INTRODUCTION

The centromere is a highly complex structure that is central tomany essential activities during cell division. For example, thecentromere is the major site of sister chromatid attachment inmitosis and is responsible for maintaining this association untilanaphase onset. The centromere also dictates assembly of thekinetochore, a tri-laminar plate to which microtubules attach,connecting chromosomes to the spindle (reviewed by Pluta etal., 1995; Choo, 1997). In addition, the kinetochore is the siteof the wait anaphase checkpoint which delays anaphase untilall chromosomes are properly aligned in a bi-polar fashion onthe metaphase plate (Li and Nicklas, 1995; Rieder et al., 1995).Knowledge of the molecular makeup of the kinetochore, andhow individual components of the kinetochore interact withone another, is central to our understanding of how thisstructure functions to ensure the accurate segregation ofchromosomes in mitosis and meiosis.

The first molecular components of the mammaliancentromere/ kinetochore were identified with autoimmune serafrom patients with the scleroderma spectrum disease CREST(Moroi et al., 1980). Centromere proteins (CENPs) A, B, andC are detected at centromeres throughout the cell cycle and areall likely to interact with centromeric DNA (Sullivan andGlass, 1991; Saitoh et al., 1992; Sullivan et al., 1994). CENP-

A appears to be a centromere specific core histone (Sullivan etal., 1994). CENP-B binds to a 17 bp DNA sequence incentromeric alpha-satellite DNA (Pluta et al., 1992), but is notessential for centromeric function as homozygous CENP-Bnull mice are viable (Hudson et al., 1998). CENP-C is locatedat the inner kinetochore plate, and has been shown to beimportant for the stability of the trilaminar kinetochore and forthe viability of mouse embryos (Tomkiel et al., 1994; Kalitsiset al., 1998). Autoimmune sera have recently defined anotherconstitutive component of the centromere called CENP-G thatassociates with a specific sequence of alpha-satellite DNA (Heet al., 1998); the molecular identity of CENP-G is as yetunknown. CENP-H is the latest addition to the class of proteinsthat constitutively associate with centromeres (Sugata et al.,1999). The precise function of CENP-H within the centromereremains unknown.

In addition to the proteins that are constitutively bound tocentromeric heterochromatin, a second class of proteinsassociates with the centromere/kinetochore only transiently.Many of these proteins have been demonstrated byimmunoelectron microscopy to be integral components of themature trilaminar kinetochore, which is not visible untilmitosis. These proteins are in fact recruited to the centromerein a sequential order at discrete times prior to or early inmitosis, suggesting that kinetochores are assembled in a series

1939Journal of Cell Science 113, 1939-1950 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1417

HZwint-1 (H uman ZW10 interacting protein-1) wasidentified in a yeast two hybrid screen for proteins thatinteract with HZW10. HZwint-1 cDNA encodes a 43 kDaprotein predicted to contain an extended coiled-coildomain. Immunofluorescence studies with sera raisedagainst HZwint-1 protein revealed strong kinetochorestaining in nocodazole-arrested chromosome spreads.This signal co-localizes at the kinetochore with HZW10,at a position slightly outside of the central part of thecentromere as revealed by staining with a CREST serum.The kinetochore localization of HZwint-1 has beenconfirmed by following GFP fluorescence in HeLa cellstransiently transfected with a plasmid encoding aGFP/HZwint-1 fusion protein. In cycling HeLa cells,

HZwint-1 localizes to the kinetochore of prophase HeLacells prior to HZW10 localization, and remains at thekinetochore until late in anaphase. This localizationpattern, combined with the two-hybrid results, suggeststhat HZwint-1 may play a role in targeting HZW10 tothe kinetochore at prometaphase. HZwint-1 was alsofound to localize to neocentromeres and to the activecentromere of dicentric chromosomes. HZwint-1 thusappears to associate with all active centromeres, implyingthat it plays an important role in correct centromerefunction.

Key words: Kinetochore, Centromere, HZW10, HZwint-1

SUMMARY

HZwint-1, a novel human kinetochore component that interacts with HZW10

Daniel A. Starr 1,*, Richard Saffery 2, Zexiao Li 1, Amanda E. Simpson 1, K. H. Andy Choo 2, Tim J. Yen 3 andMichael L. Goldberg 1,‡

1Section of Genetics and Development, Cornell University, Ithaca, NY 14853, USA2The Murdoch Childrens Institute, Royal Children’s Hospital, Flemington Road, Parkville, Melbourne, Australia, 30523Fox Chase Cancer Center, Philadelphia, PA 19111, USA*Present address: Department of MCD Biology, The University of Colorado, Boulder, CO 80309, USA‡Author for correspondence (e-mail: mlg11@cornell.edu)

Accepted 28 March; published on WWW 10 May 2000

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of separable intermediary steps (reviewed by Craig et al.,1999). Topoisomerase II, likely to play a role separating theintertwined chromatin of sister chromatids (Shamu andMurray, 1992), may be one of the earliest markers for assemblyof the mitotic kinetochore, as it is recruited to centromeres atthe onset of centromeric heterochromatin condensation duringthe late S-G2 period (Rattner et al., 1996). The mitoticcheckpoint kinase hBUB1 appears to assemble at the nascentkinetochore sometime later in G2 (Jablonski et al., 1998). Thisevent overlaps with or is immediately followed by theappearance of CENP-F, a 367 kDa protein consisting mostlyof an extended coiled-coil domain which has been postulatedto play a role in the early steps of kinetochore assembly (Chanet al., 1998; Liao et al., 1995). Subsequently, MCAK,hBUBR1, and CENP-E (described below) appear at thekinetochore in sequential order from prophase throughprometaphase (Chan et al., 1998; Liao et al., 1995; Wordemanand Mitchison, 1995; Jablonski et al., 1998). At the EM level,CENP-B and CENP-C are localized to the centromericheterochromatin and the inner kinetochore plate, respectively,while hBUBR1, CENP-F and CENP-E are localized to theouter kinetochore plate and the fibrous corona (reviewed byCraig et al., 1999).

