Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF, 907K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Starr, D. A. Articles by Goldberg, M. L. Search for Related Content PubMed PubMed Citation Articles by Starr, D. A. Articles by Goldberg, M. L. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Protein UniGene Substance via MeSH Social Bookmarking                            What's this?

© The Rockefeller University Press, 0021-9525/1998//763 $5.00
The Journal of Cell Biology, Volume 142, Number 3, , 1998 763-774

ZW10 Helps Recruit Dynactin and Dynein to the Kinetochore

Daniel A. Starr^*, Byron C. Williams^*, Thomas S. Hays, and Michael L. Goldberg^*

^* Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703; and Departments of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108

Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, our results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset.

Key Words: ZW10 • dynein • dynamitin • rough deal • kinetochore

Abbreviations used in this paper: CCD, charge-coupled device; Dhc, dynein heavy chain; GAL4, galactose metabolism regulatory gene 4; rod, rough deal gene; SD, synthetic minimal media; X-Gal, 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside.

Address all correspondence to Michael L. Goldberg, Section of Genetics and Development, Cornell University, Ithaca, NY 14853-2703. Tel.: (607) 254-4802. Fax: (607) 255-6249. E-mail: mlg11{at}cornell.edu

THE kinetochores elaborated by the centromeres of eukaryotic chromosomes play three major roles during mitosis and meiosis (for review see Pluta et al., 1995). First, the kinetochores serve as the mechanical link allowing the chromosomes to attach to the dynamic plus ends of microtubules of the spindle apparatus. Second, the kinetochores contain microtubule motor activities that are probably responsible for poleward movements of the chromosomes during prometaphase (Rieder and Alexander, 1990), for at least some aspects of chromosomal movements accompanying congression to the metaphase plate (Rieder and Salmon, 1994), and for the poleward forces exerted on chromosomes during anaphase A (Nicklas, 1989). Finally, the kinetochores are intimately involved in the elaboration of a "wait anaphase" checkpoint control that ensures cells will not enter anaphase until all chromosomes are properly oriented at the metaphase plate (Li and Nicklas, 1995; Nicklas et al., 1995; Chen et al., 1996; Li and Benerza, 1996; Taylor and McKeon, 1997). These three kinetochore functions may not in fact be fundamentally distinct. For example, recent evidence suggests that the kinesin-related microtubule motor centromere-associated E protein (CENP-E) may act by tethering kinetochores to the plus ends of disassembling microtubules during chromosome congression (Yen et al., 1992; Lombillo et al., 1995; Duesbery et al., 1997; Wood et al., 1997; Yao et al., 1997).

Cytoplasmic dynein is one of three microtubule motor proteins currently known to localize to the kinetochore of mammalian chromosomes (Pfarr et al., 1990; Steuer et al., 1990; Wordeman et al., 1991); the two others are CENP-E (see above) and mitotic centromere-associated kinesin/ Xenopus kinesin-central motor 1 (MCAK/XKCM1), a member of the KIF2 subfamily of plus end–directed kinesins (Walczak et al., 1996; Wordeman and Mitchison, 1995). It has been extremely difficult to determine the importance of dynein's association with the kinetochore because dynein is required for many intracellular processes. For example, a complex of cytoplasmic dynein and the protein NuMA at the spindle poles has recently been demonstrated to be essential for proper assembly of the mitotic spindle (Merdes et al., 1996). Disruption of this activity would be particularly likely to mask possible effects of the perturbation of dynein at the kinetochore. Thus, microinjection of anti-dynein into cells induces spindle collapse (Vaisberg et al., 1993), whereas depletion of dynein from Xenopus or HeLa cell extracts disrupts aster formation or spindle pole assembly (Verde et al., 1991; Gaglio et al., 1996; Heald et al., 1996). Moreover, in Drosophila melanogaster, recent mutational analysis of dynein function has revealed defects in centrosome behavior and spindle morphogenesis during the nuclear divisions of the early syncytial embryo (Robinson, J.R., E.J. Wojcik, M. Sanders, M. McGrail, and T.S. Hays, manuscript in preparation). Some role for cytoplasmic dynein in mitotic chromosome movements has been inferred from studies of transfected tissue culture cells that overexpress dynamitin, the p50 component of the dynactin complex that may help target dynein to intracellular cargoes (Echeverri et al., 1996). In these cells with excess dynamitin, both dynein and dynactin are no longer associated with the kinetochores, and the chromosomes do not align properly at the metaphase plate (Echeverri et al., 1996). However, as these authors point out, the observed difficulties in chromosome behavior may be indirect effects of distortions of the spindle that also occur in these cells. Because of these complications, the significance of dynein's localization at the kinetochore remains highly controversial. Does this microtubule motor in fact play any role in attaching the chromosomes to spindle fibers, in moving the chromosomes along these microtubules, or in the wait anaphase checkpoint?

In this report, we establish a connection between dynein and ZW10, a kinetochore component conserved in most if not all multicellular eukaryotes (Starr et al., 1997). Null mutations in the Drosophila gene l(1)zw10 (hereafter abbreviated zw10) encoding the fly ZW10 protein disrupt chromosome segregation during mitosis and both meiotic divisions. Mitotic missegregation in zw10 mutants produces many aneuploid cells and consequent lethality to the organism (Smith et al., 1985; Williams et al., 1992). Although in zw10 mutants the chromosomes congress normally to the metaphase plate, defects are first detected during anaphase of the cell cycle where the separation and poleward movements of sister chromatids (during mitosis and meiosis II) or of homologous chromosomes (during meiosis I) occur asynchronously. As a result, some lagging chromatids or chromosomes remain behind in the vicinity of the former metaphase plate during anaphase. Related effects can be phenocopied in Caenorhabditis elegans embryos by injection of antisense RNA of the nematode ZW10 homologue into gonads (Starr et al., 1997).

ZW10 proteins in Drosophila and HeLa cells display a similar and intriguing cell cycle-dependent intracellular distribution. ZW10 protein first becomes localized to the kinetochore at prometaphase, but then appears to move onto the kinetochore microtubules of the spindle at metaphase, and then back to the kinetochore at anaphase (Williams et al., 1992; Williams and Goldberg, 1994; Williams et al., 1996; Starr et al., 1997). Interestingly, the pattern of ZW10 localization with respect to each chromosome's kinetochores is influenced by the presence or absence of tension across the centromere. During metaphase of the first meiotic division in Drosophila spermatocytes, ZW10 remains at the kinetochore of univalents that are attached only to a single spindle pole, but appears in the same cell to move from the kinetochores of bivalent chromosomes under bipolar tension onto the attached kinetochore microtubules (Williams et al., 1996). This observation suggests that ZW10 may act as part of, or immediately downstream of, the wait anaphase tension–sensing checkpoint. In further support of a possible relationship between ZW10 and the anaphase onset signaling mechanism, sister chromatids in zw10 mutants often separate precociously in the presence of microtubule-depolymerizing drugs, in contrast to their behavior in wild-type (Smith et al., 1985; Williams et al., 1992).

In this paper, we show that mutations in the Drosophila zw10 gene prevent the association of dynein heavy chain (Dhc)¹ with the kinetochores of both meiotic and mitotic chromosomes. Interestingly, our studies also demonstrate that dynein's kinetochore localization is influenced by tension across the centromere. We further present evidence suggesting that the function of ZW10 in the targeting of dynein to the kinetochore is mediated by direct interactions of ZW10 with dynamitin, the p50 subunit of dynactin. Because zw10 mutations appear specifically to disrupt dynein at the kinetochore but not elsewhere in the cell, the phenotype caused by zw10 mutations provides information important to understanding dynein's role at the kinetochore.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cytological Analysis of Meiosis and Mitosis in Drosophila
The Drosophila stocks used in these experiments have been previously described in Williams et al. (1996) and Murphy and Karpen (1995).

