1 Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan
2 Chiba University Graduate School of Science, 1-33 Yayoi-cho, Inage-ku, Chiba, Chiba 263, Japan
*Author for correspondence (e-mail: niwa{at}kazusa.or.jp)
Accepted April 5, 2001
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SUMMARY |
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Key words: Centromere, Telomere, Fission yeast, Spindle pole body
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INTRODUCTION |
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Alterations in chromosome distribution and conformation are not independent of the preceding condition. There are some cases, however, where such dependency is more apparent. For example, the fact that each chromosome occupies a confined space, a territory, or compartment in an interphase nucleus probably reflects the process of decondensation of condensed chromosomes produced in the previous mitosis. Likewise, the successful condensation of chromosomes might be dependent on such territorialization. The Rabl orientation is an example of a more intimate relation to the previous event, because this orientation is established during mitotic anaphase and maintained through subsequent interphase with decondensed chromosomes. In Drosophila larvae, the Rabl orientation persists early in the G1 phase but, in the late G1 phase, it resolves into a non-Rabl configuration, apparently through a process in which the role of heterochromatin is dominant (Csink and Henikoff, 1998). This suggests that the lack of observations of the Rabl orientation in many species, including human tissue cells, is attributable to the short duration of the Rabl configuration after mitosis.
We are interested in the mechanisms involved in the formation of the Rabl configuration, using the fission yeast Schizosaccharomyces pombe. In vegetatively proliferating fission yeast cells, centromeres are grouped in a tight cluster, which is formed near the nuclear periphery beneath the spindle pole body (SPB; the centrosome equivalent structure in yeast), and telomeres are positioned apart from the SPB. The clustering of centromeres is disrupted only during the early phase of mitosis and re-established in anaphase (Funabiki et al., 1993). Thus, the Rabl orientation is maintained stably throughout interphase in this yeast. It is not known if the Rabl configuration is a prerequisite for successful mitosis in this yeast.
Depending on the presence of opposite mating types of cells and nutritional starvation, haploid fission yeast cells are arrested in the G1 phase and conjugate to form zygotes containing a diploid nucleus, and then immediately proceed into meiosis/sporulation (Yamamoto et al., 1997). Before the formation of the diploid nucleus in the zygote, there is a dynamic rearrangement of chromosomes in which telomeres and centromeres switch their positions in the nucleus. This switching occurs in a two-step process (Chikashige et al., 1997). First, before conjugation under the influence of the sex pheromone, telomeres gather at the SPB where centromeres are also clustered. In the next step, as a conjugation-related process, centromeres are released from the SPB to form the bouquet arrangement. Thus, the bouquet-like arrangement is formed in fission yeast before entry into meiotic prophase. The bouquet configuration is also formed directly from diploid cells without intervening conjugation, although it is not known whether this is a two-step process. The mechanisms underlying telomere clustering remain to be elucidated. When zygotes are placed in rich media before commitment to meiosis, they enter into the vegetative cell cycle of diploid cells. At some stage during this return-to-growth process (RTG), chromosomes must conform to the Rabl orientation. The present study investigated whether this reformation of the Rabl orientation is dependent on the first mitosis in RTG and, if not, the mechanism involved in the chromosomal rearrangement.
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MATERIALS AND METHODS |
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Culture conditions
All incubations were at 25 or 26°C, unless otherwise indicated. Cell conjugation was induced as described previously (Miyata et al., 1997). Cells were cultured in MEB-Gal to a cell concentration of 1x107/ml. Cells were washed once with 0.1% glucose and resuspended in MSM at a concentration of 6x107/ml and incubated for approximately 6 hours until the appropriate number of zygotes was produced. The MSM culture was diluted in YE at 5x106/ml (time 0 of the RTG). When the cdc25 mutant was used, the YE culture was transferred to 35°C, 2 hours after the nutritional shift. 100 µl of thiabendazole (Sigma) solution (30 mg/ml in dimethyl sulfoxide (DMSO); Nacalai, Kyoto) was added to 30 ml of the YE culture at the time of the temperature shift. For controls, the same amount of DMSO was added.
To monitor diploid formation of the zygotes, individual zygotes were separated on a YE plate and incubated at 26°C for 4 days (for the cdc25 mutant 22°C for 6 days). Ploidy of each colony was tested using a flowcytometric method (FACScan) as well as the phloxin B plating method (Moreno et al., 1991).
