This page has been archived and is no longer updated
Evolutionary conservation between budding yeast and human kinetochores
Author: Katsumi Kitagawa
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"� 2001 Macmillan Magazines Ltd 678 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio REVIEWS The molecular mechanisms that ensure the accurate segregation of chromosomes during mitosis are funda- mental to the conservation of chromosomal EUPLOIDY in eukaryotic organisms. Accurate chromosome segrega- tion requires functional domains within the chromoso- mal DNA, as well as the coordinated activity of many proteins within the cell cycle. The kinetochore ? centromeric DNA and associat- ed proteins ? and its regulating system are essential for the segregation of chromosomes during mitosis. The kinetochore provides the point of attachment to the mitotic spindle, and is the site through which comple- tion of metaphase is sensed by the cell-cycle regulatory machinery, which coordinates the synchronous separa- tion of chromosomes at the onset of anaphase (BOX 1). The kinetochore of the budding yeast Saccharomyces cerevisiae is the best characterized, and in this review we discuss the conservation and diversity of kinetochore function between budding yeast and humans. Humans versus yeast Classic cytological studies in ?larger? eukaryotic cells (mammalian cells, amphibian cells and insect cells, for example) have provided a detailed description of spin- dle dynamics and chromosome movements during mitosis and meiosis. Although these studies have set the stage for our understanding of kinetochore biolo- gy, a combination of biochemical and genetic approaches has been required in various organisms to understand the actual mechanisms that underlie chro- mosome segregation. Observations of chromosome dynamics in ?smaller? eukaryotes ? such as budding yeast ? have been more difficult, and are therefore less well documented, owing to the very small sizes of the individual chromosomes and the inherent difficulty of visualizing them under the microscope. Nevertheless, these ?simpler? organisms are excellent model systems for identifying and analysing the molecular components of chromosome segregation, including those required for kinetochore structure and function. An important challenge in the field has been to determine the extent to which the dynamics of chromosome movements in evolutionarily distant organisms, such as budding yeast and humans, are similar. The logical extension of this question is to define the extent to which the molecular mechanisms are conserved. Kinetochore behaviour Budding yeast kinetochores. The behaviour of kineto- chores during cell-cycle progression in S. cerevisiae has been monitored by three groups, either by tagging with the green fluorescent protein (GFP) and/or by fluores- cence in situ hybridization (FISH) 1?3 . These groups have found that the sister kinetochores separate transiently in the very early spindle at early S phase, whereas the sister arms remain associated (FIG. 1a). (In budding yeast, the centromeric DNA is duplicated in early S phase 4 .) Sister EVOLUTIONARY CONSERVATION BETWEEN BUDDING YEAST AND HUMAN KINETOCHORES Katsumi Kitagawa* and Philip Hieter ?� Accurate chromosome segregation during mitosis requires the correct assembly of kinetochores ? complexes of centromeric DNA and proteins that link chromosomes to spindle microtubules. Studies on the kinetochore of the budding yeast Saccharomyces cerevisiae have revealed functionally novel components of the kinetochore and its regulatory complexes, some of which are highly conserved in humans. *Department of Molecular Pharmacology, St Jude Children?s Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105-2794, USA. ? Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. � Centre for Molecular Medicine and Therapeutics, Vancouver, British Columbia, Canada V5Z 4H4. Correspondence to K.K. e-mail: katsumi.kitagawa@ stjude.org EUPLOIDY An entire set of chromosomes is represented in integer increments (haploid, one set; diploid, two sets; triploid, three sets). � 2001 Macmillan Magazines Ltd NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 679 REVIEWS of bipolar attachment, or in response to spindle dam- age, a ?wait anaphase? signal is generated, which is trans- mitted through the mitotic spindle-checkpoint pathway (BOX 1) to cell-cycle regulators. Over the past three decades, much has been learned about the DNA sequence elements (determinants in cis) and proteins (determinants in trans) that comprise the budding yeast and mammalian kinetochores. At the start of these studies, it was unclear how relevant the analysis of budding yeast kinetochore structure and function would be to our understanding of human kinetochore biology. In this review, we compare the molecular components that are known, at present, to be required for kinetochore function in budding yeast and mammals, and consider them as three sets of subcom- ponents (FIG. 2): first, the chromosomal DNA?innercentromeres repeatedly split and re-associate before finally parting and moving to spindle poles shortly before the onset of anaphase proper, indicating that kinetochores in yeast and humans behave more similar- ly to one another than had previously been suspected. These groups also observed that the kinetochores are closely connected with the spindle pole bodies (SPB) ? the yeast equivalent of the mammalian microtubule- organizing centre (MTOC) ? throughout the cell cycle (FIG. 1a). In budding yeast, the SPB is embedded within the nuclear envelope, and the nuclear envelope does not break down. There seems to be no authentic anaphase- A movement in budding yeast; however, it has been proposed that the decrease in the distance between the kinetochores and the poles (the anaphase-A event) might take place in telophase after full spindle elonga- tion 5,6 . Furthermore, all budding yeast kinetochores cluster together at the spindle poles through inter- phase 1,7 . The anaphase-B event is very similar in the budding yeast and humans. Human kinetochores. The behaviour of kinetochores during cell-cycle progression has been observed in mammalian cells, which contain relatively large kineto- chore structures 8 . In human cells, the centromeric ALPHOID DNA is duplicated in late S phase (FIG. 1).After nuclear-envelope breakdown, kinetochores associate with kinetochore microtubules that connect chromo- somes to the centrosome (the MTOC in humans). Kinetochore?microtubule interactions take place only during mitosis. Bi-orientation of chromosomes (attach- ment of sister centromeres to opposite poles by means of kinetochore microtubules; BOX 2) is established at metaphase, when transient separation and reassociation of kinetochores occurs (FIG. 1b). During anaphase, kinetochores move along the spindle microtubules, and sister chromosomes are thereby pulled to opposite poles (anaphase A; FIG. 1b). After this event, elongation of pole-to-pole micro- tubules increases the distance between the two spindle poles, further separating the sister-chromatid sets (anaphase B; FIG. 1b). Humans versus yeast. In both yeast and mammalian cells, complete bipolar attachment of all chromosomes must occur before anaphase begins. Before completion Box 1 | The spindle checkpoint Cell-cycle checkpoints are cellular control systems that sense the completion of a particular event before allowing another event to proceed 106 . The spindle checkpoint is a surveillance system that can delay mitosis in response to either defective spindle organization, or a failure of the chromosomes to attach correctly to the spindle 107 . How does this checkpoint work? The