The following techniques were used to localize various molecules within Drosophila spermatocytes. Larval, pupal, or adult testes were dissected in 0.7% NaCl and then placed in a small drop of PHEMT (60 mM Pipes, 25 mM HEPES, pH 7.0, 10 mM EGTA, 4 mM MgSO₄, 0.5% Triton X-100) for 2 min. The testes were subsequently transferred to 4 µl of PHEMT + 3.7% formaldehyde on a coverslip and then immediately squashed on an inverted slide. The squashed testes were left on the slides for 10 min to allow fixation, after which the slide was immersed in liquid nitrogen and the coverslip was removed. The slide was then incubated in methanol for 20 min at –20°C, and the squash subsequently rehydrated in several changes of PBT (2.6 mM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl, 8.1 mM Na₂HPO₄, 0.02% NaN_3, 0.1% Triton-X100) at room temperature. To detect Dhc, either the anti-Dhc monoclonal antibody P1H4 (McGrail and Hays, 1997) at a 1:2,000 dilution in PBT or the rabbit anti-Dhc polyclonal antibody (Hays et al., 1994) at a dilution of 1:30 in PBT, was incubated with the squashed, fixed preparation overnight at 4°C. The samples were next washed in PBT 3 times for 5 min each at room temperature and then incubated overnight at 4°C with secondary antibody: either a 5-µg/ml dilution of TRITC-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc., Oak Grove, PA) if the primary reagent was the P1H4 monoclonal antibody, or with a 7.5-µg/ml dilution of TRITC-conjugated goat anti–rabbit IgG if the primary antibody was the polyclonal anti-Dhc antibody. In experiments where Dhc localization was examined in zw10 or rod mutants, wild-type control testes were placed side by side on the same slide as the mutant testes. For simultaneous localization of ZW10 and Dhc, affinity-purified rabbit anti-ZW10 polyclonal antibodies (Williams et al., 1992) at a dilution of 1:120 in PBT were mixed with the anti-Dhc P1H4 monoclonal antibody diluted as above. The secondary antibodies in this double-staining protocol were FITC-conjugated anti-rabbit IgG (The Jackson Laboratory, Bar Harbor, ME) or 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3- propionic acid (BODIPY-FL)– conjugated goat anti–rabbit antibody (both at 7.5 µg/ml; Molecular Probes Inc., Eugene, OR) to detect ZW10 antigen and the TRITC-conjugated anti-mouse IgG secondary antibody diluted as above to follow Dhc. After incubation with secondary antibody, all slides were washed in PBT for 2 h, stained with Hoechst 33258 (0.5 µg/ml in PBS; Sigma Chemical Co., St. Louis, MO) for 15 min, dried, and then mounted in glycerol + 2% n-propyl gallate to attenuate photobleaching.

To analyze mitotic figures in larval neuroblasts, larval brains were fixed, squashed, and then stained for immunofluorescence exactly according to Williams and Goldberg (1994), except that visualization of Dhc was performed as described above for testis preparations.

Images were collected using a charge-coupled device (CCD) camera (KAF1400 chip; 5 MgHz controller; Princeton Laboratories, Inc., Princeton, NJ) attached to a fluorescence microscope (model BX50; Olympus America, Lake Success, NY). Images were collected and processed with the Metamorph Imaging System (version 3.0; Universal Imaging Corporation, West Chester, PA). Alternatively, immunostained testes preparations were also observed using an ImagePointR CCD camera (Photometrics, Tucson, AZ) connected to a Zeiss Axioskop (Carl Zeiss, Inc., Oberkochen, Germany) using IPLab Spectrum software (Signal Analytics Co., Vienna, VA). All images were converted to Photoshop format (Adobe Systems Inc., Mountain View, CA). Final images were produced on a dye sublimation printer (Codonics NP1600; Cleveland, OH).

Two-hybrid Screen
A yeast two-hybrid interaction screen (Fields and Song, 1989) was preformed using the kit developed and provided to us by S. Elledge and colleagues (Baylor College of Medicine, Houston, TX), essentially following their published protocols (Bai and Elledge, 1996). The entire coding region of HZW10 was amplified by PCR using primers with 5' NcoI and 3' BamHI restriction site overhangs. The PCR product was then digested with NcoI and BamHI and cloned in frame and downstream of the galactose metabolism regulatory gene 4 (GAL4) DNA-binding domain (residues 1–147) in the pAS2 vector. This "bait" fusion construct (pAS2/ HZW10) was transformed into the host yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 + URA3::GAL-lacZ, LYS2::GAL[UAS]-HIS3 cyh^r). A human B cell cDNA library cloned downstream of the GAL4 transcription activation domain in the vector pACT1 (provided by S. Elledge) was transformed into Y190 + pAS2/ HZW10 as previously described (Bai and Elledge, 1996), and the transformed cells were plated onto synthetic minimal media (SD)-Trp, Leu, His + 25 mM 3-amino-1,2,4-triazole (3-AT; Sigma Chemical Co.). Transformation efficiency was determined by plating a small aliquot on SD-Trp, Leu plates. After 3–7 d, large Trp⁺ (presence of bait construct), Leu⁺ (presence of library prey construct), His⁺ (reporter turned on) colonies were streaked to fresh plates and colony filter lifts were made and tested for lacZ activity by a 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal) assay as previously described (Bai and Elledge, 1996).

Several criteria were used to screen against false positives. First, potential positives were streaked on SD-Leu, grown at 30°C for 2–3 d, and then streaked on SD-Leu + 2.5 mg/ml cycloheximide to select against pAS2/ HZW10. Once the bait plasmid was removed, a second X-Gal assay was preformed to identify false positives. Second, additional nonbait-specific false positives were identified by mating colonies with the potential positive pACT plasmids to the yeast strain Y187 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 URA3::GAL-lacZ) with bait constructs encoding CDK2, SNF1, lamin, or p53 in pAS1. Diploids, selected for growth on SD-Trp, Leu, were assayed for X-Gal activity.

Plasmids containing prey constructs for pAS2/HZW10 bait-dependent positives were isolated from the host yeast as previously described (Bai and Elledge, 1996), and electroporated using an Escherichia coli pulser (Bio-Rad Laboratories, Hercules, CA) into E. coli XL1-blue (Stratagene, La Jolla, CA). Positives were sequenced using the pACT forward 5' primer (Bai and Elledge, 1996) by the ddNTP chain termination method with the Sequenase kit (United States Biochemical, Cleveland, OH) and potential identity was determined by a BLAST search of GenBank (Altschul et al., 1990).

To exchange bait and prey, the NcoI/BamHI restriction fragment from the dynamitin/pACT plasmid was isolated and cloned into the bait vector pAS2, whereas the NcoI/BamHI PCR fragment of HZW10 was cloned into the prey vector pACTII. In addition, fragments of dynamitin were cloned by PCR with restriction site overhangs into pACTII, whereas fragments of HZW10 were cloned in a similar manner into pAS2 (see Fig. 6 for the exact size of each fragment). These constructs were tested in the two-hybrid system in the host strain Y190 by following the activity of lacZ in X-gal assays in the combinations described in the text and Fig. 6.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Determination of the interaction domains of dynamitin and HZW10. (A) Various NH2- and COOH-terminal deletions of dynamitin (the p50 subunit of dynactin) were constructed and put into the prey vector and tested for their ability to interact with HZW10 bait in the two-hybrid system. Two-hybrid activity was measured by β-galactosidase activity. +++ was arbitrarily set as the strength of the interaction (intensity of blue) between the entire dynamitin protein and HZW10. The coil-coil (coil) and helix-turn-helix (HTH) domains of dynamitin as identified by Echeverri et al. (1996) are marked. (B) Various parts of the HZW10 protein were cloned into the prey vector and tested in the two- hybrid system with dynamitin as bait. Interaction of the entire HZW10 protein with dynamitin is set as +++.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Dynein Localizes to Kinetochores during Meiosis in Drosophila Males
Despite ample evidence for the association of dynein with the kinetochores of mammalian mitotic chromosomes (Pfarr et al., 1990; Steuer et al., 1990; Wordeman et al., 1991), previous attempts to visualize dynein at the kinetochores of mitotic chromosomes in Drosophila have been unsuccessful (Hays et al., 1994). We reasoned that this failure could be due to the use of dynein at other intracellular locations such as the spindle microtubules or spindle poles, which might have obscured the observation of dynein at the kinetochores in Drosophila mitotic cells. Because Drosophila primary spermatocytes are much larger than all nonsyncytial mitotic cells, with spindles over four times the size of those of other cell types, we thought it possible that the association of dynein with the kinetochores in spermatocytes might be apparent if other dynein signals were dispersed in the large volume of these cells. Meiosis in Drosophila males is easily observed cytologically, and the behaviors of chromosomes, kinetochores, and microtubules throughout both meiotic divisions have been extensively characterized (Cenci et al., 1994; Williams et al., 1996). We thus used monoclonal and polyclonal antibodies against the Dhc to examine the localization of cytoplasmic dynein through the male meiotic cell divisions by immunofluorescence. To further lower backgrounds due to cytoplasmic Dhc, we preextracted the testes with the nonionic detergent Triton X-100 before fixation, as was done by the investigators who described the association of Dhc with mammalian kinetochores (e.g., Pfarr et al., 1990; Steuer et al., 1990; Echeverri et al., 1996).