Live observation of spindle elongation
BG990 was transformed with pDQ105 (-tubulin-GFP) and induced for conjugation in MSM medium. Cells were transferred to YE supplemented with 100 µM of thiamine and incubated for 4 hours at 26°C. Cell suspension in the same medium was put on a coverslip-bottomed petri dish and observed with a fluorescence microscope. Images of GFP-labelled spindles were taken every 30 seconds at 25-26°C, with occasional manual focusing. To see spindle elongation in vegetatively proliferating cells, diploid cells were established from the same strain and subjected to live analysis under the same condition. The length of a spindle was measured when both of the spindle ends were in focus or near in focus.
Indirect immunostaining and fluorescent in situ hybridization
The procedures used in this study were a slight modification of those described previously (Funabiki et al., 1993; Chikashige et al., 1997). Glutaraldehyde and formaldehyde (final concentration of 0.2% and 3%, respectively) were added to a cell culture containing 1-5x108 cells, followed by incubation for 1 hour at the same temperature of the culture. Fixed cells were incubated twice in 1 ml of PEM (100 mM PIPES, 1 mM EGTA, 1 mM MgSO4), containing 100 mM glycine to quench unreacted aldehyde groups. Cells were digested with Zymolyase-100T (Seikagaku) and Lysing Enzymes from Trichoderma harzianum (Sigma) at 36°C for approximately 20 minutes. Digested cells were incubated in PEM containing 1% Triton X-100 for a few minutes at room temperature and then treated with RNase A. For indirect immunostaining, rabbit anti-Sad1 antibody (Okazaki et al., 2000) and anti--tubulin monoclonal antibody, TAT1 (Woods et al., 1989) were used as primary antibodies, and Oregon green (Molecular Probes, Eugene, OR) or Cy3- (Jackson Laboratory, West Grove, PA) conjugated anti-mouse IgG and Cy3- or Cy5-conjugated anti-rabbit IgG were used as secondary antibodies. When desired, FISH was performed for the immunostained cells, using Cy3- or Cy5-labeled DNA probes produced from cosmid 212 (for subtelomeric regions of chromosomes I and II; Funabiki et al., 1993, cosmid 1228 (for the centromere-proximal region of chromosome II) (Mizukami et al., 1993), and pRS140 plasmid (for the centromeric repeats) (Funabiki et al., 1993). Chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI).
Microscopy
The Delta Vision System used in the present study was previously described (Shimanuki et al., 1997). For the observation of each nucleus, optical sectioning was performed at intervals of 0.2 µm, computational removal of out-of-focus signals was performed with these images using a deconvolution function integrated in the system. Images presented in the figures were obtained by projecting relevant sections of the images on a single plane.
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RESULTS |
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Chromosome dynamics in the returning process
The chromosome behaviour was examined using FISH. A cosmid DNA (cos212) was used as a telomere probe that recognized telomeres of chromosomes I and II, and pRS140 DNA was used as a probe for all of the centromeres (Funabiki et al., 1993). First, we verified that the telomeres made a single cluster near the SPB in the zygotes produced in the nitrogen-depleted medium (Fig. 5A). Telomeres remained in a single cluster, probably near the same SPB, until 3 hours after the shift. At 4 hours, 65% of the zygotes still contained a single cluster, but the remaining cells contained 2 to 3 telomere signals (Fig. 5B). Centromeres, by contrast, were observed as multiple (usually 3 to 4) foci at the time of the nutritional shift (Fig. 5C,D). Only a small population of the centromeres was located near the SPB (see Fig. 5D). After transfer to the rich medium, as the Sad1-bodies emerged and increased in number, the majority of the centromeres were associated with either the Sad1-bodies or the SPB (Fig. 5E,F).