metaphase-to-anaphase transition and exit from mitosis are initiated in cells by a ubiquitin-mediated proteolysis complex called the ?cyclosome? or ?anaphase-promoting complex? (APC; FIG. 4). During mitosis, the APC is thought to ubiquitylate components that are responsible for sister-chromatid cohesion (triggering anaphase), and also cyclin B (causing exit from mitosis as a result of breakdown of the maturation-promoting factor), resulting in rapid degradation of these targets by the proteasome 108,109 . The spindle checkpoint operates by communicating with the APC machinery so that anaphase will not occur. Figure 1 | Kinetochore function in budding yeast and mammals. The kinetochore is essential for chromosome segregation in both yeast and mammalian cells. a | Budding yeast. The chromosome is attached at its centromere (red) to the spindle pole body (SPB; yellow) through the kinetochore microtubules (aqua). The DNA is replicated in S phase, and the sister chromatids remain together until cohesion is lost and they are pulled to opposite poles during mitosis (M). Elongation of pole-to-pole microtubules (green) increases the distance between the two spindle poles. b | Mammalian cells. Kinetochore?microtubule interactions occur only during mitosis. The kinetochores connect the centromeres to the mammalian equivalent of the SPB, the microtubule- organizing centre (MTOC), and remain attached as the sister chromatids are pulled to opposite poles. G1 Early S Late S G2/M G2 Pro-metaphase Metaphase Anaphase A Anaphase BAnaphase B ab Centromere Kinetochore microtubulesMTOC/SPB Pole-to-pole microtubules ALPHOID DNA ?-satellite DNA; highly repetitive satellite DNA. � 2001 Macmillan Magazines Ltd 680 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio REVIEWS plex 19 , which contains four main proteins of 110, 64, 58 and 29 kDa in size. These proteins are encoded by the NDC10 (also called CTF14 or CBF2), CEP3 (also called CBF3B), CTF13 and SKP1 genes, respectively 19?26 .All four proteins are essential for viability, and temperature- sensitive mutations in any one of them abolish the CDEIII-binding activity of the CBF3 complex. In addi- tion, Ndc10?GFP has been found to localize not only at kinetochores, but also along the spindle, indicating that Ndc10 might also function at the spindles 1 . The Skp1 protein and its interaction partner Sgt1 are required for the assembly of the CBF3 complex through activation of Ctf13 (REFS 27,28). Skp1 and Sgt1 also seem to be compo- nents of the SCF UBIQUITIN-LIGASE COMPLEX 28?30 , although the connection between kinetochore and SCF function is still unclear. Human centromeres. The human kinetochore is the tril- aminar proteinaceous structure that interfaces with chromosomes at highly repeated alphoid DNA (171-bp) arrays of 2?4 Mb 31 (see also BOX 3). Several years ago, two independent groups, Harrington et al. 32 and Ikeno et al. 33 , succeeded in reconstituting a functional human centromere from cloned alphoid DNA. This break- through is analogous to the classic functional analyses used in the identification of budding yeast centromeric DNA 9?11 . However, in human cells, the established mini- chromosomes became larger than the introduced alphoid DNA. By using FISH with chromosome paint or satellite probes, Ikeno and colleagues 33 suggested that the established mini-chromosomes had not acquired large amounts of non-alphoid DNA sequences from endogenous chromosomes. Nevertheless, the possibility still remains that non-alphoid DNA might be present and could be required for centromere formation. kinetochore protein interface (FIG. 2a); second, the inner kinetochore?mitotic spindle interface (FIG. 2b); and final- ly, the kinetochore protein?cell cycle regulatory machinery interface (FIG. 2c). We conclude that the mole- cular understanding of the less-complex budding yeast kinetochore provides an excellent framework for under- standing the more complex kinetochores of humans. The DNA?inner kinetochore protein interface Yeast centromeres. In S. cerevisiae, the centromeric DNA sequence is a 125-base-pair (bp) region that contains three conserved elements ? CDEI, CDEII and CDEIII (REFS 9?11; FIG. 3a). The CDEI 8-bp sequence is bound by the centromere-binding factor 1 (Cbf1 ? also known as Cp1, Cpf1 or Cep1), which contains a helix?loop?helix DNA-binding domain, and mediates both transcriptional regulation and chromosome segregation 12,13 . Neither CDEI nor Cbf1 is essential for viability 12,13 . The 78?86-bp region of CDEII is composed of (A+T)-rich DNA, and seems to act as a spacer between the conserved CDEI and CDEIII DNA elements 14 . Mutations in the gene encoding a protein called Cse4 ? which is a histone-H3 variant that associates with cen- tromeric DNA in vivo ? reveal a genetic interaction with an insertion mutation in the CDEII DNA ele- ment 15,16 . This indicates that there might be a physical interaction between these components. Only CDEIII (25 bp) is essential, and point muta- tions within CDEIII abolish centromere function 17,18 . CDEIII interacts with the multi-protein CBF3 com- Box 2 | Kinetochores and cohesion In eukaryotes, sister chromatids remain connected to one another from S phase until the onset of anaphase. This cohesion is essential for the separation of sister chromatids to opposite poles of the cell at the correct time during mitosis, and it also allows chromosome segregation to take place long after duplication is complete. A multi-subunit complex called cohesin is essential for connecting the sister chromatids. In budding yeast, the cohesin complex consists of Smc1, Smc3, Scc1 (also known as Mcd1) and Scc3 during mitosis (although Rec8 replaces Scc1 in cells undergoing meiosis). All of these proteins are highly conserved between yeast and humans 110,111 . Several reports show that the protease Esp1 cleaves Scc1 to induce sister- chromatid segregation, indicating that cleavage of cohesin might control sister- chromatid separation (reviewed in REFS 110,111). Two independent groups have detected the association of Scc1 with centromeres and with discrete sites along chromosome arms. Tanaka and colleagues 112 showed that the association of Scc1 with a centromere depends on Mif2, Ndc10 and Cse4 (but note that Scc1 is not required to maintain the association of Ndc10 with a centromere). Megee and colleagues 113 showed that an ectopically placed centromere directs the binding of Scc1 to 2-kilobase (kb) regions that flank the centromere in a sequence-independent manner. Therefore, the centromere is a cis-acting cohesion factor, which is essential for the maintenance as well as the establishment of this cohesion domain. However, the cohesin complex cannot resist the consequent force that leads to sister-centromere splitting and chromosome stretching. Several reports have shown that the function of cohesin is conserved among eukaryotes; however, in vertebrates, the bulk of cohesin dissociates from chromosome arms during prophase, perhaps as a result of chromosome condensation (note that there is no such condensation in yeast). A small amount of cohesin remains on chromosomes, predominantly around centromeres (reviewed in REF. 110). However, despite the interesting relationship between the kinetochore and cohesin, the molecular link between these complexes has not yet been defined. Figure 2 | Kinetochore organization. The kinetochore can be thought of as three sets of subcomponents. a | The chromosomal DNA?inner kinetochore protein interface. Further details of this interface are shown in FIG. 3. b | The inner kinetochore?mitotic spindle interface. c | The kinetochore protein?cell cycle machinery interface. APC, anaphase-promoting complex; CEN, centromeric DNA; SCF, SCF ubiquitin-ligase complex. Cohesins Cohesins CEN a Cohesins Cohesins SCF?? Inner kinetochore Outer kinetochore Spindle components APC b c Spindle checkpoint Anaphase progression Motors SCF UBIQUITIN-LIGASE COMPLEX An E3 enzyme that targets ubiquitin to cell-cycle- regulatory proteins (for example, Sic1, Clns), using an F-box protein as a specificity factor. SCF refers to ?Skp1/Cul1/F-box protein?. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 681 REVIEWS Human homologues for various components of the yeast CBF3 complex ? Ndc10, Cep3 and Ctf13 ? have not been found so far in the public databases. However, both Skp1 and Sgt1 are functionally conserved between budding yeast and humans 28,43 . Human SKP1 can com- plement the yeast skp1 temperature-sensitive allele, skp1-11, but not the skp1 null mutant 43 (K.K. et al., unpublished data). However, human SGT1 can rescue the yeast sgt1 null mutant, indicating that the function of SGT1 is probably highly conserved 28 . Although it is not yet known whether the human homologues of Skp1 and Sgt1 have a role in kinetochore function, ten Hoopen and colleagues 44 reported that a specific anti- body against a barley homologue of yeast Skp1 strongly labelled the centromeres of barley and field-bean metaphase chromosomes. At the chromosomal DNA?inner kinetochore pro- tein interface (FIG. 2a), we conclude that the cis elements (centromeric DNA) and ?direct? trans elements (the DNA-binding proteins) do not seem to be highly con- served between budding yeast and humans. The inner kinetochore?mitotic spindle interface As shown in FIG. 2b, there must be a protein network from the inner kinetochore to the mitotic spindle. The complete set of proteins that provide this linkage has not been established yet in either humans or bud- ding yeast. Yeast cells. In budding yeast, in vivo crosslinking chro- matin-immunoprecipitation (CHIP) methods have recently revealed several new kinetochore proteins that Historically, human centromeres have been located using antibodies (from patients with autoimmune dis- ease) against the centromere-protein (CENP) antigens, which are human kinetochore proteins. CENPA and CENPB (FIG. 3b) are thought to have structural roles in kinetochore formation. CENPA is a histone-H3 variant, and experiments both in vivo and in vitro indicate that it is probably a centromeric nucleosome component 34?36 . CENPB binds to alphoid DNA and forms a dimer 36?38 ; however, the function of CENPB at kinetochores is still obscure because knockout mice are viable and seem to maintain normal centromeres 39?41 . Humans versus yeast. The Cse4 and CENPA proteins are structurally and biochemically similar 15,16,42 , and have similar roles in kinetochore function in budding yeast and humans. However, when Stoler and co-work- ers 15 attempted to rescue either temperature-sensitive or null alleles of cse4 by high expression of CENPA, they were unsuccessful. In addition, transient-expres- sion experiments in mammalian cell lines show that, although an epitope-tagged version of Cse4 (Cse4?haemagglutinin) is transported into the mam- malian nucleus, it does not localize to the centromere (REF. 15 and K. F. Sullivan, unpublished data). It is unclear whether these negative results indicate that the two proteins are not orthologues (functionally identi- cal) in their respective organisms. We believe it more likely that they represent true orthologues, but that they cannot complement ?cross-species?, owing to the evolution of key protein-interaction surfaces over a long period of time (800?1,000 million years). Figure 3 | The DNA?inner kinetochore protein interface. a | Yeast. The 125-base-pair (bp) centromeric (CEN) sequence contains three conserved elements. The first is the 8-bp CDEI sequence. This is bound by a protein called Cbf1, which mediates transcriptional regulation and chromosome segregation. The 78?86-bp CDEII sequence is potentially bound by a histone-H3 variant, Cse4. The 25-bp CDEIII sequence is bound by the centromere-binding factor 3 (CBF3) complex, which consists of four proteins: Ndc10, Cep3, Ctf13 and Skp1. b | Humans. The human CEN sequence may be bound by the histone-H3 variant CENPA, which is structurally and biochemically similar to yeast Cse4, in addition to the CENPB dimer. The function of CENPB is not yet known, as knockout mice are viable. CENA, centromere protein A; CENB, centromere protein B. Sgt1 Ndc10 Ndc10Cse4 ? CBF3 complex CDE I CDE II CDE III 8 bp 78?86 bp 25 bp CEN DNA CENPA CENPB CENPA CENPB High-order repeat (0.17?6 kb) 200?9,000 kb a Yeast b Human Alphoid DNA 171 bp Cep3 Skp1 Ctf13 Cbf1 CHIP (in vivo crosslinking chromatin- immunoprecipitation methods). After live cells are chemically crosslinked, extracted and mechanically sheared, chromatin fragments (crosslinked DNA?protein complexes) are immunoprecipitated. � 2001 Macmillan Magazines Ltd 682 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio REVIEWS activity mediates kinetochore function 48 . Bir1,a ?bac- ulovirus inhibitor of apoptosis repeat? (BIR MOTIF) protein, was isolated by the two-hybrid system as another pro- tein that interacts with Ndc10, and subsequent genetic analyses of bir1 mutants support a role in kinetochore function 50 . The recent determination of the close proximity of kinetochores and SPBs has raised a question as to whether some of the previously defined SPB proteins could, in fact, be kinetochore proteins or proteins that link both complexes. Two independent groups, Janke and colleagues 51 , and Wigge and Kilmartin 52 ,have shown that Ndc80, Nuf2, Spc24 and Spc25, which were previously described as components of the SPB, bind to centromeric DNA in vivo by the CHIP assay, and func- tion as kinetochore proteins. Very recently, He and col- leagues 53 used live-cell imaging and the CHIP assay to show that Spc19 and Spc34 (SPB proteins), Cin8 (a molecular motor), and Dam1, Stu2 and Bik1 (micro- tubule-associated proteins) are kinetochore subunits. Human cells. In human cells, CENPC is localized towards the outer centromere, in contrast to the inner- kinetochore localization of CENPB 54 . CENPC might have a role in the connection between centromeric chro- matin and the kinetochore. Studies analysing the molec- ular components associated with centromeres on stable dicentric chromosomes showed that, whereas both active and inactive centromeres contained CENPB, only active centromeres contained CENPC 55,56 . Antibody-injection associate with centromeric DNA specifically, although not necessarily directly. Meluh and Koshland 45 were the first to use the CHIP assay to show that Mif2 binds to centromeric DNA in vivo, possibly through CBF3 com- ponents. Hyland and colleagues 46 have used the CHIP assay to show that Ctf19 binds to centromeric DNA in vivo. Ortiz and colleagues 47 have also shown that Ctf19, Mcm21 and Okp1 bind to centromeric DNA in vivo using the CHIP assay, and that these proteins form a protein complex, which has been called the ?outer-kine- tochore complex?. Each component has also been shown to associate with CBF3 components by the two-hybrid system and/or immunoprecipitation 47 . In addition, the Mtw1 protein has been shown to bind centromeric DNA in an Ndc10-dependent manner 1 . Goshima and Yanagida 1 showed that Mtw1 and the sequences that lie 1.8 and 3.8 kb from CEN3 (the centromere of chromo- some III) and CEN15 (the centromere of chromosome XV), respectively, co-localize near the SPBs (the cen- tromeres and the SPBs are distinguishable), and that Mtw1 co-localizes with Ndc10, except for the extra spindle localization of Ndc10. Two more proteins ? Slk19 and