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2 Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201
3 Department of Biomedical Sciences, University at Albany, State University of New York, Albany, NY 12222
4 Dipartimento di Genetica e Biologia Molecolare, Universitá di Roma La Sapienza, 00185 Roma, Italy
Address correspondence to Michael L. Goldberg, Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703. Tel.: (607) 254-4802. Fax: (607) 255-6249. email: MLG11{at}cornell.edu
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Abstract |
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Key Words: Drosophila; cell cycle; kinase; chromosome condensation; nuclear protein
Abbreviations used in this paper: dsRNA, double-stranded RNA; gwl, greatwall; NEB, nuclear envelope breakdown; RNAi, RNA interference.
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Introduction |
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Several preconditions must be fulfilled to allow chromosomes to condense. First, DNA replication must be completed; for example, Drosophila mutants in DNA replication factor genes show defects in chromosome condensation (Krause et al., 2001; Pflumm and Botchan, 2001). Second, several cell cycle regulators must become activated. The first visible stages of condensation during late G2 or early prophase are mediated by cyclin A-CDK, but not by cyclin B-CDK. Cyclin A-CDK activity increases throughout G2, and injection of cyclin A-CDK into G2 cells rapidly induces chromosome condensation (Furuno et al., 1999). In contrast, cyclin B-CDK is inactive and stays in the cytoplasm during these early condensation stages (Rieder and Cole, 1998; Pines and Rieder, 2001). Later aspects of chromosome condensation do require cyclin B-CDK. Late in prophase, the nuclear accumulation and activation of cyclin B-CDK1 triggers nuclear envelope breakdown (NEB), and also mediates subsequent chromosome condensation. In particular, cyclin B-CDK1 phosphorylates and activates the 13S condensin complex (Kimura et al., 1998; Sutani et al., 1999).
Here, we describe a novel Drosophila gene, greatwall (gwl), that is required for proper chromosome condensation. The Greatwall protein is conserved in insects and vertebrates, and contains a kinase domain bifurcated by a long stretch of unrelated amino acids. Greatwall kinase is found in the nuclei of interphase cells, but is distributed throughout mitotic cells. Mitotic chromosomes in gwl mutants are notably undercondensed in the euchromatin. Surprisingly, condensin, phosphorylated histone H3, and topoisomerase II are recruited to these abnormally condensed chromosomes. Mutant cells have major delays in cell cycle progression. The period of chromosome condensation before NEB is much longer than normal. After NEB, mutant cells display a delay in metaphase that is dependent on the spindle checkpoint.
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Results and discussion |
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The most obvious phenotype associated with gwl mutations is chromosome undercondensation. This is apparent in mutant larval brains whose chromosomes were stained with either orcein or Hoechst 33258 (Fig. 1), and is most visible in brains treated with colchicine, which arrests cells under conditions that promote chromosome condensation. In these chromosomes, the euchromatin is highly undercondensed, whereas the heterochromatin remains more compacted. Many aberrantly condensed chromosomes were nonetheless clearly composed of two sister chromatids.
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More detailed observations showed that gwl cells are defective in two aspects of cell cycle progression (Table S1 and Table S2). First, the fraction of cells in late G2/prophase was elevated, indicating that NEB is delayed. Second, as evidenced by the very low frequency of anaphases, mutant cells in which NEB has occurred are subsequently delayed in entering anaphase.
To better understand the gwl phenotype, we examined the division of live neuroblasts in time-lapse microscopy using newly described methods that maintain culture viability for >12 h and that provide excellent four-dimensional resolution (Fleming and Rieder, 2003). To allow simultaneous visualization of chromosome condensation, we generated mutant and control flies expressing GFP-tagged histone H2AvD (Clarkson and Saint, 1999), and filmed cultured neuroblasts with both epifluorescence and differential interference contrast microscopy.
Live imaging of gwl neuroblasts revealed delays in several mitotic phases (Fig. 3 and Table S3). Prophase in mutant neuroblasts lasted >10 times longer than in controls. After NEB, mutant cells were somewhat slower than controls (2 times) to reach metaphase, and 20% of the mutant cells never achieved a stable metaphase. The duration of metaphase was also longer in gwl mutants than in controls (>4 times), and this does not take into account the 17% (2/12) of mutant cells that reached metaphase but failed to enter anaphase during the recording period. Thus, our results with both fixed and live material present a consistent view of the mutant's difficulties in mitotic progression.
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Mutations in gwl do not block chromosome replication or activate a caffeine-sensitive G2 checkpoint
The phenotypes described thus far might possibly be secondary consequences of DNA replication problems; similar defects are associated with mutations in many Drosophila genes encoding DNA replication proteins (Pflumm and Botchan, 2001). We used several techniques to show that DNA replication is substantially complete in G2 mutant cells before mitosis. First, as seen in Fig. 1, many gwl mutant chromosomes appear as two complete sister chromatids late in mitosis. Second, we analyzed BrdU incorporation in whole brains (Fig. S3). Mutant brains, even from animals with the null gwl2970 allele, showed strong BrdU incorporation. In contrast, BrdU incorporation is virtually abolished in the brains of DNA replication mutants (Krause et al., 2001; Pflumm and Botchan, 2001). Third, we quantified the amount of DNA in individual cells by integrating Hoechst 33258 fluorescence signals in cells also stained for phosphohistone H3 to discriminate mitotic stages. In both wild-type and gwl brains, many interphase cells and all mitotic cells had roughly twice the DNA of the interphase cells with the lowest DNA content (unpublished data). Together, these results reveal that DNA replication in gwl mutants is globally normal, but we cannot exclude that subtle S phase defects might occur.
An alternative explanation for the mutant phenotype is that absence of Greatwall might activate a G2/early prophase checkpoint that reverses the early stages of chromosome condensation and delays NEB (Pines and Rieder, 2001; Mikhailov and Rieder, 2002). Many checkpoints of this type monitor DNA damage and are mediated by the ATM and ATR kinases; when activated, these checkpoints inhibit cyclin B-CDK via the inhibition of Cdc25 phosphatase (for review see Abraham, 2001). Thus, we filmed live gwl neuroblasts treated with levels of caffeine that disrupt these checkpoints (De Marco and Polani, 1981). If such a checkpoint was activated in gwl mutants, caffeine should shorten the period of chromosome condensation before NEB. In fact, we saw exactly the opposite: many caffeine-treated mutant prophase cells never underwent NEB (unpublished data).
The spindle checkpoint delays anaphase onset in gwl mutants
As already described in this paper, gwl mutant cells are also delayed in a metaphase-like state after NEB. This arrest almost certainly results from spindle checkpoint activity caused by improper metaphase chromosome condensation. First, because sister chromatids remain connected in gwl cells after NEB, the cells have not entered anaphase (unpublished data). Second, all gwl cells staining for phosphohistone H3 have elevated cyclin B levels, as expected if the checkpoint is active (Fig. S4, AH). Third, the checkpoint protein Bub1 accumulates on gwl mutant kinetochores, as is true in wild-type cells before anaphase onset (Fig. S4, IT). That the metaphase arrest in gwl mutants is due to the spindle checkpoint is consistent with published reports showing that improper chromosome condensation or DNA damage can disrupt the centromere/kinetochore and delay satisfaction of the checkpoint (Pflumm and Botchan, 2001; Garber and Rine, 2002; Mikhailov et al., 2002).
gwl encodes an evolutionarily conserved, putative protein kinase
Fig. S5 details our assignment of the gene CG7719 (protein kinase-like 91C) as gwl. All five gwl alleles contain missense or nonsense mutations in CG7719. The four missense alleles affect conserved amino acids in the serine/threonine protein kinase catalytic domain of the encoded protein, whereas the null allele gwl2970 has a nonsense mutation that truncates the protein. We verified this assignment by depleting the CG7719 protein from Drosophila tissue culture cells using RNA interference (RNAi). Chromosomes from cells treated with CG7719 double-stranded RNA (dsRNA) were highly undercondensed (Fig. 4), reminiscent of the chromosomes in gwl mutant brains. This undercondensation is strikingly similar to that observed when fly tissue culture cells were treated with dsRNA for two condensin complex components (Gluon [XCAP-C] and Barren [XCAP-H]; Fig. 4 EH). Greatwall-depleted cells also showed a 23-fold increase in the mitotic index and a threefold decrease in the percentage of anaphase/telophase cells (unpublished data), consistent with the mitotic delays in gwl brains. Western blotting with antibody to the CG7719 product established that the dsRNA-treated cells are indeed Greatwall deficient (Fig. S6). Of interest, immunostaining of dsRNA-treated cells showed that topoisomerase II localizes normally to chromosomes in cells lacking Greatwall and cohesin components (Fig. S7).
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Possible functions of the Greatwall kinase
Mitotic progression in gwl mutants is much slower than normal, with delays during both late G2/prophase and the metaphaseanaphase transition. Mutations in gwl also cause improper chromosome condensation. In live recordings, chromosomes remain poorly condensed throughout a prolonged prophase, eventually condensing rapidly just before NEB (Fig. 3). However, because the undercondensed chromosomes seen in fixed, colchicine-treated brains (Fig. 1) are from arrested metaphase-like cells, we conclude that the condensation before NEB is incomplete and does not proceed further during prometaphase/metaphase. Incomplete chromosome condensation could disrupt centromere structure, preventing satisfaction of the spindle checkpoint and thus delaying anaphase onset. The aberrations seen in the few residual anaphases in mutants could be caused by the tangling of incompletely condensed chromosomes.
What is the primary defect in gwl mutants? Our results argue strongly that the mutant phenotypes are not secondary consequences of DNA damage or incomplete chromosome replication. Thus, we imagine that Greatwall is directly involved either in chromosome condensation or in the basic cell cycle machinery. Though problems in chromosome condensation during G2/M could trigger an ATM/ATR-independent checkpoint that delays the commitment to mitosis (Goldstone et al., 2001; Pearce and Humphrey, 2001; Rui and Tse-Dinh, 2003), we do not favor this hypothesis. Greatwall is unlikely to play a structural role in chromosome condensation, as it does not appear to associate with chromosomes (Fig. S8), and because cells contain <5,000 Greatwall molecules as estimated by Western blots (unpublished data). Furthermore, chromosome undercondensation in gwl mutants reflects neither a lack of histone H3 phosphorylation nor a failure of condensin or topoisomerase II to associate with chromosomes (Fig. 2 and Fig. S7).
Instead, we believe that Greatwall helps activate cell cycle regulators that prepare interphase cells for entry into mitosis (Pines and Rieder, 2001). Our preliminary efforts to investigate Greatwall in Xenopus egg extracts suggest that Greatwall is needed to establish high cyclin B-CDK activity during mitotic entry. This hypothesis explains many aspects of the gwl phenotype, given that cyclin B-CDK phosphorylates the 13S condensin complex during mitosis (Kimura et al., 1998; Sutani et al., 1999).
In summary, though the importance of Greatwall to chromosome condensation and mitotic progression is clear, the biochemical function of this novel kinase remains to be determined. We are currently trying to identify potential substrates for Greatwall phosphorylation, and other proteins with which Greatwall might associate.
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Materials and methods |
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Cytology and RNAi
Mitosis in larval brains was examined as described previously (Bentley et al., 2002). Mouse anti-lamin antibody was the gift of P. Fisher (State University of New York at Stony Brook, Stony Brook, NY; Stuurman et al., 1995), rabbit anti-Bub1 was provided by C. Sunkel (Universidade do Porto, Porto, Portugal; Basu et al., 1999), and rabbit anti-Barren was from H. Bellen (Baylor College of Medicine, Houston, TX; Bhat et al., 1996). To generate anti-Greatwall antibodies, sequences from a gwl cDNA clone (LD35132) encoding aa 371846 were inserted into the pMAL-C2 vector (New England Biolabs, Inc.). Rabbit sera against the resultant Greatwall-maltose binding protein fusion made in Escherichia coli were affinity purified as in Bentley et al. (2002).
Observations of living Greatwall-H2AvD-GFP neuroblasts were performed according to Fleming and Rieder (2003). In some cases, isolated brains were exposed to 50 µM colchicine at RT for 1.5 h before dissection. For caffeine treatment, brains were incubated with 100 µM caffeine in neuroblast culture medium throughout the filming.
We followed the protocols of Somma et al. (2002) to deplete Greatwall, Barren, and Gluon from Drosophila Kc cells by RNAi and to examine the phenotypic consequences. Rabbit polyclonal anti-topoisomerase II antibody was the gift of D. Arndt-Jovin (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Gemkow et al., 2001).
Online supplemental material
The supplemental material contains eight figures and three tables that document the Greatwall phenotype, the specificity of the anti-Greatwall antibody, and the intracellular localization of the Greatwall protein. The online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200310059/DC1.
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Acknowledgments |
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Work in the laboratory of M.L. Goldberg was supported by National Institutes of Health grant GM48430; work in the laboratory of C.L. Rieder was supported by National Institutes of Health grant GM40198.
Submitted: 14 October 2003
Accepted: 23 December 2003
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