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Unordinary features of the first mitosis
As noted above, when cells reached a certain length in the RTG, a short spindle was formed and then it elongated to drive chromosome segregation. The first mitosis showed several unusual features compared with ordinary mitoses in vegetatively proliferating diploid cells. The spindle dynamics in live cells with GFP-tagged microtubules was observed. In ordinary diploid mitoses, the elongation of a spindle proceeded in the three-phase mode (Nabeshima et al., 1998), the second phase, corresponding to the metaphase when the elongation paused, occurred when the spindle was approximately 3.5 µm and lasted for about 8 minutes (Fig. 10A). Spindle elongation in the first mitosis also showed three phases, however, the spindle length of the second phase varied from 3.5 to 8 µm and its duration also varied from 3 to 25 minutes (Fig. 10B). Perhaps related with the longer metaphase spindle, the first mitosis was missing the U-shape stage (Toda et al., 1981), which would be seen in early anaphase in an ordinal mitosis. Furthermore, we found that even after the formation of a spindle, telomere cluster could be maintained in about 40% of the cells that contained a spindle less than 4 µm in length, although, in these cells, the cluster was often released from the SPB. This indicated that the telomere cluster could be sustained without the associated SPB. When the spindle became more than 4 µm long, however, all of the telomere clusters were disrupted into multiple subclusters.
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DISCUSSION |
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Jin et al. (2000) recently showed that centromeres are clustered as a rosette around the SPB in interphase nuclei of Saccharomyces cerevisiae, and this configuration could be formed de novo without the passage through mitotic anaphase (Jin et al., 2000). In the budding yeast, even in interphase nuclei there are intranuclear microtubules that appear to connect the SPB with centromeres. Since such intranuclear microtubules are missing in the fission yeast, mechanisms involved in de novo clustering of centromeres are likely to differ in these yeasts. Nevertheless, the presence of such mechanisms that allow de novo reformation of Rabl-like configuration in two different organisms strongly suggests the importance of organized chromosome arrangement for successful chromosome maintenance and transmission.
The centromere clustering observed in the present study is reminiscent of the de novo formation of telomere clustering during the early phase of meiosis (Dernburg et al., 1995; Scherthan et al., 1996; Bass et al., 1997). This is a key step towards formation of the bouquet arrangement, which is important for the pairing and recombination of homologous chromosomes (Loidl, 1990; Dernburg et al., 1995; Roeder, 1997; Niwa et al., 2000). In mouse spermatogenesis, scattered telomeres move to the periphery of the nucleus and then gather beneath the centrosome to form the telomere cluster (Scherthan et al., 1996). This is similar to the formation of telomere clusters in fission yeast, because in both cases the site of telomere clustering is close to the MTOC. In maize meiosis de novo, telomere clustering has been clearly shown using 3D microscopic observation (Bass et al., 1997). In all of these cases, the mechanism underlying the clustering is not known, but there is circumstantial evidence suggesting that cytoplasmic microtubules are involved (Dernburg et al., 1995; Scherthan, 1997; Dawe, 1998).
An important finding of the present study is that the Sad1 protein is located in multiple sites on the periphery of the nucleus during RTG. In the ordinary mitotic cell cycle, the localization of the Sad1 protein is confined to the SPB (Hagan and Yanagida, 1995). The functional significance of this unusual multiplication of the Sad1 localization, or the formation of the Sad1-bodies, was immediately inferred from the fact that the Sad1-body was often associated with centromere(s) as well as with one of the cytoplasmic microtubules. It is conceivable that de novo centromere clustering is driven by the microtubular system. Consistent with this notion, we demonstrated that thiabendazole, a microtubule destabilizing agent, strongly interferes with centromere clustering in the RTG process. We have also shown that the effect of the drug was against the gathering of the centromere-associated Sad1-bodies but not against the maintenance of the centromere clustering.
The Sad1 protein belongs to the SUN-domain protein family, identified in a wide range of species (Malone et al., 1999; M. Shimanuki, unpublished), which is required for spindle formation in fission yeast and for nuclear migration in the nematode. The Sad1 protein contains a putative membrane-spanning segment and, when mildly overexpressed, it localizes to the nuclear periphery in addition to the SPB (Hagan and Yanagida, 1995). This situation resembles the present result, although it differs in one important aspect. In Sad1 overexpression, none of the subsidiary Sad1 signals were associated with microtubules (Hagan and Yanagida, 1995), whereas Sad1-bodies produced in the RTG process often associated with microtubules. At present, there is only limited information regarding the components of the Sad1-body. Further studies will be needed to elucidate the structure and function of the Sad1-body as well as the SPB. They also will benefit from understanding how chromosomes are rearranged within the nucleus, in response to extranuclear signals that might be conveyed though the SPB.
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ACKNOWLEDGMENTS |
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