Several molecules transiently associated with thecentromere/kinetochore govern mechanical activities ofchromosomes during mitosis. CENP-E is a kinesin like proteinthat is essential for chromosome alignment both in vitro andin vivo (Scharr et al., 1997; Wood et al., 1997).Mechanistically, CENP-E is thought to be important forestablishing stable kinetochore microtubule attachmentsthrough its amino-terminal motor domain (Lombillo et al.,1995; Scharr et al., 1997). Mitotic centromere-associatedkinesin (MCAK), another member of the kinesin superfamily,has been serendipitously localized to the mitotic centromereand is needed for anaphase chromosome segregation(Wordeman and Mitchison, 1995; Maney et al., 1998). TheXenopus homolog of MCAK (XKCM1) destabilizesmicrotubules, and might thus help to depolymerizemicrotubules at the kinetochore during anaphase A (Walczaket al., 1996; Desai et al., 1999). Finally, cytoplasmic dyneinand its associated multisubunit complex dynactin are alsofound at kinetochores (Steuer et al., 1990; Pfarr et al., 1990;Wordeman et al., 1991; Echeverri et al., 1996; Karki et al.,1998). Recent evidence suggests that dynein and dynactin arerequired for the fidelity of chromatid separation or movementsat anaphase onset (Starr et al., 1998; see below).

A group of evolutionarily conserved checkpoint proteinsalso associates transiently with the kinetochore. Theseproteins, which include Mad1, Mad2, Bub1, Bub3, Skp1, andp55CDC were originally identified by genetic screens in yeastsas components of the spindle assembly checkpoint, whicharrests the cell cycle prior to anaphase if the mitotic spindle isdamaged (reviewed by Skibbens and Hieter, 1998; Gorbsky etal., 1999; Zachariae and Nasmyth, 1999). Through theconcerted action of these molecules, even a single kinetochoreunattached to the spindle generates a signal that can preventcells from entering anaphase until all chromosomes areproperly aligned on the spindle (Rieder et al., 1995; Li andNicklas, 1995).

Finally, the human kinetochore proteins HZW10 and HRODwere identified because of their homology with the Drosophila

kinetochore proteins ZW10 and ROD. Mutations in theDrosophila genes encoding either protein yield the samephenotypes, as the ZW10 and ROD proteins function togetherin a complex (D. A. Starr, F. Scaerou, R. Karess, and M. L.Goldberg, manuscript in preparation). Null mutations in eithergene cause a high level of aneuploidy due to defects atanaphase, when chromatids are often seen lagging at themetaphase plate (Smith et al., 1985; Karess and Glover, 1989;Williams et al., 1992; Starr et al., 1997; Scaerou et al., 1999).ZW10 and HZW10 display an intriguing pattern of cell cycledependent distribution: they localize to the kinetochore atprometaphase, the kinetochore and kinetochore microtubulesat metaphase, and the kinetochore at anaphase onset (Williamsand Goldberg, 1994; Williams et al., 1996; Starr et al., 1997).As is the case with CENP-C and CENP-E, which provideessential kinetochore functions, ZW10 and HZW10 associateonly with the functional centromere of dicentric chromosomes(Williams and Goldberg, 1994; Sullivan and Schwartz, 1995;Faulkner et al., 1998). In Drosophila, ZW10 also binds to‘neocentromeres’ that lack conventional centromericsequences but that nonetheless function as centromeres(Williams et al., 1998); the same is true for HZW10 and humanneocentromeres (Saffery et al., 2000). These results suggestthat the binding of ZW10 to centromeres reflects centromereactivity but is not directed by any specific centromeric DNAsequence. Furthermore, recent evidence in Drosophilasuggeststhat ZW10 senses or responds to tension across individualchromosomes. ZW10 fails to localize to microtubules of aunivalent at metaphase of meiosis I (which is only attached toa single pole), while in the same cell, ZW10 can be found onkinetochore microtubules attached to bivalents under bi-polartension (Williams et al., 1996). At least one function of ZW10is to recruit dynein to the kinetochore through a directinteraction with dynamitin, the p50 subunit of dynactin (Starret al., 1998). This interaction with a microtubule motor at thekinetochore suggests how ZW10 might help to sense tensionacross chromosomes.

To further characterize HZW10, we used the yeast two-hybrid screen (Fields and Song, 1989) to search for additionalmolecules at the kinetochore that might interact withHZW10. We then raised antibodies against the six partialcDNA sequences that encoded potential interactors thatexhibited no sequence homologies with known proteins incomputer databases. Immunofluorescence studies using theseantibody preparations revealed a novel component of themammalian kinetochore, which we call HZwint-1 (forHuman ZW10 interacting protein). Antibodies to HZwint-1labeled kinetochores of HeLa cells strongly during mitosis.This result was verified when we found that a GFP/HZwint-1 fusion protein also localized to kinetochores in transfectedcells. In cycling HeLa cells, HZwint-1 binds to thekinetochores during prophase prior to the arrival of HZW10,and remains detectable on the kinetochore until late inanaphase, after HZW10 staining is lost. The pattern ofkinetochore localization exhibited by HZwint-1 suggests thatone of its functions might be to mediate or regulate theassociation between HZW10 and kinetochores. Finally, byshowing that HZwint-1 associates with neocentromeres andwith the active (but not the inactive) centromere of dicentricchromosomes, we suggest that HZwint-1 is essential forproper centromere function.

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1941HZwint-1, a novel kinetochore component

MATERIALS AND METHODS

Two hybrid screenA yeast two hybrid interaction screen (Fields and Song, 1989) wasperformed using the kit developed and kindly provided to us by S.Elledge and his colleagues (Baylor College of Medicine, Houston,TX), essentially following their published protocols (Bai and Elledge,1996). HZW10 was fused in frame and downstream of the DNAbinding domain of GAL4 (residues 1-147) in the pAS2 bait vector(Starr et al., 1998). The HZW10 bait was transformed into the yeasthost strain Y190; these cells were transformed with a human B cellcDNA library cloned downstream of the GAL4transcription activationdomain in the vector pACT. Details on the procedure of the screenand the elimination of false positives are described by Bai and Elledge(1996) and for this specific screen by Starr et al. (1998). Plasmidsencoding the strongest interactors were isolated, sequenced, andanalyzed by blastn searches of GenBank (Altschul et al., 1990; seeTable 1).

To exchange bait and prey, the 1.3 kb BglII restriction fragmentfrom the initially isolated HZwint-1/pACT prey plasmid was clonedinto the BamHI site of the bait vector pAS2. HZW10 was cloned intothe prey vector pACTII as previously described (Starr et al., 1998). Inaddition, six cDNA fragments encoding different regions of HZW10were cloned into pAS2 (Starr et al., 1998) to map the HZwint-1interaction domain of HZW10 (see Table 2 for the exact size of eachfragment). These constructs were tested in the two hybrid system inthe host strain Y190 by assaying the activity of lacZ in X-gal assays(Bai and Elledge, 1996) in the combinations described by the text andin Table 2.