The results of this analysis, which were consistent for two different anti-Drosophila Dhc preparations (refer to Materials and Methods), are shown in Fig. 1. Dhc did not localize to discrete intracellular structures in mature primary spermatocytes before prophase I (stages M1a–M1b according to the stage designations of Cenci et al. [1994]; data not shown). However, as the bivalents condense during prometaphase I (stage M2), bright Dhc staining appeared at two separate sites on each bivalent, at the positions of the kinetochores (Fig. 1, a and d). At this stage, each kinetochore is shared by the two sister centromeres in each dyad comprising the bivalent (Goldstein, 1981; Church and Lin, 1982). The Dhc staining often assumed a hemispherical character (Fig. 1 d, arrow), reflecting the shape of the kinetochore visible in electron micrographs (Lin and Church, 1982). Dhc exactly colocalizes with the kinetochore component ZW10 during prometaphase I (Fig. 1, a–c). Further evidence that Dhc indeed associates with the kinetochores during prometaphase is presented below, where we show that Dhc association with chromosomes is correlated with the ability of DNA sequences in those chromosomes to assemble functional kinetochores, and that Dhc is bound to the kinetochore in Drosophila mitotic cells arrested in prometaphase.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Dhc localization in Drosophila spermatocytes. Blue, DNA; red, Dhc; green, ZW10 protein. (a–c) A prometaphase I figure illustrating the presence of Dhc at the kinetochores of the bivalents. In addition, the colocalization of Dhc and ZW10 is indicated by the yellow signals in c, representing overlap of the sites of Dhc (seen alone in a) and ZW10 staining (seen alone in b). (d) Another prometaphase I figure with Dhc at the kinetochores of the chromosomes in the bivalents. Arrow, a hemispherical zone of Dhc staining on one of the kinetochores of the small fourth chromosome bivalent. (e and f) Metaphase I figures with decreased Dhc staining at the kinetochores (arrows) and with Dhc staining near the poles (e) or along kinetochore microtubules (f). (g and h) Anaphase I figures with Dhc concentrated near the poles. (i) Telophase I figure with Dhc excluded from the reforming nuclei but present at the centrosomes associated with the daughter nuclei. Bar, 10 µm.

The pattern of Dhc localization during the second meiotic division was very similar to that observed during the first division. Dhc occupied the sister kinetochores of each prometaphase II chromosome (data not shown). During metaphase II, some kinetochore staining was visible, concomitant with increased staining along the spindle. At anaphase II, staining in the vicinity of the poles, possibly including some residual kinetochore signals, was visible.

Localization of Dynein to the Kinetochore Correlates with Centromere Activity but Not with Specific Centromeric Sequences
In an effort to determine which sequences at the centromere are required for the localization of Dhc to the kinetochore, we asked whether Dhc would associate with Drosophila minichromosomes containing relatively short, defined DNA sequences. G. Karpen and colleagues (Molecular Biology and Virology Laboratory, The Salk Institute, La Jolla, CA) have described the minichromosome Dp(1;f)1187 (Dp1187) and its derivatives Dp8-23 and 238, all of which are deleted for most of the X chromosome, but nonetheless retain a functional centromere within 1 Mb of X chromosome centric heterochromatin. These minichromosomes also contain 290 kb of noncentromeric sequences from the tip of the X, including subtelomeric heterochromatin and euchromatin (collectively referred to as subtelomeric DNA). As shown in Fig. 2, Dhc is targeted to these minichromosomes during prometaphase I in primary spermatocytes.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Dhc binds to acentric minichromosomes. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, and i) and Dhc (b, d, f, h, and j) in primary spermatocytes containing minichromosomes or their deleted derivatives. In all panels, arrows point to the position of the minichromosome(s). (a and b) Dp1187. Other full-length derivatives of Dp1187 (Dp8-23 and 238) also bind Dhc (data not shown). (c and d) 31E. This deleted minichromosome contains all centromeric heterochromatin, but none of the subtelomeric DNA in Dp1187. (e and f) J21A. This deleted minichromosome contains all the subtelomeric DNA, but only approximately half the centromeric heterochromatin of Dp1187. As the stocks containing the various minichromosomes have not been continuously selected for maintenance of minichromosome number, and because transmission of smaller minichromosomes such as J21A through the male and female is imperfect, some spermatocytes in some individuals contain more than one copy of the minichromosome. (g and h) 19C. This deleted minichromosome is similar to J21A, but has lost 150 kb more centromeric heterochromatin. (i and j) 26C. This deleted minichromosome is only 285 kb in length and contains only the subtelomeric DNA, and thus none of the centromeric heterochromatin, of Dp1187. Details on the physical maps of all these minichromosomes can be found in Murphy and Karpen (1995) and in Williams et al. (1998). Bar, 10 µm.

238

Dhc Localization Responds to Bipolar Orientation of Bivalents
For bivalents to achieve a stable bipolar orientation during the first meiotic metaphase, tension must be exerted across the bivalent from opposite poles of the spindle. In at least some if not all cell types, all chromosomes must be subjected to this tension before the cell progresses into anaphase (Nicklas et al., 1995). Because the Dhc signal at the kinetochores appeared to decrease in metaphase spermatocytes (refer to Fig. 1, e and f), we entertained the possibility that the association of Dhc with the kinetochores might be lessened by the presence of spindle tension. If this were the case, it might then be imagined that Dhc acts either to help measure bipolar tension, or as part of the system that transduces the measurement of tension to the eventual disjunction of homologous chromosomes at onset of anaphase I. To investigate these hypotheses, we analyzed the distribution of Dhc in primary spermatocytes containing monooriented chromosomes (univalents). In these studies, we used two compound chromosomes, the attached X-Y (Y) and the compound 4th (C[4]RM), as univalents. Such compound chromosomes, in which homologues are attached to a single centromere, behave as univalents because they do not possess a pairing partner (Yamamoto, 1979; Church and Lin, 1982). As univalents can attach to only a single pole during prometaphase I and metaphase I, they are not subject to normal forces of bipolar tension (Ault and Lin, 1984; Ault and Nicklas, 1989). As a result, they cannot attain a stable metaphase orientation, and oscillate along the spindle from one pole to the other, eventually becoming randomly incorporated into daughter nuclei (Church and Lin, 1982).

Fig. 3 displays the staining of the kinetochores of these univalent chromosomes with anti-Dhc relative to the kinetochores of the second and third chromosomes in the same spermatocytes, which pair normally as bivalents. During prometaphase I, levels of kinetochore staining on univalents and bivalents were essentially identical (138 univalents scored in 13 testes; Fig. 3, a and b). However, clear differences were observed at metaphase I (Fig. 3, c–f). Although staining of the bivalent kinetochores was quite weak, the kinetochores of the univalents (when clearly separated from the bivalents) in the same cells showed intense anti-Dhc signals (44 univalents scored in 5 testes). By comparing prometaphase and metaphase figures in the same preparation, it appears that this difference is due to the loss of signal from bivalent kinetochores between prometaphase and metaphase, whereas the levels of Dhc association with univalent kinetochores seem little changed between these points of the cell cycle (Fig. 3). In summary, these observations indicate that although bipolar orientation and/or forces are not needed for the initial localization of Dhc to the kinetochore, they are necessary for the redistribution of Dhc at metaphase. We presume that this redistribution involves in part the movement of Dhc from the kinetochores to the kinetochores microtubules of chromosomes subjected to bipolar tension.