Plc1 ? have also separately been shown 48,49 to bind to centromeric DNA in vivo by the CHIP assay. SLK19 genetically interacts with KAR3 and CIN8 (both of which encode kinesin- related motor proteins), and a GFP-tagged Slk19 local- izes to kinetochores 49 . Plc1 also physically binds Ndc10 48 . Plc1 is the yeast homologue of phospholipase C, although it is not known whether its phospholipase Box 3 | Epigenetic effects on kinetochore assembly In budding yeast, kinetochores assemble on centromeric DNA only. However, in rare cases in humans and the fruitfly Drosophila melanogaster, kinetochores can assemble at positions that lack centromeric DNA. This position is called the ?neocentromere? (reviewed in REF. 114). In humans, du Sart and colleagues 115 analysed the neocentromere in a marker chromosome by restriction and polymerase-chain-reaction mapping, and showed that the neocentromere has the same structure as DNA derived from normal chromosomes. So, a human kinetochore can assemble on DNA that is not normally centromeric. Drosophila centromeres (420 kb) consist of non-repetitive core sequences and several highly repetitive elements, both of which are necessary for kinetochore formation. Williams and colleagues 116 analysed several mini- chromosomes that lacked the 420-kb centromeric DNA sequence, but were still more stable than acentric chromosome fragments. DNA mini-chromosomes without centromeres seemed to acquire neocentromeres, indicating that functional Drosophila kinetochores can assemble on DNA that is not normally centromeric. How is kinetochore assembly controlled in the absence of a specific centromeric DNA sequence? One hypothesis is that epigenetic control is used. Although there is no direct molecular evidence to indicate that a specific epigenetic event is required for the formation of the neocentromere, there is some evidence in the fission yeast Schizosaccharomyces pombe for epigenetic control of the centromere. The fission yeast centromere (40?100 kb) consists of a central core of 4?7 kb, surrounded by roughly 5 kb of repeated DNA elements. This arrangement is structurally similar to that of the fly centromere, although there is no sequence similarity between human, fly and fission yeast centromeres. Ekwall and colleagues 117 observed that the transient drug treatment that induces histone hyperacetylation induces a heritable hyperacetylated state in centromeric chromatin, and causes chromosome loss. This increased loss persists in daughter cells even after the drug is removed. Assembly of fully functional centromeres might, therefore, be partly imprinted in the underacetylated state of centromeric chromatin. Centromeric repetitive sequences in these organisms might be needed only for the efficient establishment of kinetochores. In nature, it is rare that kinetochores get assembled on naked DNA. However, it might be important for us to understand the epigenetic mechanism to establish artificial chromosomes more efficiently in humans. What about the budding yeast? Tanaka and colleagues 112 noted that Scc1, an essential cohesin protein (see BOX 2), associates with authentic centromeres, but not with recently activated centromeres when ndc10-1 ?/? cells are shifted to the restrictive temperature, or when centromeric DNA is mutated. Therefore, the chromatin structure that recruits Scc1 to centromeres might be epigenetically inherited. BIR MOTIF A motif found in the ?inhibition of apoptosis? (IAP) proteins. It is essential for interaction of the IAP proteins with pro- apoptotic proteins, including the caspase family of death proteases. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 683 REVIEWS cated mutant proteins in mammalian cells blocks cell- cycle progression mainly at the G2/M transition 62 .In addition, two-hybrid interaction data show that CENPF interacts with CENPE, a kinetochore-microtubule motor protein 63 . Three microtubule-dependent motors, CENPE, dynein and mitotic-centromere-associated kinesin (MCAK) have been specifically localized to mammalian centromeres 64?67 . Both CENPE and dynein co-localize experiments, analysis of conditional CENPC knockouts in chicken cells, and analysis of CENPC-knockout mice, have shown that CENPC is essential for mitotic chro- mosome segregation 57?59 . CENPF assembles onto kinetochores during late G2 and is seen on every chromosome by the onset of prophase 60,61 . During mitosis, CENPF is associated with the outer kinetochore plate and remains there through metaphase 60 . Overexpression of amino-terminally trun- Table 1 | Budding yeast kinetochore and spindle-checkpoint components Saccharomyces Function/domain Human protein Homology Conservation cerevisiae protein (localization) CBF3 complex Ndc10 CBF3 component - - - Cep3 CBF3 component, - - - zinc-finger domain Ctf13 CBF3 component, - - - F-box protein Skp1 CBF3 component, p19/SKP1 3.3e-52 C, RN Ctf13 activation, (centrosome) SCF component CBF3 regulator Sgt1 Ctf13 activation, SGT1 1e-21 R SCF component Ipl1 Ndc10 kinase IAK1 (mouse) 3e-73 S (mouse)* (spindle pole) Glc7 Ndc10 phosphatase? PP1 (PPP1cc) 1e-162 ND Other inner kinetochore Cse4 Centromeric histone H3 CENPA (centromere) 5e-16 F, CN Cbf1 CDEI-binding protein, TFE3? 3e-05 ND MET-gene regulation Outer kinetochore and others Mif2 Inner/outer kinetochore? CENPC (centromere) 0.31 ND Ctf19 Outer kinetochore - - - Mcm21 Outer kinetochore - - - Okp1 Outer kinetochore CENPF? (centromere) 0.94 ND Slk19 Outer kinetochore? CENPF? (centromere) 3.5e-07 ND Mtw1 Outer kinetochore? - - - Plc1 Phospholipase C PLC-?1 5.2e-57 R (soybean) Ndc80 Outer kinetochore? HEC1 (centromere) 2e-16 R Nuf2 SPB?/Outer kinetochore? HNUF2R 2.4 ND Spc24 SPB?/Outer kinetochore? - - - Spc25 SPB?/Outer kinetochore? - - - Spc19 SPB?/Outer kinetochore? - - - Spc34 SPB?/Outer kinetochore? - - - Dam1 Microtubule-binding protein - - - Stu2 Microtubule-binding protein Ch-TOG 2.6e-27 ND Bik1 Microtubule-binding protein CLIP170 (centromere) 1.5e-11 ND Cin8 Kinesin-related protein HKSP 1e-85 ND Spindle checkpoint Bub1 Ser/Thr protein kinase BUB1 (centromere) 1e-42 F Bub3 WD-repeat protein BUB3 (centromere) 4e-30 F Mad1 Coiled?coil domain MAD1 (centromere/ 0.0081 F centrosome?) Mad2 MAD2L1 ? 6e-39 F, C (Caenorhabditis elegans) (centromere/ CN (Xenopus centrosome) laevis) Mad3 Similar to Bub1, but not BUBR1 (centromere) � 4e-21 F to kinase domain Mps1 Ser/Thr protein kinase PYT/TTK1 (centromere) 9.5e-62 F * The species in parentheses indicates the result with the homologous protein. For example, C (Caenorhabditis elegans) means that the C. elegans homologue complements the yeast mutant. ? Overproduction of human Mad2 suppresses the thiabendazole sensitivity of cpf1-null mutants. � BUBR1 is the best hit for the BLAST search with Mad3; however, some people categorize BUBR1 as a Bub1 homologue, because the Bub1 kinase domain is conserved in BUBR1 but not in Mad3 63,118 . C, complementation of the lethality of the mutant; CBF, centromere-binding factor; CN, complementation was negative; F, function is conserved; ND, not done; R, rescue of the lethality of the null mutant; RN, rescue was negative; S, synthetic dosage lethality; SCF, Skp1/Cul1/F-box protein; SPB, spindle pole body. � 2001 Macmillan Magazines Ltd 684 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio REVIEWS anaphase B 49 . This case is similar to that of CENPF, CENPE, dynein and the inner-centromere proteins (INCENPs), which also associate with the spindle mid- zone and resulting MIDBODY in human cells 60,64,77,78 . These human proteins are called ?kinetochore passenger pro- teins? and, although their function in the midzone is unknown, it might be conserved in budding yeast