Isolation and characterization of HZwint-1 cDNAsTo determine the size of the HZwint-1 mRNA, a northern blotcontaining RNAs from several human cancer cell lines (Clonetech,Palo Alto, CA) was probed with the 1.3 kb BglII insert from HZwint-1/pACT according to the manufacturer’s protocol. Because the resultsof the northern blot indicated that the 1.3 kb BglII insert was smallerthan the 1.8 kb HZwint-1 mRNA, the same BglII insert was used toprobe a HeLa cell S3 Unizap cDNA library (Stratagene, La Jolla, CA)to find longer HZwint-1 cDNAs. Both strands of the longest cDNA(clone 41-22) were sequenced at the BioResource Center (CornellUniversity, Ithaca, NY). In addition, Blastn searches (Altschul, et al.,1990) were performed on the EST databases at NCBI(http://www.ncbi.nlm.nih.gov) and TIGR (http://www.tigr.org) toidentify HZwint-1 ESTs. The predicted open reading frame wasanalyzed for potential coiled-coil domains by the COILS program

version 2.1 using the MTIDK matrix and a window of 28 (Lupas etal., 1991; http://ulrec3.unil.ch).

Antibodies against the HZwint-1 proteinThe BglII insert of HZwint-1/pACT, which encodes the carboxy 177residues of HZwint-1, was cloned into the BamHI site of the Histagged vector pQE32 (Qiagen, Chatsworth CA). This construct wasexpressed in E. coli XL1-Blue cells (Stratagene), and protein waspurified from a nickel agarose column under denaturing conditionsaccording to the manufacturer’s protocols (Qiagen). The fusionprotein was further purified by SDS-PAGE, and approximately 100µg of purified His-tagged protein was injected three times into tworats at two week intervals. The identical procedure was used toproduce antibodies against His-tagged versions of polypeptidesencoded by the five other novel open reading frames identified in thetwo hybrid screen (see Table 1).

We prepared a different HZwint-1 fusion protein for affinitypurification of the rat anti-HZwint-1 antisera. The entire predictedopen reading frame of HZwint-1 was amplified by PCR with EcoRIand BamHI overhangs, and cloned into the EcoRI and BamHI sites ofthe pMAL-c2 vector (New England BioLabs, Beverly, MA), placingit downstream and in frame to sequences encoding the maltosebinding protein (MBP). The MBP/HZwint-1 fusion protein wasexpressed in E. coli XL1-Blue cells (Stratagene), and purified fromextracts of these cells on an amylose resin column (New EnglandBioLabs) according to the manufacturer’s protocol.

Whole serum from a rat which showed the strongest immuneresponse to His-tagged HZwint-1 fusion protein was affinity purifiedon a column of MBP/HZwint-1 fusion protein covalently bound toAffi-Gel 15 resin (Bio-Rad, Hercules, CA). Serum was incubated withthe column, and after extensive washes in PBS (140 mM NaCl, 2.7mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4), PBS + 0.5 MNaCl, and PBS + 0.5% TritonX-100, the antibodies that remainedbound to the column were eluted with 50 mM dimethyl amine (pH11.5). The eluted antibody was immediately neutralized in 1 M Tris(pH 7.5), and extensively dialyzed against PBS.

To test the specificity of antibodies, western blots were performed onwhole HeLa cell extracts that were separated by SDS-PAGE on a 10%acrylamide gel and electroblotted (Bio-Rad) onto an Immobilon-Pmembrane (Millipore, Bedford, MA) in Towbin buffer (15% methanol,25 mM Tris, 192 mM glycine, pH 8.3). Membranes were blocked one

Table 2. HZwint-1 interacts with HZW10 in the yeast twohybrid system

β-GalactosidaseBait construct* Prey construct‡ activity

HZW10 (all) None -HZwint-1 None -None HZW10 -None HZwint-1 -Lamin HZwint-1 -p53 HZwint-1 -CDK2 HZwint-1 -SNF1 HZwint-1 -HZW10 (all) HZwint-1 ++HZW10 (1-316) HZwint-1 -HZW10 (257-537) HZwint-1 -HZW10 (468-end) HZwint-1 +HZW10 (468-694) HZwint-1 -HZW10 (580-694) HZwint-1 -HZW10 (580-779) HZwint-1 -HZwint-1 HZW10 (all) ++HZwint-1 HZwint-1 +++

*The bait construct is fused to the DNA binding domain of GAL4 in thevector pAS2.

‡The prey construct is fused to the GAL4 activation domain in pACT orpACTII.

Table 1. Two hybrid positives with HZW10 as bait# of isolates Identification of gene GenBank

2 Dynamitin (p50 subunit of dynactin) U507331* HZwint-1 AF0676561* Homolog of Xenopusmitotic phosphoprotein MP43 U950972* Novel; THC 2245581* Novel; THC 2084261* Novel; mouse TC 551851 Novel; THC 90030, orthologs in prokaryotes1 155 kDa subunit of human SWI/SNF complex U666153 SRB7 subunit of RNApolymerase II U468371 Ubiquitin carboxy-terminal hydrolase 7310461 Beta-3-endonexin (binds to cytoplasmic domain of U37139

integrin)3 NAC polypeptide binding protein X809091 Member of SNARE Golgi complex U490991 Jun activation domain binding protein U659281 p31 component of the ER X94910

*These clones were used to raise antibodies in rats.

http://www.ncbi.nlm.nih.govhttp://www.tigr.orghttp://ulrec3.unil.ch

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hour in TBST (20 mM Tris, 137 mM NaCl, 0.2% Tween, pH 7.6) +5% dry milk, and incubated overnight at room temperature with affinity-purified primary antibody at a dilution of 1:100. After washing in TBST,horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (at adilution of 1:5000; Jackson ImmunoResearch Laboratories, WestGrove, PA) was incubated with the blot for one hour. Signals weredetected using the ECL kit (Amersham, Arlington Heights, IL).

Tissue culture and immunofluorescenceHeLa cells were arrested in mitosis after overnight incubation in 1µg/ml nocodazole (Sigma). Chromosome spreads were prepared andfixed as described by Starr et al. (1997). Asynchronous HeLa cellswere grown on coverslips, fixed in 4% paraformaldehyde forimmunofluorescence as previously described (Starr et al., 1997).Samples were incubated for 45 minutes at room temperature withprimary antibodies to HZwint-1, HZW10 or human CREST at thefollowing dilutions in PBS: crude and pre-immune rat anti-HZwint-1sera at 1:250, affinity purified rat anti-HZwint-1 at 1:25, affinitypurified rabbit anti-HZW10 at 1:250 (Starr et al., 1997), and humanCREST serum 2187 (a gift from J. B. Rattner, University of Calgary,AB, Canada) at 1:5000. Images were collected using a charge-coupleddevice camera (KAF1400 chip; 5 MgHz controller; PrincetonLaboratories, Princeton, NJ) attached to an Olympus BX50microscope equipped with epifluorescence optics (Olympus America,Lake Success, NY) driven by Metamorph Imaging System 3.0software (Universal Imaging Corporation, West Chester, PA). Allimages were converted to Adobe Photoshop (Adobe Systems Inc.,Mountain View, CA) where some were pseudocolored.