View larger version (137K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Dhc localization is tension dependent. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, and e) and Dhc (b, d, and f) in primary spermatocytes containing two univalents: X^Y and C(4)RM (refer to text). Arrows, positions of these univalents; the univalent with less DNA is C(4)RM, the univalent with more DNA in the same panel is X^Y. The remaining DNA staining is due to the large autosomal bivalents that are present in the same spermatocytes. (a and b) Prometaphase I. All kinetochores, including those on the bivalents and those on the univalents, stain with equal intensity. Note that there is only a single spot of Dhc localization associated with the univalents but two spots associated with each bivalent, consistent with the number of kinetochores they contain. (c–f) Metaphase I. Univalents have a much brighter intensity of Dhc staining. Arrowheads, bivalent chromosomes situated on the metaphase plate. Bar, 10 µm.

The results of these experiments, shown in Fig. 4, clearly demonstrate that the ZW10 protein is needed for the association of Dhc with kinetochores. In all zw10 mutant prometaphase I figures examined (n = 104 from 15 testes), the Dhc localization at the kinetochores seen in wild-type (as in Fig. 1, a and d) was eliminated (Fig. 4, a–d). Additionally, no Dhc staining was visualized on kinetochores or kinetochore microtubules at metaphase I and metaphase II (Fig. 4, e–h). The elimination of Dhc staining in zw10 mutants appears to be specific to the kinetochore, since Dhc still inhabited the polar areas during metaphase (Fig. 4, e–h) and anaphase (data not shown). Thus, the absence of the ZW10 protein affected Dhc localization to kinetochores but not to the spindle poles during metaphase and anaphase.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Dhc fails to localize to kinetochores in zw10 or rod mutant spermatocytes. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, i, and k) and Dhc (b, d, f, h, j, and l) in zw10S1/Y mutant testes (a–h) or in rod X–5 mutant testes (i–l). (a–d; i and j) Prometaphase I; (e and f) Metaphase I; (k and l) Anaphase I; (g and h) Metaphase II. Arrows, residual Dhc staining near the poles. Kinetochore localization of Dhc is absent at all stages. Bar, 10 µm.

To see if ZW10 was also required for Dhc localization at the kinetochores of mitotic chromosomes, we examined the localization of Dhc in Drosophila brain neuroblast cells. Dhc localization in these mitotic cells appeared to closely reflected its behavior in meiotic cells, including its localization to kinetochores and the spindle (data not shown), but a high cytoplasmic background hampered our observations. To improve the cytology, we therefore examined Dhc in wild-type larval brain neuroblasts arrested in a prometaphase-like state with the microtubule poison colchicine, and swollen with hypotonic solution (Gatti and Goldberg, 1991). In these cells, Dhc was clearly localized at the sister kinetochores of the duplicated chromosomes (Fig. 5, a–f). Note that because these preparations were not preextracted with Triton X-100 before fixation, the kinetochore localization of Dhc cannot be only a detergent-induced artifact. When zw10 mutant brains were stained under exactly the same conditions, however, Dhc protein was absent from the kinetochore, and was only uniformly dispersed throughout the cell (Fig. 5, g–j). Similar results were observed in the neuroblasts of animals carrying mutations in rod (Fig. 5, k and l). Thus, ZW10 (as well as the product of the rod gene) is required to recruit Dhc to the kinetochore in both meiosis and mitosis.

View larger version (100K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Dhc fails to localize to the kinetochore in zw10 or rod mutant larval brain neuroblasts. Two views of the same field are presented to show the relationship between the distribution of DNA (a, c, e, g, i, and k) and Dhc (b, d, f, h, j, and l) in neuroblasts from brain preparations treated with colchicine and swelled in hypotonic solution as described in Materials and Methods. (a–f) Wild-type (Oregon-R). (g–j) zw10S1/Y. (k and l) rod X–5. Arrows, clear Dhc staining at the kinetochore regions in wild-type neuroblasts. Dhc in zw10 and rod mutant brains is absent at the kinetochore. Bar, 10 µm.

Dhc and Dynamitin Colocalize with ZW10 at the Kinetochore
As argued more fully in the Discussion below, the results reported in this paper support a model in which ZW10 targets the dynactin complex to the kinetochore through a direct interaction with dynamitin, and that the dynactin complex in turn recruits dynein to the kinetochore. If this hypothesis is valid, one would expect that all three proteins would be found in close association at the kinetochore. We have already shown in Fig. 1, a–c that in Drosophila, Dhc and ZW10 colocalize at the kinetochores during prometaphase of meiosis I; this is also true during mitosis and meiosis II in the fly (data not shown). A similar finding was also obtained for human cells: Fig. 7, a–d depicts the results of immunofluorescence experiments demonstrating that dynamitin and HZW10 also completely colocalize to the kinetochores in HeLa cell chromosome spreads from cells arrested by nocodazole treatment. Echeverri et al. (1996) have previously shown that several components of dynactin and dynein complexes colocalize at the kinetochore of similarly treated HeLa cells. It should be noted that substantial evidence shows that the location of these three molecules is in fact at the kinetochore (or its fibrous corona) rather than in centromeric heterochromatin (Wordemann et al., 1991; Starr et al., 1997).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Colocalization of dynamitin and HZW10. (a–d) A chromosome spread from a HeLa cell arrested in mitosis by nocodazole. (e–h) A HeLa cell in prometaphase. (i–l) A HeLa cell in metaphase. The panels in the first column of each row show staining with anti-dynamitin (a, e, and i); in the second column, staining with anti HZW10 (b, f, and j); and in the third column staining with Hoechst 33258 to visualize DNA (c, g, and k). In the column at the right (d, h, and l), dynamitin staining is shown in red, and HZW10 staining in green; the overlap between these signals is yellow. Bars, 5 µm.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
ZW10 Is Required for the Association of Dynein with the Kinetochore
Several previous publications have documented the presence of dynein at the kinetochores of chromosomes in mammalian tissue culture cells (Pfarr et al., 1990; Steuer et al., 1990; Wordeman et al., 1991; Echeverri et al., 1996; Dujardin et al., 1998). In this paper, we have found that Dhc also associates with the kinetochores of mitotic and meiotic chromosomes in Drosophila. All previous work has used detergent preextraction to remove background staining due to high concentrations of dynein in the cytoplasm in order to visualize dynein at the kinetochore. Although we have used a similar procedure to see Dhc at the kinetochore in Drosophila primary spermatocytes (Figs. 1–4), we have also been able to detect Dhc at the kinetochore of mitotic chromosomes that have not been subjected to detergent preextraction, demonstrating that the association of dynein with the kinetochore is not artefactually induced by this method of preparation.

We have presented a number of lines of evidence supporting the hypothesis that one role played by the wild-type zw10 gene product is to recruit dynein to the kinetochore. ZW10 and Dhc colocalize to the kinetochores of fly and human chromosomes (refer to Fig. 1 and Fig. 7) in a fashion that is identically influenced by tension across the chromosomes (refer to Fig. 3). Both proteins are found at the kinetochores elaborated by minichromosome derivatives that lack centromeric sequences but that display neocentric activity (refer to Fig. 2). Both proteins fail to associate with the kinetochore in rod mutant spermatocytes (refer to Fig. 4, i–l) and neuroblasts (refer to Fig. 5, k and l). Yeast two-hybrid experiments show that human ZW10 protein and dynamitin, the p50 subunit of dynactin complex, can interact with each other directly (refer to Table I and Fig. 6).

The most compelling evidence pointing to a role for the ZW10 protein in dynein localization to the kinetochore is documented in Fig. 4, a–d and Fig. 5, g–j, which show that in zw10 mutant spermatocytes and neuroblasts, Dhc fails to localize to the kinetochore at levels detectable by immunofluorescence. Of course, because of significant backgrounds of anti-Dhc staining (presumably reflecting the use of Dhc in many intracellular contexts), we cannot exclude the possibility that an undetectable small fraction of Dhc is retained at the prometaphase kinetochore in zw10 mutants. The same caveat also applies in our studies demonstrating that Dhc is not kinetochore associated in rod mutant testes and brains (refer to Fig. 4, i–l and Fig. 5, k and l). These latter results indicate that the large majority of the binding of Dhc to the kinetochore is dependent upon the arrival of ZW10 protein at the same location. This is because the ZW10 protein is present in rod mutant cells in normal amounts but is not present at the kinetochore (Williams and Goldberg, 1994). The requirement for both ZW10 and ROD proteins would be most easily explained if these two polypeptides were complexed with each other. Indeed, in collaboration with F. Scaerou and R. Karess (CNRS Centre de Génétique Moléculaire, Gif-sur-Yvette, France), we have recently obtained evidence for the existence of such a complex, which requires the participation of both proteins for its localization to the kinetochore (our manuscript in preparation).

