolecular complex assembled at each centromere in eukaryotes. It provides a chromosomal attachment point for the mitotic spindle, linking the chromosome to the microtubule, and functions in initiating, controlling, and monitoring the movements of chromosomes during mitosis. The kinetochore of animal cells contains two regions: the inner kinetochore, which is tightly and persistently associated with centromeric DNA sequences throughout the cell cycle, and the outer kinetochore, which contains many dynamic protein components that interact with microtubules only during mitosis. The stable propagation of eukaryotic cells requires that each chromosome is accurately duplicated and faithfully segregated. During mitosis, attaching, positioning, and bi-orientating kinetochores with the spindle microtubules play essential roles in chromosome segregation and genomic stability (see Refs. 1Fukagawa T. Exp. Cell Res. 2004; 296: 21-27Crossref PubMed Scopus (39) Google Scholar, 2Amor D.J. Kalitsis P. Sumer H. Choo K.H. Trends Cell Biol. 2004; 14: 359-368Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 3Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, 4Maiato H. DeLuca J. Salmon E.D. Earnshaw W.C. J. Cell Sci. 2004; 117: 5461-5477Crossref PubMed Scopus (323) Google Scholar). Centromere-associated protein E (CENP-E) 3The abbreviations used are: CENP-E, centromere-associated protein E; Hs, Homo sapiens; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MBP, maltose-binding protein; GFP, green fluorescent protein; aa, amino acids; X-α-gal, 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside; siRNA, small interfering RNA; ACA, anti-centromere antibodies. is a microtubule-based kinesin motor protein located on the outer kinetochore and is responsible for a stable microtubule-kinetochore attachment (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar). CENP-E participates in the chromosome movements from prometaphase to anaphase (6Yen T.J. Compton D.A. Wise D. Zinkowski R.P. Brinkley B.R. Earnshaw W.C. Cleveland D.W. EMBO J. 1991; 10: 1245-1254Crossref PubMed Scopus (300) Google Scholar). It moves toward and tethers kinetochores to microtubule “plus” ends (7Wood K.W. Sakowicz R. Goldstein L.S.B. Cleveland D.W. Cell. 1997; 91: 357-366Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), helping mono-oriented chromosomes align at the metaphase plate before bi-orientation (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar, 6Yen T.J. Compton D.A. Wise D. Zinkowski R.P. Brinkley B.R. Earnshaw W.C. Cleveland D.W. EMBO J. 1991; 10: 1245-1254Crossref PubMed Scopus (300) Google Scholar, 7Wood K.W. Sakowicz R. Goldstein L.S.B. Cleveland D.W. Cell. 1997; 91: 357-366Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 8Schaar B.T. Chan G.K. Maddox P. Salmon E.D. Yen T.J. J. Cell Biol. 1997; 139: 1373-1382Crossref PubMed Scopus (268) Google Scholar). During this process, its location on the kinetochore depends on BUBR1 and CENP-F (9Chan G.K. Schaar B.T. Yen T.J. J. Cell Biol. 1998; 143: 49-63Crossref PubMed Scopus (227) Google Scholar). Unattached kinetochores are also the sensing apparatus for the mitotic checkpoint, which delays anaphase onset until all kinetochores have properly attached to spindle microtubules (10Rieder C.L. Schultz A. Cole R. Sluder G. J. Cell Biol. 1994; 127: 1301-1310Crossref PubMed Scopus (449) Google Scholar, 11Li X. Nicklas R.B. Nature. 1995; 373: 630-632Crossref PubMed Scopus (470) Google Scholar). In this context, CENP-E as a kinetochore attachment sensor is also involved in a spindle checkpoint signaling pathway. It activates BUBR1-related checkpoint signaling in a microtubule-dependent manner (12Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 13Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (119) Google Scholar), and CENP-E/BUBR1 interaction is quite important in controlling this spindle checkpoint (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar, 12Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 13Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (119) Google Scholar). Thus, CENP-E is necessary for microtubule-kinetochore conjugation, chromosome congression, and spindle checkpoint activation. Homo sapiens (Hs) NUF2 is a kinetochore protein that forms a stable complex with HEC1, HsSPC24, and HsSPC25 (named the HsNDC80 complex). Depletion of HsNUF2 by RNA interference results in a strong prometaphase block with an active spindle checkpoint, which is correlated with low but detectable MAD2 at kinetochores that have no or few stable kinetochore microtubules (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar). HEC1 was originally identified as a retino-blastoma protein-associated protein. Microinjection of anti-HEC1 antibodies into cultured cells disrupts mitotic progression (15Chen Y. Riley D.J. Chen P.L. Lee W.H. Mol. Cell. Biol. 1997; 17: 6049-6056Crossref PubMed Scopus (115) Google Scholar). Both the Nuf2-Ndc80 complex and its human homolog, NUF2-HEC1, have been shown to be important for stable microtubule-kinetochore attachment, chromosome alignment, and also spindle checkpoint activation in mitosis (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar, 16He X. Rines D.R. Espelin C.W. Sorger P.K. Cell. 2001; 106: 195-206Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17Wigge P.A. Kilmartin J.V. J. Cell Biol. 2001; 152: 349-360Crossref PubMed Scopus (275) Google Scholar, 18Martin-Lluesma 2002; PubMed Scopus Google Scholar, T. T. Y. T. J. Cell Sci. 2003; PubMed Scopus Google Scholar). been that depletion of the localization of CENP-E to the kinetochore 2002; PubMed Scopus Google Scholar, J. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar), the in the CENP-E and the that the CENP-E amino Y. D. X. Sci. Scopus Google Scholar), we a for kinetochore that the kinetochore localization of CENP-E the its as and a yeast two-hybrid in which the kinetochore protein HsNUF2 was identified as of Our the interaction between HsNUF2 and In addition, that HsNUF2 specifies the kinetochore localization of CENP-E and is essential for a stable microtubule-kinetochore Cell and and cells as in with and at in at with for with phosphate-buffered and cultured in for for of for cells for to In was to the cell to cells in of was as X. Cleveland D.W. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). to HsNUF2 was from and used at a for and at a for to HEC1 was from and used at a for and at a for antibodies to glutathione S-transferase and maltose-binding protein from Cell and used at a for to green fluorescent protein was from the C-terminal of human CENP-E was as Y. D. X. Sci. Scopus Google Scholar). human CENP-E and HsNUF2 or into and The used for in and in the and in the DNA activation and in and in in and and in in two-hybrid as Y. J. A. H. J. Y. X. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). two CENP-E and into the of to a with of the domain. The into with the and the was by and an that with the C-terminal the and with the HeLa on and onto on from and with and to of the interaction was by the into with that on for and of and in and glutathione and and as X. X. X. R. J.G. X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). pulldown with and with was with cell of cells with a for and for at The with and for in by for or onto for a interaction between HsNUF2 and CENP-E, HsNUF2 on was used as an for with The with before in The on and the was with by of with into yeast of that on onto to the domain of HsNUF2 interaction with with and on and with and that two to the CENP-E cells at prometaphase as The cells with and was to and for with protein and with and before to for the These protein to the cell and for at the with and with by and onto to RNA the the of the HsNUF2 (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar), HEC1 2002; PubMed Scopus Google Scholar), and CENP-E 2002; PubMed Scopus Google by a of and at for was as the for and with and in a plate and to the HeLa cells onto in and cells with HEC1, or CENP-E and as and as B.T. Chan G.K. Maddox P. Salmon E.D. Yen T.J. J. Cell Biol. 1997; 139: 1373-1382Crossref PubMed Scopus (268) Google Scholar). and antibodies GFP, HEC1, and used at of and a with and of kinetochore protein was a on an with a The from a with The between kinetochores with was as the between the as (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar). of HsNUF2 and CENP-E the tension the kinetochore, we to the of cells the for (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar). The between kinetochores with was as of the of protein was as by D. J. Cell Sci. 2004; 117: PubMed Scopus Google Scholar). In the from at kinetochore from cells and The at each kinetochore to for in or The of cells as a of the from cells with a HsNUF2 a contains an microtubule-based motor a and a C-terminal domain. It been the of CENP-E with kinetochore BUBR1 and of which are to be involved in CENP-E on kinetochores (9Chan G.K. Schaar B.T. Yen T.J. J. Cell Biol. 1998; 143: 49-63Crossref PubMed Scopus (227) Google Scholar). that the interaction between CENP-E and BUBR1 is important in spindle checkpoint activation (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar). It been that CENP-E BUBR1 and activates its and that the spindle checkpoint is as CENP-E a microtubule (12Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 13Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (119) Google Scholar). These indicate that CENP-E is a kinetochore protein in the spindle checkpoint signaling and tension to spindle checkpoint activation. in a human of CENP-E, and we a of amino in the Y. D. X. Sci. Scopus Google and this that with the CENP-E C-terminal of CENP-E used as to a HeLa the yeast two-hybrid as Y. J. A. H. J. Y. X. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). was on a and we identified by and we identified interacting of that of the of HsNUF2 that of HsNUF2 the of interaction between HsNUF2 and CENP-E and to further the that the interaction between we of yeast and to yeast two-hybrid to the binding this we used to the of HsNUF2 from a HeLa cell and we into the the domain responsible for the interaction between HsNUF2 and CENP-E, we a of HsNUF2 to for interaction binding we that The of HsNUF2 is located between and the of CENP-E is located between and we to the association between CENP-E and this a of as in The HsNUF2 each with the of the CENP-E into yeast cells. used as a for shown in yeast that a region to the of HsNUF2 to the CENP-E C-terminal Thus, yeast two-hybrid that the of CENP-E with the in the binding region of we of HsNUF2 on the of HsNUF2 with CENP-E C-terminal into yeast cells. the region of HsNUF2 to the CENP-E C-terminal and but its interaction to be to that of and which that the of HsNUF2 is required for CENP-E binding the interaction between CENP-E and HsNUF2 in yeast two-hybrid we a pulldown protein as an to the binding of the HsNUF2 protein and its this HsNUF2 and its in cells to HsNUF2 as in of mitotic cell and its with shown only HsNUF2 and to CENP-E on on the as by of the of the region and abolished the binding of HsNUF2 to Thus, that the HsNUF2 to CENP-E and that of HsNUF2 are essential for binding the HsNUF2 protein to the CENP-E protein we a pulldown in which HsNUF2 was on and used as an to in shown in and HsNUF2 and a interaction between CENP-E and HsNUF2 in was by a interaction between HsNUF2 and Thus, we that HsNUF2 with CENP-E its C-terminal HsNUF2 a with the the interaction between CENP-E and HsNUF2 in pulldown and CENP-E forms a complex with we used to CENP-E and from of HeLa cells. with a of CENP-E, and HsNUF2 that HsNUF2 was of HsNUF2 a the of a complex of HsNUF2 and CENP-E, the of HsNUF2 to CENP-E in the was that with a of CENP-E HsNUF2 was with and and no was in of the Thus, we that HsNUF2 and CENP-E a complex in HsNUF2 forms a complex with CENP-E, HsNUF2 also interact with CENP-E in this and to that the HsNUF2 domain responsible for CENP-E binding in also interaction in we used to and from of HeLa cells to HsNUF2 and with a of and CENP-E that CENP-E was with HsNUF2 and and was CENP-E was by that in and in HsNUF2 and HEC1 CENP-E to the to is an important of the HsNDC80 centromere complex J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar). the of HsNUF2 on the localization of CENP-E to the kinetochore and on the of mitotic checkpoint we to HsNUF2 and HEC1 by into HeLa cells. that of HeLa cells with for of the shown in with that the caused of HsNUF2 protein at was as the of as of HEC1 and CENP-E was also a as by HsNUF2 and HEC1 a stable and we the of HEC1 and HsNUF2 on the localization of CENP-E to the HeLa cells on in and with with to the of prometaphase cells. of the of kinetochore to the of microtubules (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar, J. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar), we used to HeLa cells to the in CENP-E cells from for we the and an In HsNUF2 and HEC1 with CENP-E at the prometaphase kinetochores In cells in which HEC1 been the of HsNUF2 reduced of HEC1 was reduced to of its HsNUF2 reduced to that HEC1 is required for kinetochore localization of with a J.G. Y. P. J. Hickey J.M. Salmon E.D. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). in cells in which HEC1 been the of CENP-E detectable at kinetochores CENP-E of HEC1 was reduced to of its CENP-E reduced to we the of In cells in which HsNUF2 been the of HEC1 and CENP-E reduced of HsNUF2 was reduced to of its HEC1 reduced to CENP-E reduced to that HsNUF2 is required for kinetochore localization of CENP-E and the of CENP-E on the kinetochore localization of HsNUF2 and In HsNUF2 and HEC1 with CENP-E at the prometaphase kinetochores In cells in which CENP-E been the of HEC1 and CENP-E CENP-E of CENP-E was reduced to of its HEC1 of the the of HsNUF2 reduced to that CENP-E is required for kinetochore localization of HsNUF2 and HEC1, with a J.G. Y. P. J. Hickey J.M. Salmon E.D. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). Thus, we that HEC1 and HsNUF2 are required for stable association of CENP-E with the kinetochore and that HsNUF2 HEC1 to an HsNUF2 the of forms a complex with HEC1, HsSPC24, and HsSPC25 D.J. 2003; 17: PubMed Scopus Google Scholar, R. H. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). HsNUF2 is the for CENP-E localization to the kinetochore, of which contains in the of HsNUF2 the association of CENP-E with the In this as a as the HsNUF2 this HeLa cells with HsNUF2 siRNA, by a of the was used to cells in prometaphase as shown in of the CENP-E been from the kinetochore in HsNUF2 cells. with CENP-E at the kinetochore that the HsNUF2 is for the kinetochore localization of of the kinetochore localization of CENP-E in the cells of HsNUF2 shown in of that of in the cells the CENP-E from to of its that interaction the kinetochore localization of CENP-E with HsNUF2 to a have that CENP-E and the complex are essential for microtubule-kinetochore (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar, 16He X. Rines D.R. Espelin C.W. Sorger P.K. Cell. 2001; 106: 195-206Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, J.G. Y. P. J. Hickey J.M. Salmon E.D. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). between the kinetochores by been used as an for the tension the kinetochore X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar). In this aberrant microtubule attachment to the kinetochore, in which tension is the the of microtubule in the cells of HEC1, CENP-E, or HsNUF2 and HEC1, we this in kinetochore in which kinetochores in the in cells and cells shown in depletion of HEC1, or CENP-E in in chromosome at the which is with Refs. X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar, J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar, and 18Martin-Lluesma 2002; PubMed Scopus Google Scholar). kinetochores a of the between kinetochores in in and in cells. depletion of HEC1 and HsNUF2 further of the depletion of HsNUF2 and CENP-E in of the that of CENP-E and HsNUF2 the tension the this we the in cells. The between kinetochores in in which kinetochore no was this in and cells was that CENP-E and HsNUF2 are required for stable microtubule-kinetochore Thus, we that CENP-E with HsNUF2 in microtubule-kinetochore The HsNDC80 complex is of HEC1, HsSPC24, and HsNDC80 is an essential kinetochore with a in chromosome and segregation during mitosis (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar, 17Wigge P.A. Kilmartin J.V. J. 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Cell. 127: Full Text Full Text PDF PubMed Scopus Google Scholar). Our of an interaction provides a between spindle microtubules and the kinetochore complex the mitotic kinesin CENP-E and that the HsNDC80 complex a between and in to a with CENP-E in microtubule of an interaction we that CENP-E the kinetochore complex with the spindle the of of the interaction and the of an interaction in microtubule-kinetochore in mitosis, we a for the interaction of CENP-E with HsNUF2 in microtubule-kinetochore The CENP-E domain and domain interact with the microtubule and the association between the microtubule and the complex a the HsNUF2 is a stable microtubule-kinetochore interaction is of an in complex which the association of HEC1 with the spindle microtubule shown in that the domain of HsNUF2 in cells the kinetochore localization of CENP-E but to that the of HsNUF2 is essential for faithful chromosome segregation. In of in but cells that the is which is reduced with that of cells but of CENP-E, HEC1, or HsNUF2 cells. that microtubule-kinetochore attachment in cells in the of contains the binding required for of the HsNDC80 is that a complex with HsNDC80 components DeLuca J. D. H. Kilmartin J. Salmon E.D. A. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). be of to interacting with HEC1, HsSPC24, and HsSPC25 HsNUF2 and with CENP-E in microtubule-kinetochore attachment in chromosome movements in mitosis. of HsNDC80 that the HsNDC80 complex two Y. T. H. T. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, Sorger P.K. Sci. A. 2005; PubMed Scopus Google Scholar, DeLuca J. D. H. Kilmartin J. Salmon E.D. A. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). but is HsNDC80 components are toward the centromere and toward the that with the complex Y. T. H. T. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar), and we have also that to the and X. These results that play a in the HsNDC80 complex to the kinetochore during mitosis. In CENP-E is a kinetochore outer plate and its the microtubule at X. Cleveland D.W. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). Thus, the of the kinetochore and The interaction that the complex be toward the microtubule and the kinetochore localization of this is with that the kinetochore localization of HsNDC80 and HsNUF2 depends on and HsSPC25 but that the R. H. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Espelin C.W. Sorger P.K. J. Cell Biol. 2004; PubMed Scopus Google Scholar). CENP-E is also an important protein in chromosome P. D. Salmon E.D. A. PubMed Scopus Google and spindle checkpoint (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (309) Google Scholar, 12Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 13Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (119) Google Scholar). interaction with BUBR1 activates its which is important in the spindle checkpoint (12Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The of microtubules by CENP-E checkpoint signaling Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (119) Google Scholar), but CENP-E is indicate that the complex is also necessary for segregation of of HsNUF2 or HEC1 to a strong prometaphase block (14DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (230) Google Scholar, Y. Riley D.J. Chen P.L. Lee W.H. Mol. Cell. Biol. 1997; 17: 6049-6056Crossref PubMed Scopus (115) Google Scholar, 16He X. Rines D.R. Espelin C.W. Sorger P.K. Cell. 2001; 106: 195-206Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17Wigge P.A. 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It is that the interaction of CENP-E with the microtubule and HsNUF2 its or BUBR1 the spindle checkpoint on or of CENP-E, HEC1, or two of to the of microtubule-kinetochore attachment and the and the spindle checkpoint the CENP-E/BUBR1 pathway. that the of CENP-E and HsNUF2 is necessary in microtubule-kinetochore attachment, and the spindle It is that all of the kinetochore outer plate interact to a kinetochore during chromosome segregation. The interaction is a of this which kinetochore components to spindle microtubule attachment in the The interaction of HEC1 with and that been Y. T. H. T. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, Chen Y. G. Lee W.H. PubMed Scopus Google also of for during the of this with