and humans, because these homologous proteins show a similar localization. Human survivin, a BIR-motif protein, is also a kine- tochore-associated passenger protein 79 . Because other BIR-motif proteins act at discrete steps to regulate apop- totic pathways 80 , it is possible that survivin links failure of mitotic checkpoint controls to apoptotic activation in human cells 79,81 . The Ndc80, Nuf2 and Bik1 proteins are conserved between budding yeast and humans. Recently 52 , the human homologues of Ndc80 and Nuf2 were shown by immunofluorescence to localize to kine- tochores of mitotic HeLa cells. In addition, the micro- tubule-binding region of CLIP170 is highly conserved in the yeast homologue, Bik1 (REF. 82). We conclude, then, that the structure and function of the inner kinetochore?mitotic spindle interface is highly conserved between budding yeast and humans. Kinetochore function and anaphase progression The kinetochore has been linked to the spindle check- point (BOX 1), but is there any evidence that components of this checkpoint are associated with kinetochore func- tion? And, if so, is there any conservation between the proteins that are involved in humans and the budding yeast proteins? Yeast cells. In budding yeast, genetic screens for ?mitotic arrest defective? (mad) and ?budding uninhibited by benzimidazole? (bub) mutants originally identified six components of the spindle checkpoint 83,84 (FIG. 4). Mad1 and Mad2 form a very tight complex 85 ; Mad2, Mad3, Bub3 and Cdc20 form a separate complex 86 . The BUB1 gene encodes a protein kinase that binds to, and phos- phorylates, Bub3 (REF. 87). The Mad1 protein shows a regulated association with Bub1 and Bub3 during the normal cell cycle, and this complex is found at consider- ably higher levels once the spindle checkpoint is activat- ed 88 . In addition, MPS1, which encodes an essential pro- tein kinase, also has a spindle-checkpoint function 89 . Mps1 can phosphorylate Mad1, and overexpression of Mps1 is enough to activate the checkpoint 90 . The kinetochore must somehow send a signal to the spindle checkpoint. Pangilinan and Spencer 91 have shown that MAD2 and BUB1 are required for the G2 delay that occurs when there is a problem with the kinetochore, caused either by a ctf13 mutation, or by the presence of a single-copy centromeric-DNA muta- tion. Interestingly, ndc10-1 mutants do not arrest dur- ing the G2/M transition at the non-permissive temper- ature 21 , even though another allele, ndc10-42 (ctf14-42), clearly causes a G2/M arrest 20 . Furthermore, NOCODAZOLE does not inhibit cell-cycle progression in ndc10-1 ?/? cells 92 . to the fibrous corona (outside the outer kineto- chore) 66,68,69 . By contrast, MCAK partially co-localizes with these motors and extends throughout the cen- tromere region 67,70 . Although extensive in vitro and in vivo experiments have investigated the motor activities of these three proteins, the precise identities and func- tion of motor activities in mitotic chromosome move- ment is still unclear (reviewed in REF. 71). The 170 kDa- cytoplasmic linker protein (CLIP170), which is a microtubule-associated protein but not a member of any motor family 72 , localizes at kinetochores, and has been shown to be important for proper chromosome congression during pro-metaphase 73 . Humans versus yeast. Is there any homology between the various proteins found at the human and yeast outer kinetochores? Two regions of yeast Mif2 that are essential for its function share homology with the two most highly conserved regions of human CENPC 74,75 (TABLE 1). Interestingly, in human cells, the herpes sim- plex virus immediate-early protein (Vmw110), a RING- FINGER protein, induces the proteasome-dependent degradation of CENPC, indicating that CENPC might be modified with SUMO-1 or a similar ubiquitin-like protein 76 .In S. cerevisiae, temperature-sensitive muta- tions 75 in MIF2 can be suppressed by high-level expres- sion of Smt3, the budding yeast homologue of SUMO- 1. So, the modification of both proteins might be conserved in budding yeast and humans. Ortiz and colleagues 47 proposed that four regions of Okp1 are homologous to CENPF. In fact, Slk19 shows higher homology to CENPF than Okp1p does (TABLE 1). It is unclear how comparable CENPF is to these kineto- chore proteins of budding yeast on a functional level. Interestingly, Slk19, like Ndc10, is left behind at the spindle midzone when the spindle poles separate in Figure 4 | Kinetochore function and anaphase progression. The spindle-assembly checkpoint delays mitosis in response to defective spindle organization or unattached kinetochores. This delay is brought about when components of the spindle-assembly checkpoint are recruited to the unattached kinetochores. These components include the Mad2 protein, which forms a tight complex with Mad1 and inhibits the anaphase-promoting complex (APC), thereby delaying mitosis. One way in which blocking the APC has this effect is by inhibition of the ubiquitin (Ub)-dependent degradation of securin. Destruction of this protein would normally allow cleavage of another protein called separase, which is involved in overcoming the cohesion between sister chromatids. APC Anaphase inhibitors Anaphase progression Spindle- assembly checkpointSpindle damage or unattached kinetochore Centromere Cohesins Mad1, Mad2, Mad3, Bub1, Bub3, Mps1 Ub Ub Ub RING-FINGER A cysteine-rich zinc-binding domain, which is thought to be required for protein?protein interactions. MIDBODY Dense structure formed during cytokinesis at the cleavage furrow. It consists of remnants of spindle fibres and other amorphous material and disappears before cell division is completed. NOCODAZOLE A microtubule-depolymerizing drug. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 685 REVIEWS found 63 . CENPE, a kinetochore kinesin motor, was found to interact with BUBR1 (a human homologue of Bub1), in a two-hybrid test and by co-immunoprecipi- tation. Using Xenopus egg extracts, CENPE was shown to be necessary for establishing and maintaining the spindle checkpoint in vitro 105 . The combination of the vertebrate and budding yeast results suggest a plausible pathway for the spindle checkpoint ? microtubule-free kinetochores recruit the Bub and Mad checkpoint proteins which, in turn, inhibit the anaphase-promoting complex (APC), thereby preventing sister chromatids from separating (FIG. 4). We conclude that the signalling pathway is high- ly and functionally conserved between budding yeast and humans. Conclusions and future prospects We have described our present knowledge of how the kinetochore is conserved between budding yeast and humans by discussing individual kinetochore compo- nents. Although this field is at an early stage in fully understanding the molecular mechanisms of kineto- chore function, we believe that the evidence indicates that the kinetochore and its regulating system are highly conserved between budding yeast and humans. Of course, there are probably human or mammalian- specific activities at the kinetochore ? methylation, for example, or the relevance of kinetochore studies and the spindle checkpoint to cancer (BOX 4).However, we believe that research into the budding yeast will continue to reveal further conserved functions at the kinetochore. We know very little about the regulation of kineto- chore activity during cell-cycle progression. What, for example, controls the timing of assembly of the kineto- chore proteins? We know that unattached or damaged kinetochores send a signal to the mitotic checkpoint to stop the cell cycle, but what is the molecular mecha- nism of the signalling from kinetochores to the