It could be argued that the depletion of Dhc from the kinetochore in zw10 or rod mutant spermatocytes is only an indirect effect of major disruptions in kinetochore structure caused by these mutations. Three observations indicate that this is unlikely to be true. First, many aspects of kinetochore function are relatively untouched by these mutations. In mutant zw10 and rod meiotic and mitotic cells, the chromosomes appear to condense appropriately and congress to the metaphase plate. At anaphase, most sister chromatids (mitosis and meiosis II) or homologous chromosomes (meiosis I) separate from each other and migrate toward the poles during anaphase. Second, other kinetochore proteins, like the those recognized by the 3F3/2 antibody (Bousbaa et al., 1997; Gorbsky and Ricketts, 1993) and Drosophila Bub1, are properly localized in zw10 and rod mutants (Basu, J., B.C. Williams, and M.L. Goldberg, manuscript in preparation). Third, we have two- hybrid evidence that the interaction between ZW10 and dynein is reasonably direct, being mediated by contacts between ZW10 and dynamitin, the p50 subunit of the dynactin complex (refer to Table I). This scenario is in accord with previous work suggesting that the dynactin complex helps target cytoplasmic dynein to appropriate intracellular sites (for review see Vallee and Sheetz, 1996).

In the yeast two-hybrid system, human ZW10 and dynamitin associate with each other to activate the transcription of two different reporter genes. We have mapped the interaction domain of dynamitin to a 22-amino acid region including part of a conserved coil-coil domain and the region immediately downstream (refer to Fig. 6). This same region has been shown to be important for dynamitin function (Echeverri, C., and R. Vallee, personal communication). Echeverri et al. (1996) demonstrated that overexpression of wild-type dynamitin disrupts the dynactin complex, leading to a number of phenotypes. However, when expressed at even low levels, dynamitin, with a small deletion including the human ZW10-interacting domain, causes the same phenotypes, including a lack of dynein at the kinetochore (Echeverri, C., and R. Vallee, personal communication). The idea that ZW10 and dynamitin interact directly with each other in the cell must nonetheless be approached with some caution. We have thus far been unable to show a direct interaction between ZW10 and dynamitin by methods other than the yeast two-hybrid system, such as coimmunoprecipitation or binding assays using in vitro–translated proteins. We believe this is because the two proteins are normally able to interact only in the context of the kinetochore, an insoluble structure. In addition, though we know that the region of dynamitin that interacts with ZW10 in the two-hybrid system is necessary for dynamitin function, it is not yet clear whether this region alone can target dynactin to the kinetochore.

Our results taken together strongly imply a model in which a complex including ZW10 and ROD arrives at the kinetochore early in prometaphase, that this complex then attracts the dynactin complex to the kinetochore by virtue of direct contacts between ZW10 and dynamitin, and finally, that dynactin in turn targets dynein to the kinetochore (Fig. 8).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Model for the ZW10/ROD-dependent targeting of dynein to the kinetochore. In this model, a complex containing ZW10 and ROD proteins, as well as potential unknown additional components (?), is associated with the fibrous corona of the prometaphase kinetochore. Direct interactions between ZW10 and the p50 subunit of the dynactin complex then bring dynactin to the kinetochore. Dynactin in turn recruits cytoplasmic dynein to the kinetochore, providing one possible contact between the kinetochore and microtubules. Our results suggest that this contact is sensitive to bipolar tension exerted across the chromosome. As described in the Introduction, additional kinetochore/microtubule interactions are undoubtedly mediated by other microtubule motors such as CENP-E (data not shown). This figure is modified from Vallee et al. (1995).

Since chromosomes in a zw10 or rod mutant cell congress to the metaphase plate (Karess et al., 1989; Williams et al., 1992), wild-type levels of dynein at the kinetochore can not be uniquely required for chromosome microtubule attachments or movements before anaphase onset. This conclusion is somewhat surprising, since it has been previously proposed that dynein at the kinetochore is involved in the initial capture of a microtubule and the rapid poleward movement observed by Rieder and Alexander (1990) (Pfarr et al., 1990; Steuer et al., 1990). The kinetics of this rapid minus end–directed movement along the side of a single microtubule match those of dynein (Rieder and Alexander, 1990). This initial poleward movement is thought to eventually facilitate the ability of kinetochores near the poles to capture the plus ends of additional microtubules. Our results do not disprove such a role for dynein in chromosome congression, but they do suggest that other microtubule motors are able to supplant dynein function in this regard.

Phenotypic analysis suggests that zw10 and rod mutations mostly interfere with the fidelity and coordination of events at anaphase onset. Sister chromatids (mitosis and meiosis II) or homologous chromosomes (meiosis I) for the most part separate at anaphase onset, and migrate towards the spindle pole in anaphase. However, some chromatids or chromosomes appear to separate from each other later than normal, and often remain in the vicinity of the metaphase plate even late in anaphase. To the extent that these phenotypes reflect the role of dynein at the kinetochore, they suggest two possibilities for dynein's function at this location. Dynein might participate in the checkpoint mechanisms that sense bipolar tension across the centromere, delaying anaphase onset until all the chromosomes are properly aligned on the metaphase plate. In this light, it is of interest that in zw10 and rod mutant, but not wild-type neuroblasts, sister chromatids separate precociously when the cells are treated with the microtubule poison colchicine. One interpretation consistent with our observations is that the lack of dynein at the kinetochore allows cells to bypass the wait anaphase checkpoint. Alternatively, dynein might be required at the kinetochore to supplement and/or coordinate other microtubule motors in moving chromosomes to the poles during anaphase. The resolution to this question may lie in a more detailed analysis of the zw10 and rod mutant phenotypes.

Submitted: 14 April 1998

Revised: 1 July 1998

We would like to dedicate this paper to the memory of B. Keller (Cornell University, Ithaca, NY). We thank Z. Li and E. Williams (both from Cornell University) for technical help, M. Serr and S. O'Rourke (both from University of Minnesota, St. Paul, MN) for the Dhc antibodies, T. Murphy and G. Karpen (both from The Salk Institute, La Jolla, CA) for the minichromosome stocks, J. Lis and C. Bayles (both from Cornell University) for assistance with CCD microscopy, and F. Scaerou and R. Karess (both from CNRS Centre de Génétique Moléculaire, Gif-sur-Yvette, France) for helpful discussion. We are indebted to C. Echeverri and R. Vallee (both from University of Massachusetts Medical Center, Boston, MA) for insightful discussions and artistic help in preparing Fig. 8.

This research was supported by a grant from the National Institutes of Health (NIH) to M.L. Goldberg (GM 48430) and by an NIH training grant (GM 07617) to the Field of Genetics and Development at Cornell University (Ithaca, NY).

D.A. Starr and B.C. Williams contributed equally to this work.