Analysis of neocentromeres and dicentric chromosomesWe used two previously described transformed lymphoblast cell linesderived directly from patients as a source of mitotically stableneocentromeres in this study (Voullaire et al., 1993, 1999; du Sart etal., 1997; Saffery et al., 2000). The first line contains the mar del(10)chromosome derived from a complex rearrangement of a normalhuman chromosome 10, which involved the deletion of the normalcentromere, followed by activation of a neocentromere at a positioncorresponding to 10q25. The second neocentromere is located on amarker chromosome which resulted from a breakage of a normalchromosome 20 at 20p11.2 followed by an inverted duplication ofthe p arm sequences and activation of a neocentromere at one of the20p12 derived regions within this chromosome. For combinedFISH/immunofluroscence studies on the 10q25 neocentromere weused an 80 kb neocentromere BAC DNA probe (designated E8),derived directly from the mar del(10) chromosome. For identificationof the Inv dup(20p) marker chromosome we used a 173 kb BAC(designated 859D4; GenBank accession number AL035668) whichlocalizes to 20p13 on the normal chromosome 20 and produces twodistinct signals on the inv dup(20p) marker following FISH analysis.FISH/immunofluorescence was carried out as previously described(Saffery et al., 2000).

For dicentric studies, double immunofluorescence was carried outusing CREST-6 antisera (anti-CENP-A, -B) in combination with anti-HZwint-1 antisera. Two cell lines carrying dicentric chromosomeswere used in this study. The first was a lymphoblast line derived froma patient containing a X:15 translocation resulting in a dicentricchromosome (Page et al., 1995) and the second was a fibroblast linederived from a patient carrying an inv dup (8)(p23.1) chromosomewith two chromosome 8 centromeres (L. E. Voullaire, unpublisheddata). Combined immunofluorescence was carried out using aprocedure previously described (Jeppesen et al., 1992).

Transfection of HeLa cellsThe entire coding region of HZwint-1 cDNA (41-22) was amplified byPCR with 5′ NheI and BglII overhangs, and a 3′ HindIII overhang. ThePCR product was then cloned into the BglII and HindIII sites of thepEGFP-C3 vector (Clontech, Palo Alto, CA) to make a mammalian

expression construct coding for a GFP/HZwint-1 (GFP: greenfluorescent protein; Chalfie et al., 1994) fusion protein under control ofthe strong immediate early promoter of human cytomegalovirus(CMV). Alternatively, the HZwint-1 PCR product was cloned into theNheI and HindIII sites of pEGFP-C3 to over-express HZwint-1 intransfected cells without GFP. The HZwint-1 constructs weretransiently transfected into HeLa cells using LipofectAMINE (LifeTechnologies) according to the manufacturer’s protocol.

Chromosome spreads were made and fixed from transfected cellsas described above to observe GFP autofluorescence. Alternatively,transfected cells were prepared for western analysis as describedabove. To detect GFP on western blots, crude GFP antibody 4179made in a rabbit (the gift of J. Cohen and T. Fox, Cornell University,Ithaca, NY) was used as a primary antibody at a 1:2000 dilution.

RESULTS

A yeast two hybrid screen for HZW10 interactorsIn order to find new components of the mammalian kinetochorethat might interact directly with HZW10, we performed aGAL4-based yeast two hybrid screen (Bai and Elledge, 1996;Fields and Song, 1989). The entire coding region of the HZW10cDNA was fused to the DNA binding domain of GAL4, and thisconstruct was transformed into yeast (see Materials andMethods). Expression of the GAL4/HZW10 fusion protein inthe transformed yeast cells was confirmed by western blotanalysis (data not shown). As required for the two hybridprocedure, the GAL4/HZW10 bait was not able by itself toactivate either of the reporter genes (HIS3 or lacZ). About750,000 cDNA inserts from a human B cell cDNA library fusedto the GAL4 transcription activation domain were screened.Positives were defined by their ability to turn on both reportergenes only in the presence of the HZW10 bait, but not withother baits (CDK2, SNF1, lamin, or p53). After eliminatingnon-bait-specific false positives, DNA sequences weredetermined from the 21 remaining interactors (see Table 1).

The DNA sequence of 12 of these cDNAs (representing 8known genes) indicated that they are likely to be false positives(Hengen, 1997), as the intracellular locations of the proteinsthey encode were inconsistent with a role in kinetochorefunction, and the biochemical functions of these proteinsappear to have little overlap with that of HZW10 (Table 1).Two cDNAs encoding dynamitin, the p50 subunit of thedynein-associated dynactin complex that is a knowncomponent of the kinetochore (Echeverri et al., 1996) wereisolated in this screen. This result supported genetic evidencethat linked dynein and ZW10 in Drosophila(Starr et al., 1998).The finding of a two hybrid interaction between HZW10 anddynamitin not only confirmed the relationship between ZW10and dynein/dynactin, but of greater importance here,demonstates the utility of the two hybrid technique foridentifying kinetochore proteins that can interact with HZW10.

One of the remaining seven positives appears to be thehuman homolog of the Xenopusmitotic phosphoprotein MP43.Little is known about this protein other than that it isphosphorylated specifically in mitosis (Stukenberg et al.,1997). Sequences from the six remaining positives(representing five separate genes) from the two hybrid screendid not correspond with proteins of known function in databasesearches (Table 1). One appeared to have orthologs inprokaryotes and was not further investigated, as this protein

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1943HZwint-1, a novel kinetochore component

would be unlikely to be a kinetochore component. Toinvestigate whether the MP43 homolog, or any of the fourremaining novel proteins, associates with the kinetochore andwith HZW10, antibodies were raised against these proteins(see Table 1). When probed against His-tagged bacteriallyexpressed protein on western blots, crude sera showed a strongimmune response in all cases (data not shown). The crude ratsera were used for immunofluorescence experiments in HeLacells to check for kinetochore localization of correspondingepitopes. Antibodies raised against one of the novel proteinswere found to stain kinetochores specifically (see below). Thisprotein was therefore renamed HZwint-1 for HZW10interactor-1. As the other four sera failed to show kinetochorestaining, we have not further pursued investigations on theauthenticity of these positives as true HZW10 interactors.