check- point? As described above, the molecular linkage of individual proteins within the supramolecular com- plexes that define kinetochores are not yet known. In These observations indicate that Ndc10 might have an important function in spindle-checkpoint signalling. Recently, Gardner and colleagues 93 showed that the spindle checkpoint is destroyed when the CBF3 compo- nents Cep3 or Ndc10 are eliminated from cells using the degron system (which generates a conditional-null mutation by specific proteolysis), indicating that the CBF3 complex is both monitored by the spindle check- point and required for checkpoint signalling. Mammalian cells. The mammalian kinetochore has been proposed as a likely candidate for the site at which the signal is generated that is sensed by the spindle- checkpoint pathway when chromosomes are not attached to the spindle. Laser ablation of both kineto- chores of the last unattached chromosome in PtK1 cells (a marsupial cell line) showed that at least one part of the spindle checkpoint is located at the kinetochore, and that the checkpoint is activated by the presence of unattached kinetochores 94 . Vertebrates versus yeast. All of the mitotic checkpoint proteins described above are highly conserved between budding yeast and humans (TABLE 1). Vertebrate homo- logues of Mad1, Mad2, Mad3, Bub1 and Bub3 bind to all kinetochores in cells that have been arrested in mito- sis by microtubule-polymerization inhibitors, and specifically localize to microtubule-free kinetochores during spindle assembly in normal cells. Furthermore, the Mps1, Mad1, Mad2, Mad3, Bub1 and Bub3 homo- logues have been shown to have a role in the spindle checkpoint 63,95?103 .In Xenopus laevis, Mps1 recruits CENPE and Mad1/Mad2 to kinetochores 102 , and Mad1 recruits Mad2 to kinetochores 97 . Bub3 might recruit both Bub1 and Mad3 to kinetochores in humans 101 . However, although the fission yeast (Schizo- saccharomyces pombe) homologue of Bub1 has been shown to localize to kinetochores 104 , it has not been shown that any component of the budding yeast check- point is localized to kinetochores. A physical link between spindle-checkpoint proteins and a kinetochore-bound motor protein has been Box 4 | Relevance to cancer mechanism Cancer cells are known for their genetic instability, and ANEUPLOIDY can result when chromosome segregation occurs erroneously. It is thought that mutations leading to increased rates of chromosome mis-segregation in certain classes of cancer might be predisposing factors that accelerate the process of tumorigenesis. By this model, the centromere and its regulatory system, which is essential for genome stability, must have an important role in providing some measure of protection against the development of cancer. Results from Bert Vogelstein?s group 118 indicate that the spindle checkpoint has an important function in human cancer. One of the main mechanisms that contributes to the development of colon cancers is thought to be improper sister-chromosome segregation, which leads to aneuploidy or chromosome instability (a ?CIN tumour?). Two independent cell lines derived from CIN tumours have been shown 118 to carry mutations in the human homologue of BUB1. Functional analysis showed that these BUBR1 mutations had dominant-negative effects: CIN cells that normally retain functional checkpoint controls were severely compromised for checkpoint function when expressing the dominant-negative allele. Complete loss of the mitotic checkpoint control results in embryonic lethality, presumably due to intolerably high rates of chromosome mis-segregation 119?122 . Recently, Michel and colleagues 123 reported that Mad2 +/? mice develop lung tumours at high rates after long latency periods. These results indicate that all the genes in the detection pathway in FIG. 2b have the potential to be cancer-related targets. So, further studies of kinetochore?spindle-checkpoint biology in both budding yeast and humans could shed light on our understanding of cancer. ANEUPLOIDY One or more chromosomes of a normal set of chromosomes are missing, or present in more than their usual number of copies. � 2001 Macmillan Magazines Ltd 686 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio REVIEWS notypic analyses in vivo). We suspect that such defini- tive experiments will show that the basic strategies and molecular components that define kinetochore activity and regulation have been conserved throughout eukaryotic evolution. addition, there is the question of how kinetochores link to microtubules. There might also be unknown activi- ties at kinetochores. Budding yeast is a good test-system for developing genome-wide technologies. Systematic protein?pro- tein interaction analyses using the two-hybrid tech- nique or mass spectrometry could provide insight into all these unanswered questions. Eventually, after deter- mining all the players, it would be exciting and power- ful if we could reconstitute the kinetochore dynamic activity in vitro. In this way, definitive experiments could be done to associate directly specific biochemi- cal activities of individual proteins and subcomplexes (through direct biochemical analyses in vitro) with specific cellular functions (using mutational and phe- 1. Goshima, G. & Yanagida, M. Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell 100, 619?633 (2000). 2. He, X., Asthana, S. & Sorger, P. K. Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell 101, 763?775 (2000). 3. Tanaka, T., Fuchs, J., Loidl, J. & Nasmyth, K. Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation. Nature Cell Biol. 2, 492?499 (2000). References 1?3 describe yeast kinetochore behaviour throughout the cell cycle. 4. McCarroll, R. M. & Fangman, W. L. Time of replication of yeast centromeres and telomeres. Cell 54, 505?513 (1988). 5. Guacci, V., Hogan, E. & Koshland, D. Centromere position in budding yeast: evidence for anaphase A. Mol. Biol. Cell 8, 957?972 (1997). 6. O?Toole, E. T., Winey, M. & McIntosh, J. R. High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 10, 2017?2031 (1999). 7. Jin, Q. W., Fuchs, J. & Loidl, J. Centromere clustering is a major determinant of yeast interphase nuclear organization. J. Cell Sci. 113, 1903?1912 (2000). 8. Rieder, C. L. & Salmon, E. D. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J. Cell Biol. 124, 223?233 (1994). 9. Clarke, L. & Carbon, J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287, 504?509 (1980). 10. Fitzgerald-Hayes, M., Clarke, L. & Carbon, J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29, 235?244 (1982). 11. Hieter, P. et al. Functional selection and analysis of yeast centromeric DNA. Cell 42, 913?921 (1985). 12. Baker, R. E. & Masison, D. C. Isolation of the gene encoding the Saccharomyces cerevisiae centromere- binding protein CP1. Mol. Cell. Biol. 10, 2458?2467 (1990). 13. Cai, M. & Davis, R. W. Yeast centromere binding protein CBF1, of the helix?loop?helix protein family, is required for chromosome stability and methionine prototrophy. Cell 61, 437?446 (1990). 14. Gaudet, A. & Fitzgerald-Hayes, M. Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 68?75 (1987). 15. Stoler, S., Keith, K. C., Curnick, K. E. & Fitzgerald-Hayes, M. A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9, 573?586 (1995). 16. Meluh, P. B., Yang, P., Glowczewski, L., Koshland, D. & Smith, M. M. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell 94, 607?613 (1998). 17. McGrew, J., Diehl, B. & Fitzgerald-Hayes, M. Single base- pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 6, 530?538 (1986). 18. Ng, R. & Carbon, J. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 4522?4534 (1987). 