	References

Top Abstract Materials and Methods Results Discussion References

 

Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ. Basic local alignment search tool, J Mol Biol, 1990, 215, 403–410.[Medline]

Ault JG & Lin HP. Bivalent behavior in Drosophila melanogastermales containing the In(1)sc4Lsc8RX chromosome, Chromosoma, 1984, 90, 222–228.[Medline]

Ault JG & Nicklas RB. Tension, microtubule rearrangements, and the proper distribution of chromosomes in mitosis, Chromosoma, 1989, 98, 33–39.[Medline]

Bai C & Elledge SJ. Gene identification using the two-hybrid system, Methods Enzymol, 1996, 273, 331–347.[Medline]

Bousbaa H, Correia L, Gorbsky GJ & Sunkel CE. Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster. , J Cell Sci, 1997, 110, 1979–1988.[Abstract]

Cancilla MR, Tainton KM, Barry AE, Larionov V, Kouprina N, Resnick MA, Sart DD & Choo KHA. Direct cloning of human 10q25 neocentromere DNA using transformation-associated recombination (TAR) in yeast, Genomics, 1998, 47, 399–404.[Medline]

Cenci G, Bonaccorsi S, Pisano C, Verni F & Gatti M. Chromatin and microtubule organization during premeiotic, meiotic and early postmeiotic stages of Drosophila melanogasterspermatogenesis, J Cell Sci, 1994, 107, 3521–3534.[Abstract]

Chen R, Waters JC, Salmon ED & Murray AW. Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores, Science, 1996, 274, 242–246.[Abstract/Free Full Text]

Church K & Lin HP. Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males, J Cell Biol, 1982, 93, 365–373.[Abstract/Free Full Text]

Dujardin D, Wacker UI, Moreau A, Schroer TA, Rickard JE & De Mey JR. Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment, J Cell Biol, 1998, 141, 849–862.[Abstract/Free Full Text]

Duesbery NS, Choi T, Brown KD, Wood KW, Resau J, Fukasawa K, Cleveland DW & Van de Woude GF. CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II, Proc Natl Acad Sci USA, 1997, 94, 9165–9170.[Abstract/Free Full Text]

Echeverri CJ, Paschal BM, Vaughan KT & Vallee RB. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis, J Cell Biol, 1996, 132, 617–633.[Abstract/Free Full Text]

Fields S & Song O. A novel genetic system to detect protein-protein interactions, Nature, 1989, 340, 245–246.[Medline]

Gaglio T, Saredi A, Bingham JB, Hasbani MJ, Gill SR, Schroer TA & Compton DA. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole, J Cell Biol, 1996, 135, 399–414.[Abstract/Free Full Text]

Gatti M & Goldberg ML. Mutations affecting cell division in Drosophila. , Methods Cell Biol, 1991, 35, 543–586.[Medline]

Goldstein LS. Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster. , Cell, 1981, 25, 591–602.[Medline]

Gorbsky G & Ricketts WA. Differential expression of phosphoepitope at the kinetochore of moving chromosomes, J Cell Biol, 1993, 122, 1311–1321.[Abstract/Free Full Text]

Hays TS, Porter ME, McGrail M, Grissom P, Gosch P, Fuller MT & McIntosh JR. A cytoplasmic dynein motor in Drosophila: identification and localization during embryogenesis, J Cell Sci, 1994, 107, 1557–1569.[Abstract]

Heald R, Tournebize R, Blank T, Sandaltzopoulos R, Becker P, Hyman A & Karsenti E. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopusegg extracts, Nature, 1996, 382, 420–425.[Medline]

Karess RE & Glover DM. rough deal, a gene required for proper mitotic segregation in Drosophila. , J Cell Biol, 1989, 109, 2951–2961.[Abstract/Free Full Text]

Li X & Nicklas RB. Mitotic forces control a cell cycle checkpoint, Nature, 1995, 373, 630–632.[Medline]

Li Y & Benerza R. Identification of a human mitotic checkpoint gene: hsMAD2. , Science, 1996, 274, 246–248.[Abstract/Free Full Text]

Lin HP & Church K. Meiosis in Drosophila melanogaster, III. The effect of orientation disruptor (ord)on gonial mitotic and the meiotic divisions in males, Genetics, 1982, 102, 751–770.[Abstract/Free Full Text]

Lombillo VA, Nislow C, Yen TJ, Gelfand VI & McIntosh JR. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro, J Cell Biol, 1995, 128, 107–115.[Abstract/Free Full Text]

McGrail M & Hays TS. The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila. , Development (Camb), 1997, 124, 2409–2419.[Abstract]

Merdes A, Ramyar K, Vechio JD & Cleveland DW. A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly, Cell, 1996, 87, 447–458.[Medline]

Murphy TD & Karpen GH. Location of centromere function in a Drosophilaminichromosome, Cell, 1995, 82, 599–609.[Medline]

Nicklas RB. The motor for poleward chromosome movement in anaphase is in or near the kinetochore, J Cell Biol, 1989, 109, 2245–2255.[Abstract/Free Full Text]

Nicklas RB, Ward SC & Gorbsky GJ. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint, J Cell Biol, 1995, 130, 929–939.[Abstract/Free Full Text]

Pfarr CM, Coue M, Grissom PM, Hays TS, Porter ME & McIntosh JR. Cytoplasmic dynein is localized to kinetochores during mitosis, Nature, 1990, 345, 263–265.[Medline]

Pluta AF, Mackay AM, Ainsztein AM, Goldberg IG & Earnshaw WC. The centromere: hub of chromosomal activities, Science, 1995, 270, 1591–1594.[Abstract/Free Full Text]

Rieder CL. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber, Int Rev Cytol, 1982, 79, 1–58.[Medline]

Rieder CL & Alexander SP. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells, J Cell Biol, 1990, 110, 81–95.[Abstract/Free Full Text]

Rieder CL & Salmon ED. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle, J Cell Biol, 1994, 124, 223–233.[Abstract/Free Full Text]

Smith DA, Baker BS & Gatti M. Mutations in genes controlling essential mitotic functions in Drosophila melanogaster. , Genetics, 1985, 110, 647–670.[Abstract/Free Full Text]

Starr DA, Williams BC, Li Z, Etemad-Moghadam B, Dawe RK & Goldberg ML. Conservation of the centromere/kinetochore protein ZW10, J Cell Biol, 1997, 138, 1289–1301.[Abstract/Free Full Text]

Steuer ER, Wordeman L, Schroer TA & Sheetz MP. Localization of cytoplasmic dynein to mitotic spindles and kinetochores, Nature, 1990, 345, 266–268.[Medline]

Taylor SS & McKeon F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage, Cell, 1997, 89, 727–735.[Medline]

Vaisberg EA, Koonce MP & McIntosh JR. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation, J Cell Biol, 1993, 123, 849–858.[Abstract/Free Full Text]

Vallee RB, Echeverri CJ & Vaughn KT. Targeting of cytoplasmic dynein to membranous organelles and kinetochores via dynactin, Cold Spr Harb Symp Quant Biol, 1995, 60, 803–811.[Abstract/Free Full Text]

Vallee RB & Sheetz MP. Targeting of motor proteins, Science, 1996, 271, 1539–1544.[Abstract]

Verde F, Berrez JM, Antony C & Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopuseggs: requirement for phosphorylated factors and cytoplasmic dynein, J Cell Biol, 1991, 112, 1177–1187.[Abstract/Free Full Text]

Walczak CE, Mitchison TJ & Desai A. XKCM1: a Xenopuskinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly, Cell, 1996, 84, 37–47.[Medline]

Williams BC, Gatti M & Goldberg ML. Bipolar spindle attachments affect redistributions of ZW10, a Drosophilacentromere/kinetochore component required for accurate chromosome segregation, J Cell Biol, 1996, 134, 1127–1140.[Abstract/Free Full Text]

Williams BC & Goldberg ML. Determinants of Drosophilazw10 protein localization and function, J Cell Sci, 1994, 107, 785–798.[Abstract]

Williams BC, Karr TL, Montgomery JM & Goldberg ML. The Drosophila l(1)zw10gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset, J Cell Biol, 1992, 118, 759–773.[Abstract/Free Full Text]

Williams BC, Murphy TD, Goldberg ML & Karpen GH. Neocentromere activity of structurally acentric mini-chromosomes in Drosophila. , Nat Genet, 1998, 18, 30–37.[Medline]

Wood KW, Sakowicz R, Goldstein LSB & Cleveland DW. CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment, Cell, 1997, 91, 357–366.[Medline]

Wordeman L & Mitchison TJ. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin related protein that associates with centromeres during mitosis, J Cell Biol, 1995, 128, 95–105.[Abstract/Free Full Text]

Wordeman L, Steuer ER, Sheetz MP & Mitchison T. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes, J Cell Biol, 1991, 114, 285–294.[Abstract/Free Full Text]

Yamamoto M. Cytological studies of heterochromatin function in the Drosophila melanogastermale: autosomal meiotic paring, Chromosoma, 1979, 72, 293–328.[Medline]

Yao X, Anderson KL & Cleveland DW. The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochores to spindle microtubules, J Cell Biol, 1997, 139, 435–447.[Abstract/Free Full Text]

Yen TJ, Li G, Schaar BT, Szilak I & Cleveland DW. CENP-E is a putative kinetochore motor that accumulates just before mitosis, Nature, 1992, 359, 536–539.[Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Facebook

Reddit

Technorati

Twitter

This article has been cited by other articles:

• Milks, K. J., Moree, B., Straight, A. F. (2009). Dissection of CENP-C-directed Centromere and Kinetochore Assembly. Mol. Biol. Cell 20: 4246-4255 [Abstract] [Full Text]  
• Reis, R., Feijao, T., Gouveia, S., Pereira, A. J., Matos, I., Sampaio, P., Maiato, H., Sunkel, C. E. (2009). Dynein and Mast/Orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics. J. Cell Sci. 122: 2543-2553 [Abstract] [Full Text]  
• Chan, Y. W., Fava, L. L., Uldschmid, A., Schmitz, M. H.A., Gerlich, D. W., Nigg, E. A., Santamaria, A. (2009). Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly. JCB 185: 859-874 [Abstract] [Full Text]  
• Perry, R. J., Mast, F. D., Rachubinski, R. A. (2009). Endoplasmic Reticulum-Associated Secretory Proteins Sec20p, Sec39p, and Dsl1p Are Involved in Peroxisome Biogenesis. Eukaryot Cell 8: 830-843 [Abstract] [Full Text]  
• Wainman, A., Creque, J., Williams, B., Williams, E. V., Bonaccorsi, S., Gatti, M., Goldberg, M. L. (2009). Roles of the Drosophila NudE protein in kinetochore function and centrosome migration. J. Cell Sci. 122: 1747-1758 [Abstract] [Full Text]  
• Aoki, T., Ichimura, S., Itoh, A., Kuramoto, M., Shinkawa, T., Isobe, T., Tagaya, M. (2009). Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport. Mol. Biol. Cell 20: 2639-2649 [Abstract] [Full Text]  
• Maresca, T. J., Salmon, E. D. (2009). Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity. JCB 184: 373-381 [Abstract] [Full Text]  
• Whyte, J., Bader, J. R., Tauhata, S. B.F., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., Vaughan, K. T. (2008). Phosphorylation regulates targeting of cytoplasmic dynein to kinetochores during mitosis. JCB 183: 819-834 [Abstract] [Full Text]  
• Mische, S., He, Y., Ma, L., Li, M., Serr, M., Hays, T. S. (2008). Dynein Light Intermediate Chain: An Essential Subunit That Contributes to Spindle Checkpoint Inactivation. Mol. Biol. Cell 19: 4918-4929 [Abstract] [Full Text]  
• Yamamoto, T. G., Watanabe, S., Essex, A., Kitagawa, R. (2008). SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans. JCB 183: 187-194 [Abstract] [Full Text]  
• Varma, D., Monzo, P., Stehman, S. A., Vallee, R. B. (2008). Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment. JCB 182: 1045-1054 [Abstract] [Full Text]  
• Civril, F., Musacchio, A. (2008). Spindly attachments. Genes Dev. 22: 2302-2307 [Abstract] [Full Text]  
• Gassmann, R., Essex, A., Hu, J.-S., Maddox, P. S., Motegi, F., Sugimoto, A., O'Rourke, S. M., Bowerman, B., McLeod, I., Yates, J. R. III, Oegema, K., Cheeseman, I. M., Desai, A. (2008). A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex. Genes Dev. 22: 2385-2399 [Abstract] [Full Text]  
• Inoue, M., Arasaki, K., Ueda, A., Aoki, T., Tagaya, M. (2008). N-terminal region of ZW10 serves not only as a determinant for localization but also as a link with dynein function.. GENES CELLS 13: 905-914 [Abstract] [Full Text]  
• Emanuele, M. J., Lan, W., Jwa, M., Miller, S. A., Chan, C. S.M., Stukenberg, P. T. (2008). Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly. JCB 181: 241-254 [Abstract] [Full Text]  
• Famulski, J. K., Vos, L., Sun, X., Chan, G. (2008). Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint. JCB 180: 507-520 [Abstract] [Full Text]  
• Williams, B., Leung, G., Maiato, H., Wong, A., Li, Z., Williams, E. V., Kirkpatrick, C., Aquadro, C. F., Rieder, C. L., Goldberg, M. L. (2007). Mitch a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila. J. Cell Sci. 120: 3522-3533 [Abstract] [Full Text]  
• Stehman, S. A., Chen, Y., McKenney, R. J., Vallee, R. B. (2007). NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores. JCB 178: 583-594 [Abstract] [Full Text]  
• Griffis, E. R., Stuurman, N., Vale, R. D. (2007). Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. JCB 177: 1005-1015 [Abstract] [Full Text]  
• Liang, Y., Yu, W., Li, Y., Yu, L., Zhang, Q., Wang, F., Yang, Z., Du, J., Huang, Q., Yao, X., Zhu, X. (2007). Nudel Modulates Kinetochore Association and Function of Cytoplasmic Dynein in M Phase. Mol. Biol. Cell 18: 2656-2666 [Abstract] [Full Text]  
• Zhang, D., Yin, S., Jiang, M.-X., Ma, W., Hou, Y., Liang, C.-G., Yu, L.-Z., Wang, W.-H., Sun, Q.-Y. (2007). Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles. Reproduction 133: 685-695 [Abstract] [Full Text]  
• Arasaki, K., Tani, K., Yoshimori, T., Stephens, D. J., Tagaya, M. (2007). Nordihydroguaiaretic Acid Affects Multiple Dynein-Dynactin Functions in Interphase and Mitotic Cells. Mol. Pharmacol. 71: 454-460 [Abstract] [Full Text]  
• Appenzeller-Herzog, C., Hauri, H.-P. (2006). The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function. J. Cell Sci. 119: 2173-2183 [Abstract] [Full Text]  
• Arasaki, K., Taniguchi, M., Tani, K., Tagaya, M. (2006). RINT-1 Regulates the Localization and Entry of ZW10 to the Syntaxin 18 Complex. Mol. Biol. Cell 17: 2780-2788 [Abstract] [Full Text]  
• Varma, D., Dujardin, D. L., Stehman, S. A., Vallee, R. B. (2006). Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function. JCB 172: 655-662 [Abstract] [Full Text]  
• Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. (2005). Live Imaging of Drosophila Brain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control. Mol. Biol. Cell 16: 5127-5140 [Abstract] [Full Text]  
• Kraynack, B. A., Chan, A., Rosenthal, E., Essid, M., Umansky, B., Waters, M. G., Schmitt, H. D. (2005). Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast. Mol. Biol. Cell 16: 3963-3977 [Abstract] [Full Text]  
• Dzhindzhev, N. S., Rogers, S. L., Vale, R. D., Ohkura, H. (2005). Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends. J. Cell Sci. 118: 3781-3790 [Abstract] [Full Text]  
• Hut, H. M.J., Kampinga, H. H., Sibon, O. C.M. (2005). Hsp70 Protects Mitotic Cells against Heat-induced Centrosome Damage and Division Abnormalities. Mol. Biol. Cell 16: 3776-3785 [Abstract] [Full Text]  
• Kops, G. J.P.L., Kim, Y., Weaver, B. A.A., Mao, Y., McLeod, I., Yates, J. R. III, Tagaya, M., Cleveland, D. W. (2005). ZW10 links mitotic checkpoint signaling to the structural kinetochore. JCB 169: 49-60 [Abstract] [Full Text]  
• Schmidt, D. J., Rose, D. J., Saxton, W. M., Strome, S. (2005). Functional Analysis of Cytoplasmic Dynein Heavy Chain in Caenorhabditis elegans with Fast-acting Temperature-sensitive Mutations. Mol. Biol. Cell 16: 1200-1212 [Abstract] [Full Text]  
• Wang, H., Hu, X., Ding, X., Dou, Z., Yang, Z., Shaw, A. W., Teng, M., Cleveland, D. W., Goldberg, M. L., Niu, L., Yao, X. (2004). Human Zwint-1 Specifies Localization of Zeste White 10 to Kinetochores and Is Essential for Mitotic Checkpoint Signaling. J. Biol. Chem. 279: 54590-54598 [Abstract] [Full Text]  
• Andag, U., Schmitt, H. D. (2003). Dsl1p, an Essential Component of the Golgi-Endoplasmic Reticulum Retrieval System in Yeast, Uses the Same Sequence Motif to Interact with Different Subunits of the COPI Vesicle Coat. J. Biol. Chem. 278: 51722-51734 [Abstract] [Full Text]  
• Mimori-Kiyosue, Y., Tsukita, S. (2003). "Search-and-Capture" of Microtubules through Plus-End-Binding Proteins (+TIPs). J Biochem 134: 321-326 [Abstract] [Full Text]  
• Williams, B. C., Li, Z., Liu, S., Williams, E. V., Leung, G., Yen, T. J., Goldberg, M. L. (2003). Zwilch, a New Component of the ZW10/ROD Complex Required for Kinetochore Functions. Mol. Biol. Cell 14: 1379-1391 [Abstract] [Full Text]  
• Balicky, E. M., Endres, M. W., Lai, C., Bickel, S. E. (2002). Meiotic Cohesion Requires Accumulation of ORD on Chromosomes before Condensation. Mol. Biol. Cell 13: 3890-3900 [Abstract] [Full Text]  
• Helfand, B. T., Mikami, A., Vallee, R. B., Goldman, R. D. (2002). A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization. JCB 157: 795-806 [Abstract] [Full Text]  
• Karki, S., Ligon, L. A., DeSantis, J., Tokito, M., Holzbaur, E. L. F. (2002). PLAC-24 Is a Cytoplasmic Dynein-Binding Protein That Is Recruited to Sites of Cell-Cell Contact. Mol. Biol. Cell 13: 1722-1734 [Abstract] [Full Text]  
• Tai, C.-Y., Dujardin, D. L., Faulkner, N. E., Vallee, R. B. (2002). Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function. JCB 156: 959-968 [Abstract] [Full Text]  
• Matthies, H. J.G., Baskin, R. J., Hawley, R. S. (2001). Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity. Mol. Biol. Cell 12: 4000-4012 [Abstract] [Full Text]  
• Troxell, C. L., Sweezy, M. A., West, R. R., Reed, K. D., Carson, B. D., Pidoux, A. L., Cande, W. Z., McIntosh, J. R. (2001). pkl1+and klp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis. Mol. Biol. Cell 12: 3476-3488 [Abstract] [Full Text]  
• Sullivan, B., Karpen, G. (2001). Centromere identity in Drosophila is not determined in vivo by replication timing. JCB 154: 683-690 [Abstract] [Full Text]  
• Maggert, K. A., Karpen, G. H. (2001). The Activation of a Neocentromere in Drosophila Requires Proximity to an Endogenous Centromere. Genetics 158: 1615-1628 [Abstract] [Full Text]  
• Moore, L. L., Roth, M. B. (2001). Hcp-4, a Cenp-C-Like Protein inCaenorhabditis elegans, Is Required for Resolution of Sister Centromeres. JCB 153: 1199-1208 [Abstract] [Full Text]  
• Van Hooser, A. A., Ouspenski, I. I., Gregson, H. C., Starr, D. A., Yen, T. J., Goldberg, M. L., Yokomori, K., Earnshaw, W. C., Sullivan, K. F., Brinkley, B. R. (2001). Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J. Cell Sci. 114: 3529-3542 [Abstract] [Full Text]  
• Scaerou, F., Starr, D. A., Piano, F., Papoulas, O., Karess, R. E., Goldberg, M. L. (2001). The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore. J. Cell Sci. 114: 3103-3114 [Abstract] [Full Text]  
• Habermann, A, Schroer, T., Griffiths, G, Burkhardt, J. (2001). Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles. J. Cell Sci. 114: 229-240 [Abstract]  
• King, J. M., Hays, T. S., Nicklas, R. B. (2000). Dynein Is a Transient Kinetochore Component Whose Binding Is Regulated by Microtubule Attachment, Not Tension. JCB 151: 739-748 [Abstract] [Full Text]  
• Rogers, G. C., Chui, K. K., Lee, E. W., Wedaman, K. P., Sharp, D. J., Holland, G., Morris, R. L., Scholey, J. M. (2000). A Kinesin-Related Protein, Krp180, Positions Prometaphase Spindle Poles during Early Sea Urchin Embryonic Cell Division. JCB 150: 499-512 [Abstract] [Full Text]  
• Karki, S., Tokito, M. K., Holzbaur, E. L. F. (2000). A Dynactin Subunit with a Highly Conserved Cysteine-rich Motif Interacts Directly with Arp1. J. Biol. Chem. 275: 4834-4839 [Abstract] [Full Text]  
• Ye, G.-J., Vaughan, K. T., Vallee, R. B., Roizman, B. (2000). The Herpes Simplex Virus 1 UL34 Protein Interacts with a Cytoplasmic Dynein Intermediate Chain and Targets Nuclear Membrane. J. Virol. 74: 1355-1363 [Abstract] [Full Text]  
• Saffery, R., Irvine, D. V., Griffiths, B., Kalitsis, P., Wordeman, L., Choo, K.H. A. (2000). Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins.. Hum Mol Genet 9: 175-185 [Abstract] [Full Text]  
• Starr, D., Saffery, R, Li, Z, Simpson, A., Choo, K., Yen, T., Goldberg, M. (2000). HZwint-1, a novel human kinetochore component that interacts with HZW10. J. Cell Sci. 113: 1939-1950 [Abstract]  
• Matthies, H. J.G., Messina, L. G., Namba, R., Greer, K. J., Walker, M.Y., Hawley, R. S. (1999). Mutations in the {alpha}-Tubulin 67C Gene Specifically Impair Achiasmate Segregation in Drosophila melanogaster. JCB 147: 1137-1144 [Abstract] [Full Text]  
• Purohit, A., Tynan, S. H., Vallee, R., Doxsey, S. J. (1999). Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization. JCB 147: 481-492 [Abstract] [Full Text]  
• Eckley, D. M., Gill, S. R., Melkonian, K. A., Bingham, J. B., Goodson, H. V., Heuser, J. E., Schroer, T. A. (1999). Analysis of Dynactin Subcomplexes Reveals a Novel Actin-Related Protein Associated with the Arp1 Minifilament Pointed End. JCB 147: 307-320 [Abstract] [Full Text]  
• Robinson, J. T., Wojcik, E. J., Sanders, M. A., McGrail, M., Hays, T. S. (1999). Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes during Mitosis in Drosophila. JCB 146: 597-608 [Abstract] [Full Text]  
• Basu, J., Bousbaa, H., Logarinho, E., Li, Z., Williams, B. C., Lopes, C., Sunkel, C. E., Goldberg, M. L. (1999). Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila. JCB 146: 13-28 [Abstract] [Full Text]  
• Bowman, A. B., Patel-King, R. S., Benashski, S. E., McCaffery, J. M., Goldstein, L. S.B., King, S. M. (1999). Drosophila roadblock and Chlamydomonas Lc7: A Conserved Family of Dynein-Associated Proteins Involved in Axonal Transport, Flagellar Motility, and Mitosis. JCB 146: 165-180 [Abstract] [Full Text]  
• Scaerou, F, Aguilera, I, Saunders, R, Kane, N, Blottiere, L, Karess, R (1999). The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila. J. Cell Sci. 112: 3757-3768 [Abstract]  
• Vaughan, K., Tynan, S., Faulkner, N., Echeverri, C., Vallee, R. (1999). Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends. J. Cell Sci. 112: 1437-1447 [Abstract]  
• Maney, T., Hunter, A. W., Wagenbach, M., Wordeman, L. (1998). Mitotic Centromere-associated Kinesin Is Important for Anaphase Chromosome Segregation. JCB 142: 787-801 [Abstract] [Full Text]  
• Holleran, E. A., Ligon, L. A., Tokito, M., Stankewich, M. C., Morrow, J. S., Holzbaur, E. L. F. (2001). beta III Spectrin Binds to the Arp1 Subunit of Dynactin. J. Biol. Chem. 276: 36598-36605 [Abstract] [Full Text]  
• Hirakawa, E., Higuchi, H., Toyoshima, Y. Y. (2000). Processive movement of single 22S dynein molecules occurs only at low ATP concentrations. Proc. Natl. Acad. Sci. USA 97: 2533-2537 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF, 907K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Starr, D. A. Articles by Goldberg, M. L. Search for Related Content PubMed PubMed Citation Articles by Starr, D. A. Articles by Goldberg, M. L. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Protein UniGene Substance via MeSH Social Bookmarking                            What's this?