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Bipolar Spindle Attachments Affect Redistributions of ZW10, a Drosophila Centromere/Kinetochore Component Required for Accurate Chromosome Segregation Byron C

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Bipolar Spindle Attachments Affect Redistributions of ZW10, a Drosophila Centromere/Kinetochore Component Required for Accurate Chromosome Segregation Byron C Bipolar Spindle Attachments Affect Redistributions of ZW10, a Drosophila Centromere/Kinetochore Component Required for Accurate Chromosome Segregation Byron C. Williams,* Maurizio Gatti,* and Michael L. Goldberg* *Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703; andHstituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Universit~ di Roma "La Sapienza," 00185 Rome, Italy Abstract. Previous efforts have shown that mutations (during meiosis II). During metaphase of both divi- in the Drosophila zwlO gene cause massive chromo- sions, ZW10 appears to move from the kinetochores some missegregation during mitotic divisions in several and to spread toward the poles along what appear to be tissues. Here we demonstrate that mutations in zwlO kinetochore microtubules. Redistributions of ZWl0 at also disrupt chromosome behavior in male meiosis I metaphase require bipolar attachments of individual and meiosis II, indicating that ZW10 function is com- chromosomes or paired bivalents to the spindle. At the mon to both equational and reductional divisions. Divi- onset of anaphase I or anaphase II, ZWl0 rapidly relo- sions are apparently normal before anaphase onset, but calizes to the kinetochore regions of the separating ZW10 mutants exhibit lagging chromosomes and irreg- chromosomes. In other mutant backgrounds in which ular chromosome segregation at anaphase. Chromo- chromosomes lag during anaphase, the presence or ab- some missegregation during meiosis I of these mutants sence of ZW10 at a particular kinetochore predicts is not caused by precocious separation of sister chroma- whether or not the chromosome moves appropriately tids, but rather the nondisjunction of homologs. ZW10 to the spindle poles. We propose that ZWl0 acts as part is first visible during prometaphase, where it localizes of, or immediately downstream of, a tension-sensing to the kinetochores of the bivalent chromosomes (dur- mechanism that regulates chromosome separation or ing meiosis I) or to the sister kinetochores of dyads movement at anaphase onset. variety of interactions between the kinetochores and phases of poleward and away-from-the-pole movements kinetochore microtubules (kMTs) 1 is essential for ("kinetochore directional instability"; for review see Rieder ensuring the accuracy of chromosome segregation and Salmon, 1994). Finally, the poleward migration of during cell division. (a) Kinetochores can capture microtu- chromosomes during anaphase A depends upon forces bules, initially binding to the lateral surface of a single mi- generated at the kinetochore. Congression and anaphase crotubule and later to the plus ends of several microtu- A movements are probably based upon the ability of mi- bules (Rieder and Alexander, 1990; Pawletz et al., 1994). crotubule motors at the kinetochore to couple chromo- (b) Microtubule-based motors at the kinetochore partici- somes to polymerizing or depolymerizing kMTs (Hyman pate in chromosome movements during prometaphase, and Mitchison, 1991; Lombillo et al., 1995; Desai and metaphase, and anaphase. Certain motors move the chro- Mitchison, 1995). (c) Kinetochores sense tension applied mosome along the lateral surface of the first captured mi- through kMTs across chromosomes to control at least two crotubule toward the corresponding pole (Rieder and Al- processes. First, tension stabilizes kinetochore/kMT inter- exander, 1990). The eventual congression of bioriented actions during prometaphase to ensure that sister kineto- chromosomes to the metaphase plate appears to involve chores are connected to microtubules from opposite spindle the ability of kinetochores to switch abruptly between poles (Nicklas, 1985). Second, tension at the kinetochore makes certain that anaphase onset does not occur until the spindle is properly assembled (Li and Nicklas, 1995). Address all correspondence to Michael L. Goldberg, Section of Genetics Consistent with this complexity of function, a variety of and Development, Cornell University, 425 Biotechnology Building, Ith- proteins has been found to be associated with kineto- aca, NY 14853-2703. Tel.: (607) 254-4802. Fax: (607) 255-6249. e-mail: chores (for review see Pluta et al., 1995). These include MLG1 [email protected]. proteins that are present at kinetochores throughout the 1. Abbreviations used in this paper: FISH, fluorescent in situ hybridization; cell cycle, as well as other molecules that associate with ki- kMT, kinetochore microtubule; PSCS, precocious sister chromatid separation. netochores only transiently during discrete phases of the © The Rockefeller University Press, 0021-9525/96/09/1127/14 $2,00 The Journal of Cell Biology, Volume 134, Number 5, September 1996 1127-1140 1127 cell cycle. In general, little is known about the biochemical that ZWl0 association with individual chromosomes is activities of these proteins. Notable exceptions include both correlated with proper chromosome segregation and several yeast centromere-binding proteins (Hyman et al., influenced by the presence or absence of spindle tension. 1992; Meluh and Koshland, 1995), and in mammalian cells, We discuss these results in light of the hypothesis that the microtubule motors cytoplasmic dynein (Pfarr et al., ZW10 is involved in a tension-sensing mechanism that co- 1990; Steuer et al., 1990) and CENP-E (a kinesin-like pro- ordinates chromosome separation or movement at the be- tein) (Yen et al., 1992; Lombillo et al., 1995). In contrast, ginning of anaphase. to our knowledge, only a single spindle component that is unique to kMTs has been identified. This is the "spoke" antigen, whose molecular properties remain unknown Materials and Methods (Paddy and Chelsky, 1991). We have recently found a novel protein in Drosophila Drosophila Stocks called ZWl0 that appears to migrate from kMTs, where it Descriptions of zwlO mutant alleles and the means by which zwlO mutant is localized during metaphase, to kinetochores, where it is males were generated can be found in Williams et al. (1992). Male meiotic found during anaphase of mitosis. This protein is the prod- mutants and their alleles used to study ZWI0 localization were ord5 (Miyazaki and Orr-Weaver, 1992), mei-S3327 (Kerrebrock et al., 1992), uct of the l(1)zwlO gene (hereafter zwlO). ZW10 is essen- and Dub (Moore et al., 1995) (all the kind gift of T. Orr-Weaver, White- tial for accurate mitotic chromosome segregation, as null head Institute, Boston, MA). Univalent chromosomes were studied in mutations in the zwlO gene result in very high levels of Df(1)X-1/BSw+y + .Y testes (McKee and Karpen, 1990) and in a stock car- aneuploidy in a variety of Drosophila tissues (Smith et al., rying an attached X-Y and a compound fourth chromosome (YsX.YL, 1985; Williams et al., 1992; Williams and Goldberg, 1994). ln[I]EN; C[4]RM, ci eyg; Lindsley and Zimm, 1987). Dieentric chromo- somes were examined for ZWI0 localization in a stock containing In mutant cells, mitosis seems to proceed properly through Y~.YL2Rh4.CB25-1 (Ault and Lyttle, 1988; obtained from T. Lyttle, Uni- metaphase: the metaphase spindle is normal in morphol- versity of Hawaii, Manoa). Oregon-R was used as the wild-type strain. See ogy, and all chromosomes congress to the metaphase Lindsley and Zimm (1992) for further explanation of chromosomes and plate. However, many anaphases are aberrant, with segre- genetic symbols used. gating chromosomes that are misoriented or lag at the metaphase plate. In addition, mutations in zwlO produce Orcein Squashes precocious sister chromatid separation in colchicine-arrested Cytological analysis of meiotic figures in orcein squashes was carried out mitotic cells. Overall, these phenotypes have suggested to according to Lifschytz and Meyer (1977). Testes were dissected in saline us that the movement of ZWl0 from kMTs to the kineto- solution (0.7% NaCI), transferred to aceto-orcein, and squashed gently. chores helps ensure the synchrony of sister chromatid dis- junction at mitotic anaphase onset. Immunostaining In meiosis I, sister chromatids together elaborate a sin- For simultaneous visualization of microtubules and chromosomes, testes gle kinetochore (Goldstein, 1981) and fail to separate at from zwlO mutants were fixed with methanol/acetone according to previ- anaphase. This prompted us to explore whether z~wlO ously published protocols (Cenci et al., 1994; Williams et al., 1995) and function is also needed during the first meiotic division. stained to visualize microtubules and DNA. Testes were incubated with a Examination of meiosis in zwlO mutants would allow us to monoclona] anti--a-tubulin antibody raised against native chick brain mi- crotubules (code N356; Amersham Corp., Arlington Heights, IL), which determine whether ZW10 is specifically involved in sister was used at a dilution of 1:50. After overnight incubation, slides were chromatid cohesion, or instead participates in some aspect washed in three changes of PBT (2.6 mM KCI, 1.5 mM KH2PO4, 137 mM of chromosome disjunction common to meiosis I and mi- NaCI, 8.1 mM Na2HPO4, 0.02% NAN3, 0.1% Triton X-100) for a total of tosis. We chose to perform these experiments in Dro- 15 min. The slides were then incubated with TRITC-conjugated anti- sophila spermatocytes, which offer an ideal system for ex- mouse IgG (The Jackson Laboratory, Bar Harbor, ME) overnight at 4°c to visualize tubulin localization. The slides were then rinsed in PBT for 10 amining meiosis in higher eukaryotes, due to their large rain at room temperature, stained with Hoechst 33258 at a concentration size and well-characterized series of nuclear movements of 0.5 ~g/ml in Hoechst buffer, and mounted in Hoechst citrate buffer as and spindle transformations during meiosis (Cenci et al., described by Cenci et al. (1994). Specimens were examined with a Zeiss 1994). We have also exploited these advantages of spermato- Axioskop equipped for epifluorescence with an HBO 100 W/2 mercury short arc lamp (Zeiss, Oberkochen, Germany), using the 09 filter sets (BP cytes to examine ZW10 localization during meiosis, both 450-490, FT 510, LP 420). Photographs were recorded on TMAX-100 film in wild-type cells and in cells in which chromosomal be- (Eastman-Kodak Co., Rochester, NY).
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