To verify the HZwint-1/HZW10 two hybrid interaction, wereversed the combination of the bait and the prey (Table 2).HZwint-1 was fused to the DNA binding domain of GAL4andused as bait to test for an interaction with HZW10 that wasfused to the activation domain of GAL4 (Starr et al., 1998).Regardless of the combinations that were tested, HZwint-1interacted with HZW10. Furthermore, HZwint-1 appeared tobe able to self associate, as the bait and prey HZwint-1constructs together activated the reporter genes (Table 2).

We relied on the yeast two-hybrid interactions to localize theHZwint-1 interacting domain in HZW10. HZW10 was dividedinto three roughly equally-sized parts, and each of these wascloned into the bait construct (Starr et al., 1998). Only the C-terminal third of HZW10 (amino acids 468 to the end) wasfound to interact with the HZwint-1 prey, but this interactionwas weak relative to that obtained when the full-length HZW10was used (Table 2). It was not possible to map the interactiondomain of HZW10 more precisely, suggesting that the wholeC-terminal third or multiple parts of the region are required foreven minimal binding to HZwint-1 in the two hybrid system.It is interesting to note that this same region of HZW10 hasbeen mapped as the dynamitin interaction domain, but in thatcase it was also not possible to define the interaction domainmore precisely (Starr et al., 1998).

Molecular characterization of HZwint-1Northern blot analysis revealed that the 1.3 kb HZwint-1 cDNAhybridizes to a single major RNA species approximately 1.8 kbin length (Fig. 1A). We extended the cDNA by using the 1.3 kbcDNA to probe a HeLa cell cDNA library. Three cDNAs wereisolated with inserts larger than 1.3 kb (see Fig. 1B). The endsof each cDNA were sequenced, and the longest of these cDNAs(41-22) was sequenced in its entirety on both strands. Inaddition, a search of EST databases (Adams et al., 1995)identified 14 additional cDNAs that matched the HZwint-1sequence. No EST has been found to date that extends upstreamof the 5′ end of 41-22, but the 5′ ends of many of these ESTsare very close to the 5′ end of the HZwint-1 cDNA 41-22 (Fig.1B). Although we do not know at present whether we haveidentified the 5′ end of the HZwint-1 transcript, these data implythat the 1658 bp of the 41-22 cDNA clone is nearly full-length.

The sequence of the longest HZwint-1 cDNA (41-22) has apredicted open reading frame of 277 amino acids that has acalculated mass of 32 kDa (Fig. 1D). This open reading framestarts with the ATG triplet at nucleotide 24 of the 41-22 cDNAsequence and terminates at a stop codon at nucleotide 858.

Blastp searches of protein databases identified a large regionof HZwint-1 which shares significant similarities with a varietyof coiled-coil proteins. Further analysis of the predicted openreading frame using the COILS program (Lupas et al., 1991)confirmed that a large portion of the HZwint-1 open readingframe is very likely to encode an extended coiled-coil domain(Fig. 1C).

The HZwint-1 sequence overlaps that of the previouslyidentified MPP5 gene (see Fig. 1B). MPP5 was identified as apartial cDNA that expressed a protein in E. coli that could berecognized by the MPM2 monoclonal antibody (Matsumoto-Taniura et al., 1996). Antibodies made against epitopesencoded by this MPP5 partial cDNA immunoprecipitated a 130kDa protein that was also recognized by the MPM2 antibody.These anti-MPP5 antibodies also stained the spindle inimmunofluorescence studies (Matsumoto-Taniura et al., 1996).We were therefore intrigued by the possibility that HZwint-1might be in fact be MPP5. However, we now have strongevidence at the nucleic acid and protein levels that show MPP5is not HZwint-1. (1) Comparison of the cDNAs indicated thatMPP5 RNA is transcribed from the strand of DNA that isopposite to HZwint-1. No ESTs from Blast searches, or cDNAsfrom the library screen, were identified with the same polarityas MPP5; all cDNAs with polyA tails had the same 5′-3′orientation predicted for HZwint-1 (see Fig. 1B). (2) MPP5polyclonal antibodies failed to recognize full length HZwint-1fusion protein expressed in E. coli (data not shown). (3) Thepredicted protein sequence of HZwint-1 does not contain theF-phosphoT-P-L-Q epitope recognized by the MPM2 antibody(Westendorf et al., 1994). Although our results suggest that theMPP5 cDNA clone may be artefactual, it remains possible thatthis region of genomic DNA contains two genes with oppositepolarities. In this latter scenario, the MPP5 transcript might beone of the larger RNA species very weakly detected by theHZwint-1 cDNA on northern blots (Fig. 1A).

HZwint-1 is a component of the kinetochoreOur interest in HZwint-1 stemmed from the finding that crudeantisera from two rats directed against this particular proteinstained kinetochores, while preimmune sera from the sameanimals did not. To verify that the signal at the kinetochoreobserved with these antisera was in fact due to recognition ofHZwint-1, we affinity purified the strongest of these ratantisera on a column containing a MBP/HZwint-1 fusionprotein. The specificity of the affinity purified antibodies wasmonitored by western blot analysis (Fig. 2). The affinitypurified, rat anti-HZwint-1 antibodies recognized 43 and 53kDa proteins in total HeLa cell lysates (Fig. 2, lane 4). Neitherof these proteins were recognized by pre-immune serum (notshown). We believe that the 43 kDa band is likely to representthe actual HZwint-1 protein, because it is considerably moreabundant in extracts made from cells transfected with anHZwint-1 construct driven by the strong CMV early promoter(lane 2 of Fig. 2). As further verification of the specificity ofthe antibody, we used the affinity purified anti-HZwint-1antibodies to probe extracts of HeLa cells that were transfectedwith a construct designed to overexpress a GFP/HZwint-1fusion protein (Fig. 2, lane 1). The size of the novel bandrecognized in this experiment (60 kDa) is roughly thatexpected from the addition of the GFP moiety (23 kDa) to the43 kDa band. The identity of the overexpressed GFP/HZwint-

1944 D. A. Starr and others

Fig. 1. The HZwint-1 cDNA. (A) TheHZwint-1 cDNA hybridizes strongly to anapproximately 1.8 kb transcript on anorthern blot of RNAs from six cancer celllines. Lanes 1-6 contain 2 µg of poly(A)+RNA from promyelocytic leukemia HL-60,HeLa S3, chronic myelogenous leukemiaK-562, lymphoblastic leukemia MOLT-4,Burkitt’s lymphoma Raji, and colorectaladenocarcinoma SW480 cell lines,respectively. Longer exposures (not shown)show that the HZwint-1 cDNA alsohybridizes to larger bands around 2.5 kb(this band is barely visible in lane 4) andabout 6 kb. The lower panel shows thesame filter probed for actin transcripts as aloading control. RNA size marker bandsand the known size of actin mRNA areshown at the left in kilobases. (B) HZwint-1 and MPP5 cDNAs. HZwint-1 cDNAs 41-22, 41-2, and 41-5 were cloned from aHeLa cell cDNA library. In addition, manyhuman ESTs (at least 14) with sequencesidentical to that of 41-22 have been foundin database searches. EST 42601 extendsfurthest toward the 5′end of the transcript;it starts 20 bp downstream from the 5′-most extent of cDNA 41-22. Dashed linesrepresent regions of EST 42601 which areunknown in sequence or length, and a gapin the alignment of MPP5 as determined byMatsumoto-Taniura et al. (1996).Numbering of nucleotides begin with thenucleotide at the 5′-end of 41-22 as base 1.The GenBank accession number for MPP5is X98261 and for HZwint-1 is AF067656.(C) HZwint-1 contains a large coiled-coildomain. The output from COILS (Lupas,

et al., 1991) is shown. The Y axis is the probability according to this algorithm that any portion of the protein will form a coil-coil. The X axisis the predicted amino acid sequence of the HZwint-1 protein. (D) The nucleotide sequence of the longest HZwint-1 cDNA (41-22) and thepredicted amino acid sequence of the HZwint-1 protein in the one letter code.

1945HZwint-1, a novel kinetochore component

1 band was confirmed on western blots probed with anti-GFPantibodies (data not shown). We do not at present understandthe nature of the larger 53 kDa band recognized by our anti-HZwint-1 antibodies. It may represent a larger form ofHZwint-1 undetected as adiscrete cDNA, or a secondprotein with an epitope incommon with HZwint-1.

Fig. 3 shows the resultsfrom immunofluorescenceexperiments in whichaffinity purified anti-HZwint-1antibodies stained thekinetochores of chromosomesfrom HeLa cells arrested in a metaphase-like state bythe microtubule poisonnocodazole. Centromeres weremarked in the same spreadswith a CREST serum(Fig. 3B). The HZwint-1localization pattern was found

to overlap the outer fringe of staining obtained with CRESTserum. This localization is expected for a kinetochorecomponent, and is very reminiscent of that of HZW10 (Starret al., 1997). To better understand the spatial relationshipbetween HZwint-1 and HZW10, chromosome spreads werestained with antibodies against both of these molecules.Overlap in the signals was complete, with HZwint-1 andHZW10 co-localizing to the kinetochores of these metaphase-arrested chromosomes (Fig. 3C).

To confirm that the kinetochore localization seen byimmunofluorescence with anti-HZwint-1 antibodies wereindeed due to the recognition of HZwint-1 protein (and notcross-reacting epitopes) at the kinetochore, we employed theGFP/HZwint-1 overexpression construct described above. Theconstruct was transiently transfected into HeLa cells, whichwere subsequently fixed by formaldehyde and visualized forGFP fluorescence. As expected, signal was detected at thekinetochores in chromosome spreads from HeLa cellstransfected with the GFP/HZwint-1 construct (Fig. 4A,B). Thebackground of GFP fluoresence was high, due to theoverexpression of GFP/HZwint-1 driven by the strong CMVearly promoter (see also Fig. 2, lane 1). In spite of the highbackground, the kinetochore signal was specific for HZwint-1,

Fig. 2. The HZwint-1 gene product.A western blot of whole HeLa cellextracts was probed with affinitypurified anti-HZwint-1 antibody.HeLa cells extracts were made fromcells transiently transfected withvarious expression constructs tooverexpress a GFP/HZwint-1 fusionprotein (lane 1), HZwint-1 alone(lane 2), GFP alone (lane 3), or fromuntransfected cells (lane 4). Theposition of protein standards areshown on the left.

Fig. 3. HZwint-1immunolocalizes to thekinetochores of metaphasearrested HeLa cell chromosomes.(A) A chromosome spreadstained with antibodies toHZwint-1 (red). The DNA isstained with Hoechst 33258(blue). (B-C) Chromosomespreads stained with HZwint-1(red) and CREST serum 2187 (B,green) or HZW10 (C, green).Overlap is indicated by theyellow color. The arrows point toparticular centromeric regionsenlarged in the insets. Theseinsets show that HZwint-1 islocated just to the outside of theCREST staining at thecentromere (B), and completelyco-localizes with HZW10 (C).

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as a kinetochore signal was not observed in cells transfectedwith a construct coding for GFP alone (Fig. 4C,D). It is thusclear that HZwint-1 indeed localizes to the kinetochore. Thisfinding does not eliminate the remote possibility that our anti-

HZwint-1 antibodies recognize other kinetochore componentsin addition to HZwint-1 itself.

HZwint-1 arrives at the kinetochore before HZW10The localization pattern of HZwint-1 in cells untreated withmicrotubule poisons was examined to determine its normaldistribution throughout the cell cycle. These studies could alsoaddress the potential problem that HZwint-1’s association withkinetochores in chromosome spreads might be an artifact ofthe drug treatment, as has been seen for other proteins(Compton et al., 1991). Finally, simultaneous examination ofHZwint-1 and HZW10 through the cell cycle might help usbetter understand the nature of the interactions between thesetwo proteins.

HZwint-1 was found mostly uniformly distributed within thecytoplasm of interphase cells (data not shown). HZwint-1antibodies first stained discernible structures during prophase,as the chromosomes began to condense; these signals werepresent as paired dots reminiscent of kinetochore staining (Fig.5). In a number of prophase cells (8 of 20 on one slide), strongHZwint-1 staining was visible while no HZW10 signal wasdetected at kinetochores (Fig. 5A-D). Even in the prophasecells in which HZW10 was found at kinetochores, its stainingwas much less intense than later in prometaphase. However,the level of HZwint-1 staining appeared constant in all cellswith visibly condensing chromosomes. We thus conclude thatHZwint-1 localizes to prophase kinetochores prior to HZW10.In prometaphase cells, HZwint-1 and HZW10 were found tocolocalize at kinetochores (Fig. 5E-H; Starr et al., 1997).

Although HZwint-1 and HZW10 co-localize atkinetochores in colchicine-treated cells (Fig. 3C) and inprometaphase cells (Fig. 5), the two proteins differed in theirdistribution pattern in metaphase and anaphase cells. HZwint-1 remained at the kinetochores during metaphase andthroughout anaphase (Fig. 6). This contrasts with HZW10,

D. A. Starr and others

Fig. 4. Transfected GFP/HZwint-1 protein localizes to kinetochores.HeLa cells were transiently transfected with a construct tooverexpress either a GFP/HZwint-1 fusion protein (A and B) or GFPalone (C and D). Metaphase arrested chromosome spreads were thenfixed to visualize GFP autofluorescence (A and C), and stained tovisualize DNA (B and D). Note the untransfected cell on the bottomleft of C and D. The arrows in A and B point to the chromosomeenlarged in the inset. Bar, 5 µm.

Fig. 5. HZwint-1 localizes to the prophase kinetochore before HZW10. (A-D) A prophase cell. (E-H) A cell later in prometaphase. The cellswere stained on the same coverslip with HZwint-1 antibodies (A and E), HZW10 antibodies (B and F), and Hoechst 33258 to stain DNA (Cand G). (D and H) A merged view with HZwint-1 in red, HZW10 in green, and DNA in blue. Yellow is overlap of the HZwint-1 and HZW10signals. Bar, 10 µm.

1947HZwint-1, a novel kinetochore component

where previous studies have shown that most HZW10staining is on the spindle at metaphase, though a smallfraction of HZW10 is also on the kinetochores of metaphaseand early anaphase chromosomes (Starr et al., 1997). Late inanaphase, HZW10 was absent from kinetochores and wasinstead dispersed in the cytoplasm (Starr et al., 1997), whilethe kinetochore staining of HZwint-1 in similar late mitoticfigures is readily apparent (Fig. 6).

HZwint-1 is a marker for active centromeresTo establish whether HZwint-1 staining reflects centromericfunction, we asked whether HZwint-1 localizes toneocentromeres and dicentric chromosomes. FISH/immunofluorescence on two marker chromosomes containingneocentromeres (chromosome 10 and 20 derived) clearlydemonstrated the association of HZwint-1 with bothneocentromeres (Fig. 7A,B). Neither of the regions on thenormal chromosome 10 or 20 from which the neocentromereswere derived showed HZwint-1 staining, correlating HZwint-1 localization with centromere activity rather than withspecific DNA sequences. In addition, HZwint-1 associatedspecifically with the active, but not the inactive, centromereson two different dicentric chromosomes. CREST6autoimmune serum recognizing CENP-A and CENP-Bepitopes in contrast localizes to both inactive and activecentromeres on both dicentrics (Fig. 7C, and data not shown).

DISCUSSION

The two hybrid screen performed with the humanhomolog of ZW10 (HZW10) as bait has providedseveral clues to the molecular function of ZW10at the kinetochore. First, the screen identified apreviously unknown interaction of HZW10 with aknown kinetochore component, dynamitin, thep50 subunit of dynactin. This finding stimulatedus to initiate additional studies on the relationshipbetween ZW10, dynein, and dynactin. The resultsof these investigations, as reported by Starr et al.(1998), provided strong support to the model thatZW10 recruits dynein to the kinetochore throughdirect contact with dynamitin. Second, as shownhere, the two hybrid screen revealed an interactionbetween HZW10 and HZwint-1, a novelkinetochore component.

The HZwint-1 proteinAlthough we have found mouse and rat ESTs thatare clearly homologous to HZwint-1 (data notshown), extensive searches of the EST and genomicdatabases (including the essentially completegenomes of Saccharomyces cerevisiae,Caenorhabditis elegans, and Drosophilamelanogaster) have revealed no proteins related toHZwint-1 outside of mammals. It is possible thatHZwint-1 may be a relatively recently evolvedgene, but we favor an alternative explanation for ourinability to detect non-mammalian HZwint-1homologs: that the protein may be conserved at thestructural, but not at the amino acid sequence, level.

As illustrated in Fig. 1C, a large portion of HZwint-1 is predictedto fold in three-dimensional space as an extended coiled-coil.Structural conservation with putative HZwint-1 homologs mayhave been masked in computer searches because of the strongsimilarities between all coiled-coil proteins (Odgren et al.,1996). In this light, it is striking that a number of kinetochoreand centromere components have been previously shown to haveextensive coiled-coil domains including CENP-F, CENP-E,CLIP-170, INCENP, and MAD1 (Yen et al., 1991; Pierre et al.,1992; Mackay et al., 1993; Liao et al., 1995; Hardwick andMurray, 1995). Coiled-coil domains have been predicted to formfibrous structures or to be protein-protein binding sites (reviewedby Parry and Steinhart, 1992), so proteins with these domainsmay be well-suited to the stepwise assembly of the molecularlycomplex centromere/kinetochore.

Potential roles of HZwint-1 at the kinetochoreAntibody raised against HZwint-1 stains the kinetochores ofchromosomes in spreads of nocodazole-arrested HeLa cells (Fig.3). Anti-HZwint-1 signals partially overlap and are just outsideof the sites recognized by a centromere-specific antibody (Fig.3B). Moreover, HZwint-1 completely co-localizes with HZW10(Fig. 3C), as expected given the two hybrid data indicating aninteraction between HZW10 and HZwint-1 (Table 1). HZW10in turn co-localizes with CENP-E (Starr et al., 1997), which hadpreviously been shown by immuno-electron microscopy to be acomponent of the kinetochore’s outer plate and the fibrouscorona (Cooke et al., 1997; Yao et al., 1997). Based on these

Fig. 6.HZwint-1 immunolocalization in mitotic HeLa cells. (A and B) A HeLa cellin metaphase. (C and D) A HeLa cell in anaphase. The cells were stained withHZwint-1 antibodies (A and C) and Hoechst 33258 to visualize DNA (B and D).Bar, 5 µm.

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colocalization experiments, we believe it most likely thatHZwint-1 is a component of the outer kinetochore plate or ofthe fibrous corona, but clearly, the precise position of this proteinat the kinetochore will require future immunolocalization studiesat the electron microscope level. The conclusion that HZwint-1is concentrated at the kinetochore was confirmed by expressinga GFP/HZwint-1 fusion protein in transiently transfected HeLacells. Fig. 4 shows that the the GFP/HZwint-1 fusion protein, asdetected by GFP fluorescence, localizes to the kinetochore inthese cells. Further verification of HZwint-1’s identity as akinetochore protein comes from the examination of itslocalization in mitotically cycling cells. Anti-HZwint-1antibodies stain paired dots in early prometaphase cells in apattern reminiscent of other kinetochore proteins (Pluta et al.,1995; Choo, 1997), and HZwint-1 remains at the kinetochoresthroughout mitosis (Figs 5, 6).

The mitotic kinetochore appears to be constructed in astepwise manner to form the tri-laminar plate visible byelectron microscopy. hBUB1 and CENP-F are the earliesttransient kinetochore components to localize to thekinetochore, followed by MCAK, then a number of proteinsincluding HZW10 and hBubR1, and finally CENP-E (Liao etal., 1995; Jablonski et al., 1998; Chan et al., 1998). Fig. 5shows that HZwint-1 associates with kinetochores relatively

early in prometaphase, before HZW10. It remains to be seenif HZwint-1 arrives at kinetochores prior to MCAK, CENP-F,or hBUB1. The findings that HZwint-1 preceeds HZW10 to thekinetochore and that HZwint-1 and HZW10 interact in theyeast two hybrid assay together suggest that HZwint-1 may bepart of a scaffold to which HZW10 binds. Along these lines,it is interesting to note that HZwint-1 is predicted to fold in alarge coiled-coil domain (Fig. 1C), as do several other scaffoldproteins of the kinetochore (discussed above).

Given our model that HZwint-1 may target HZW10 to thekinetochore, it may be difficult to confirm the two hybridinteraction between HZwint-1 and HZW10 by biochemicalmeans such as co-immunoprecipitations or in vitro pull downassays. If HZW10 and HZwint-1 interact only at thekinetochore (a large insoluble structure), soluble complexescontaining the two proteins would not be formed. Indeed,initial attempts to co-immunoprecipitate HZwint-1 andHZW10 from extracts were not successful (data not shown). Itis also conceivable that a modification of HZwint-1 or HZW10may be necessary for the interaction, making it difficult toreconstitute an association in vitro.

A role for HZwint-1 as a scaffolding protein specifically ofthe outer kinetochore plate is further supported by our findingthat HZwint-1 stays on the kinetochore at metaphase, when

D. A. Starr and others

Fig. 7. HZwint-1 staining correlates with centromere activity. Localization of HZwint-1 to the 10q25 (A) or 20p12 (B) neocentromere.Combined FISH/ immunofluorescence on chromosomes using anti-HZwint-1 (green), DAPI (blue), and a BAC DNA probe (red) either derivedfrom the 10q25 neocentromere (Ai) or from region 20p13 adjacent to the 20p12 neocentromere (Bi). Magnification is 1000x. HZwint-1 andFISH signals show no overlap on the normal chromosome 10 or 20 (Ai and Bi, arrows). In contrast, the FISH and anti-HZwint-1 signalscolocalize at the neocentromere of mar del(10) (Ai, arrowhead). The inv dup(20p) chromosome (Bi, arrowhead) shows two distinct FISHsignals, one of which is closely apposed to the 20p12 neocentromere containing the HZwint-1 signal. Note the absence of HZwint-1 staining onthe duplicated 20p12 region on the same chromosome. Accompanying insets show enlarged views of mar del(10) or inv dup(20p): (ii)combined FISH/HZwint-1 signals, (iii) HZwint-1 alone, (iv) FISH signal alone, and (v) DAPI staining for DNA. (C) HZwint-1 localizes to theactive but not the inactive centromere on an X:15 dicentric chromosome (Ci, arrowhead). Double immunofluorescence using CREST6antiserum (red), anti-HZwint-1 antisera (green), and DAPI (blue); magnification at ×1000. Insets: (ii) HZwint-1 plus CREST6, (iii) anti-Zwint-1 only, (iv) CREST6 only, and (v) DAPI staining only.

1949HZwint-1, a novel kinetochore component

HZW10, dynactin, and dynein appear to migrate off thekinetochore and onto the spindle (Pfarr et al., 1990; Steuer etal., 1990; Echeverri et al., 1996; Starr et al., 1997). These lattermolecules are likely to be part of the fibrous corona, which hasbeen observed to stretch out or extend along kinetochoremicrotubules at metaphase (Rieder, 1982). HZwint-1 is likelyto be more centrally positioned within the kinetochore thanthese other proteins, as it does not display this behavior (Fig.6). This interpretation is also consistent with the observationthat HZwint-1 remains at the kinetochore throughout anaphase,until the mitotic kinetochore is disassembled and all transientproteins are removed from the kinetochore. Interestingly,although other predicted kinetochore scaffold proteins such asCENP-F are degraded at the end of mitosis (Liao et al., 1995),it appears that HZwint-1 remains at constant levels throughoutthe cell cycle (data not shown).

Although the precise role of HZwint-1 has yet to bedetermined, its localization to human neocentromeres (Fig.7A,B) and its specific association with active, but not inactive,centromeres on dicentric chromosomes (Fig. 7C) clearlyimplicate HZwint-1 in kinetochore function. For all testedcases, kinetochore-associated proteins for which a function hasbeen demonstrated similarly bind to neocentromeres (Safferyet al., 2000) and localize specifically to the active centromereof dicentric chromosomes (Sullivan and Schwartz, 1995;Faulkner et al., 1998). In contrast, CENP-B, produced by agene non-essential for mouse viability (Hudson et al., 1998),fails to bind neocentromeres (Saffery et al., 2000).

We speculate, based on the yeast two-hybrid interactionbetween HZwint-1 and HZW10, and on previous resultsimplicating HZW10 in the targeting of dynein and dynactin tokinetochores, that HZwint-1 will also prove to be involved inthe localization of dynein, and perhaps other motor proteins,to the kinetochore. Future analysis of HZwint-1 and HZW10should allow us to test this and other possibilities for HZwint-1’s role in kinetochore function.

We thank Dr R. Karess and F. Scaerou (CNRS, France) forcommunication of unpublished results, J. Hittle and the Lab AnimalFacility at Fox Chase Cancer Center for technical help producing theantibodies, L. G. Shaffer and L. E. Voullaire for the X:15 and inv(8)(p23.1) dicentric cell lines, and C. Bayles for assistance withmicroscopy. We are indebted to Dr B. Williams (Cornell University),and to Drs G. Chan and S. Jablonski (both of Fox Chase CancerCenter) for helpful advice and discussions. This work was supportedby NIH grant GM48430 to M.L.G.; work in the laboratory of T.J.Y.was supported by funds from the NIH, ACS, LSA Scholar’s Award,core grant CA06927, and an appropriation from the Commonwealthof Pennsylvania. R.S and K.H.A.C were supported by grants from theNHMRC, Ausindusry, and Amrad Co., Australia.

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