19. Lechner, J. & Carbon, J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64, 717?725 (1991). 20. Doheny, K. F. et al. Identification of essential components of the S. cerevisiae kinetochore. Cell 73, 761?774 (1993). 21. Goh, P. Y. & Kilmartin, J. V. NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae. J. Cell Biol. 121, 503?512 (1993). 22. Jiang, W., Lechner, J. & Carbon, J. Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore. J. Cell Biol. 121, 513?519 (1993). 23. Lechner, J. A zinc finger protein, essential for chromosome segregation, constitutes a putative DNA binding subunit of the Saccharomyces cerevisiae kinetochore complex, Cbf3. EMBO J. 13, 5203?5211 (1994). 24. Strunnikov, A. V., Kingsbury, J. & Koshland, D. CEP3 encodes a centromere protein of Saccharomyces cerevisiae. J. Cell Biol. 128, 749?760 (1995). 25. Connelly, C. & Hieter, P. Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell 86, 275?285 (1996). 26. Stemmann, O. & Lechner, J. The Saccharomyces cerevisiae kinetochore contains a cyclin-CDK complexing homologue, as identified by in vitro reconstitution. EMBO J. 15, 3611?3620 (1996). 27. Kaplan, K. B., Hyman, A. A. & Sorger, P. K. Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation. Cell 91, 491?500 (1997). 28. Kitagawa, K., Skowyra, D., Elledge, S. J., Harper, J. W. & Hieter, P. SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol. Cell 4, 21?33 (1999). References 25?28 show that the highly conserved proteins Skp1 and Sgt1 are required for activation of the yeast kinetochore and components of the SCF ubiquitin-ligase complex. 29. Feldman, R. M., Correll, C. C., Kaplan, K. B. & Deshaies, R. J. A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor. Cell 91, 221?230 (1997). 30. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J. & Harper, J. W. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209?219 (1997). 31. Murphy, T. D. & Karpen, G. H. Centromeres take flight: ?- satellite and the quest for the human centromere. Cell 93, 317?320 (1998). 32. Harrington, J. J., Van Bokkelen, G., Mays, R. W., Gustashaw, K. & Willard, H. F. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nature Genet. 15, 345?355 (1997). 33. Ikeno, M. et al. Construction of YAC-based mammalian artificial chromosomes. Nature Biotechnol. 16, 431?439 (1998). 34. Warburton, P. E. et al. Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr. Biol. 7, 901?904 (1997). 35. Vafa, O. & Sullivan, K. F. Chromatin containing CENP-A and ?-satellite DNA is a major component of the inner kinetochore plate. Curr. Biol. 7, 897?900 (1997). 36. Yoda, K., Kitagawa, K., Masumoto, H., Muro, Y. & Okazaki, T. A human centromere protein, CENP-B, has a DNA binding domain containing four potential ? helices at the NH 2 terminus, which is separable from dimerizing activity. J. Cell Biol. 119, 1413?1427 (1992). 37. Masumoto, H., Masukata, H., Muro, Y., Nozaki, N. & Okazaki, T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J. Cell Biol. 109, 1963?1973 (1989). 38. Kitagawa, K., Masumoto, H., Ikeda, M. & Okazaki, T. Analysis of protein?DNA and protein?protein interactions of centromere protein B (CENP-B) and properties of the DNA?CENP-B complex in the cell cycle. Mol. Cell. Biol. 15, 1602?1612 (1995). 39. Kapoor, M. et al. The cenpB gene is not essential in mice. Chromosoma 107, 570?576 (1998). 40. Perez-Castro, A. V. et al. Centromeric protein B null mice are viable with no apparent abnormalities. Dev. Biol. 201, 135?143 (1998). 41. Hudson, D. F. et al. Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights. J. Cell Biol. 141, 309?319 (1998). 42. Yoda, K. et al. Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc. Natl Acad. Sci. USA 97, 7266?7271 (2000). 43. Bai, C. et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263?274 (1996). 44. ten Hoopen, R., Manteuffel, R., Dolezel, J., Malysheva, L. & Schubert, I. Evolutionary conservation of kinetochore protein sequences in plants. Chromosoma 109, 482?489 (2000). 45. Meluh, P. B. & Koshland, D. Budding yeast centromere composition and assembly as revealed by in vivo cross- linking. Genes Dev. 11, 3401?3412 (1997). Pioneered the techniques of crosslinking and chromatin immunoprecipitation for analysing the binding of centromere proteins in vivo in budding yeast. 46. Hyland, K. M., Kingsbury, J., Koshland, D. & Hieter, P. Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle. J. Cell Biol. 145, 15?28 (1999). 47. Ortiz, J., Stemmann, O., Rank, S. & Lechner, J. A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13, 1140?1155 (1999). 48. Lin, H. et al. Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 3597?3607 (2000). 49. Zeng, X. et al. Slk19p is a centromere protein that functions to stabilize mitotic spindles. J. Cell Biol. 146, 415?425 (1999). 50. Yoon, H. J. & Carbon, J. Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events. Proc. Natl Acad. Sci. USA 96, 13208?13213 (1999). 51. Janke, C. et al. The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control. EMBO J. 20, 777?791 (2001). Links DATABASE LINKS Cbf1 | Cse4 | NDC10 | CEP3 | CTF13 | SKP1 | Sgt1 | CENPA | CENPB | SKP1 | SGT1 | Mif2 | Ctf19 | Mcm21 | Mtw1 | CEN3 | CEN15 | Slk19 | Plc1 | KAR3 | CIN8 | phospholipase C | Bir1 | Ndc80 | Nuf2 | Spc24 | Spc25 | Spc19 | Spc34 | Dam1 | Stu2 | Bik1 | CENPC | CENPF | CENPE | MCAK | CLIP170 | SUMO-1 | Smt3 | survivin | Mad1 | Mad2 | Mad3 | Bub3 | Cdc20 | BUB1 | MPS1 | BUBR1 | Smc1 | Smc3 | Scc1 | Rec8 | Esp1 � 2001 Macmillan Magazines Ltd NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 687 REVIEWS 52. Wigge, P. A. & Kilmartin, J. V. The Ndc80p Complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J. Cell Biol. 152, 349?360 (2001). 53. He, X., Rines, D. R., Espelin, C. W. & Sorger, P. K. Molecular analysis of kinetochore-microtubule attachment in budding yeast. Cell 106, 195?206 (2001). References 47 and 53 describe several outer- kinetochore proteins. 54. Earnshaw, W. C. & Cooke, C. A. Proteins of the inner and outer centromere of mitotic chromosomes. Genome 31, 541?552 (1989). 55. Earnshaw, W. C., Ratrie, H. & Stetten, G. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma 98, 1?12 (1989). 56. Sullivan, B. A. & Schwartz, S. Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum. Mol. Genet. 4, 2189?2197 (1995). 57. Tomkiel, J., Cooke, C. A., Saitoh, H., Bernat, R. L. & Earnshaw, W. C. CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J. Cell Biol. 125, 531?545 (1994). 58. Fukagawa, T. & Brown, W. R. Efficient conditional mutation of the vertebrate CENP-C gene. Hum. Mol. Genet. 6, 2301?2308 (1997). 59. Kalitsis, P., Fowler, K. J., Earle, E., Hill, J. & Choo, K. H. Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death. Proc. Natl Acad. Sci. USA 95, 1136?1141 (1998). 60. Rattner, J. B., Rao, A., Fritzler, M. J., Valencia, D. W. & Yen, T. J. CENP-F is a ca. 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization. Cell. Motil. Cytoskeleton 26, 214?226 (1993). 61. Liao, H., Winkfein, R. J., Mack, G., Rattner, J. B. & Yen, T. J. CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. J. Cell Biol. 130, 507?518 (1995). 62. Zhu, X. et al. Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression. Mol. Cell. Biol. 15, 5017?5029 (1995). 63. Chan, G. K., Schaar, B. T. & Yen, T. J. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J. Cell Biol. 143, 49?63 (1998). 64. Yen, T. J. et al. CENP-E, a novel human centromere- associated protein required for progression from metaphase to anaphase. EMBO J. 10, 1245?1254 (1991). 65. Steuer, E. R., Wordeman, L., Schroer, T. A. & Sheetz, M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature 345, 266?268 (1990). 66. Pfarr, C. M. et al. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature 345, 263?265 (1990). 67. Wordeman, L. & Mitchison, T. J. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J. Cell Biol. 128, 95?104 (1995). 68. Cooke, C. A., Schaar, B., Yen, T. J. & Earnshaw, W. C. Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase. Chromosoma 106, 446?455 (1997). 69. Yao, X., Anderson, K. L. & Cleveland, D. W. The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules. J. Cell Biol. 139, 435?447 (1997). 70. Maney, T., Hunter, A. W., Wagenbach, M. & Wordeman, L. Mitotic centromere-associated kinesin is important for anaphase chromosome segregation. J. Cell Biol. 142, 787?801 (1998). 71. Maney, T., Ginkel, L. M., Hunter, A. W. & Wordeman, L. The kinetochore of higher eucaryotes: a molecular view. Int. Rev. Cytol. 194, 67?131 (2000). 72. Pierre, P., Scheel, J., Rickard, J. E. & Kreis, T. E. CLIP-170 links endocytic vesicles to microtubules. Cell 70, 887?900 (1992). 73. Dujardin, D. et al. Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment. J. Cell Biol. 141, 849?862 (1998). 74. Brown, M. T., Goetsch, L. & Hartwell, L. H. MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J. Cell Biol. 123, 387?403 (1993). 75. Meluh, P. B. & Koshland, D. Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell 6, 793?807 (1995). 76. Everett, R. D., Earnshaw, W. C., Findlay, J. & Lomonte, P. Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110. EMBO J. 18, 1526?1538 (1999). 77. Cooke, C. A., Heck, M. M. & Earnshaw, W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105, 2053?2067 (1987). 78. Karki, S., LaMonte, B. & Holzbaur, E. L. Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells. J. Cell Biol. 142, 1023?1034 (1998); erratum in 143, 561 (1998). 79. Skoufias, D. A., Mollinari, C., Lacroix, F. B. & Margolis, R. L. Human survivin is a kinetochore-associated passenger protein. J. Cell Biol. 151, 1575?1582 (2000). 80. Deveraux, Q. L. & Reed, J. C. IAP family proteins ? suppressors of apoptosis. Genes Dev. 13, 239?252 (1999). 81. Li, F. et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, 580?584 (1998). 82. Trueheart, J., Boeke, J. D. & Fink, G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol. Cell. Biol. 7, 2316?2328 (1987). 83. Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507?517 (1991). 84. Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519?531 (1991); erratum in 79, 388 (1994). 85. Chen, R. H., Brady, D. M., Smith, D., Murray, A. W. & Hardwick, K. G. The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins. Mol. Biol. Cell. 10, 2607?2618 (1999). 86. Hardwick, K. G., Johnston, R. C., Smith, D. L. & Murray, A. W. MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p. J. Cell Biol. 148, 871?882 (2000). 87. Roberts, B. T., Farr, K. A. & Hoyt, M. A. The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. Mol. Cell. Biol. 14, 8282?8291 (1994). 88. Brady, D. M. & Hardwick, K. G. Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Curr. Biol. 10, 675?678 (2000). 89. Weiss, E. & Winey, M. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J. Cell Biol. 132, 111?123 (1996). 90. Hardwick, K. G., Weiss, E., Luca, F. C., Winey, M. & Murray, A. W. Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science 273, 953?956 (1996). 91. Pangilinan, F. & Spencer, F. Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast. Mol. Biol. Cell 7, 1195?1208 (1996). 92. Tavormina, P. A. & Burke, D. J. Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes. Genetics 148, 1701?1713 (1998). 93. Gardner, R. D. et al. The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain. Genetics 157, 1493?1502 (2001). 94. Rieder, 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. 127, 1301?1310 (1994). 95. Chan, G. K., Jablonski, S. A., Sudakin, V., Hittle, J. C. & Yen, T. J. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J. Cell Biol. 146, 941?954 (1999). 96. Chen, R. H., Waters, J. C., Salmon, E. D. & Murray, A. W. Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 274, 242?246 (1996). 97. Chen, R. H., Shevchenko, A., Mann, M. & Murray, A. W. Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J. Cell Biol. 143, 283?295 (1998). 98. Jin, D. Y., Spencer, F. & Jeang, K. T. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93, 81?91 (1998). 99. Li, Y. & Benezra, R. Identification of a human mitotic checkpoint gene: hsMAD2. Science 274, 246?248 (1996). 100. Taylor, S. S. & McKeon, F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage. Cell 89, 727?735 (1997). 101. Taylor, 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. 142, 1?11 (1998). 102. Abrieu, A. et al. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 106, 83?93 (2001). 103. Fisk, H. A. & Winey, M. The mouse mps1p-like kinase regulates centrosome duplication. Cell 106, 95?104 (2001). 104. Bernard, P., Hardwick, K. & Javerzat, J. P. Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis. J. Cell Biol. 143, 1775?1787 (1998). 105. Abrieu, A., Kahana, J. A., Wood, K. W. & Cleveland, D. W. CENP-E as an essential component of the mitotic checkpoint in vitro. Cell 102, 817?826 (2000). 106. Weinert, T. & Hartwell, L. Control of G2 delay by the rad9 gene of Saccharomyces cerevisiae. J. Cell. Sci. 12, S145?S148 (1989). 107. Kallio, 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. 141, 1393?1406 (1998). 108. Cohen-Fix, O., Peters, J. M., Kirschner, M. W. & Koshland, D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 10, 3081?3093 (1996). 109. Funabiki, H. et al. Cut2 proteolysis required for sister- chromatid seperation in fission yeast. Nature 381, 438?441 (1996). 110. Amon, A. Together until separin do us part. Nature Cell Biol. 3, E12?E14 (2001). 111. Nasmyth, K., Peters, J. M. & Uhlmann, F. Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288, 1379?1385 (2000). 112. Tanaka, T., Cosma, M. P., Wirth, K. & Nasmyth, K. Identification of cohesin association sites at centromeres and along chromosome arms. Cell 98, 847?858 (1999). 113. Megee, P. C., Mistrot, C., Guacci, V. & Koshland, D. The centromeric sister chromatid cohesion site directs Mcd1p binding to adjacent sequences. Mol. Cell 4, 445?450 (1999). 114. Wiens, G. R. & Sorger, P. K. Centromeric chromatin and epigenetic effects in kinetochore assembly. Cell 93, 313?316 (1998). 115. Du Sart, D. et al. A functional neo-centromere formed through activation of a latent human centromere and consisting of non-?-satellite DNA. Nature Genet. 16, 144?153 (1997). 116. Williams, B. C., Murphy, T. D., Goldberg, M. L. & Karpen, G. H. Neocentromere activity of structurally acentric mini- chromosomes in Drosophila. Nature Genet. 18, 30?37 (1998). 117. Ekwall, K., Olsson, T., Turner, B. M., Cranston, G. & Allshire, R. C. Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 91, 1021?1032 (1997). 118. Cahill, D. P. et al. Mutations of mitotic checkpoint genes in human cancers. Nature 392, 300?303 (1998). The first report that mutations are found in mitotic- checkpoint genes in human cancers. 119. Basu, J. et al. Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J. Cell Biol. 146, 13?28 (1999). 120. Dobles, M., Liberal, V., Scott, M. L., Benezra, R. & Sorger, P. K. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101, 635?645 (2000). 121. Kalitsis, P., Earle, E., Fowler, K. J. & Choo, K. H. Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes Dev. 14, 2277?2282 (2000). 122. Kitagawa, R. & Rose, A. M. Components of the spindle- assembly checkpoint are essential in Caenorhabditis elegans. Nature Cell Biol. 1, 514?521 (1999). 123. Michel, L. S. et al. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409, 355?359 (2001). "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment