Neuroblasts in gwl mutants have undercondensed chromosomes that nonetheless bind phosphohistone H3 and condensin
We isolated a mutation in Drosophila
that causes neuroblasts in third instar larval brains to display irregular chromosome condensation and a high mitotic index indicative of mitotic arrest (see Materials and methods). We named the gene identified by this mutation greatwall
because the mutant phenotype suggests a role in protecting chromosome structure. Later, we found that four uncharacterized mutants (Kitamoto et al., 2000
) were allelic to the original gwl
mutation and caused identical mitotic defects.
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 (), 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.
Figure 1. Chromosome condensation defects in gwl mutant brain cells. Wild-type (A and D), gwl716 mutant (B and E), and gwl2970 mutant (C and F) brains were treated with colchicine, and the chromosomes were stained with either orcein (A–C) or Hoechst 33258 (more ...)
Surprisingly, gwl mutant chromosomes still reacted with antibodies directed against phosphohistone H3 and the condensin component Barren (the fly homologue of XCAP-H; ). The signal intensities on gwl chromosomes were roughly similar to those seen in wild type, but Barren and phosphohistone H3 were more diffuse on the undercondensed mutant chromosomes.
Figure 2. Chromosomes from gwl mutants contain the condensin component Barren and phosphohistone H3. Wild-type (A–D) and gwl2970mutant (E–H) neuroblasts stained for DNA (A and E), phosphohistone H3 (B and F), and Barren (C and G). (D and H) Merge (more ...)
Progression through mitosis is delayed or arrested in gwl mutants
Cytological examination of fixed brains from larvae homozygous or hemizygous for all five gwl
alleles revealed defective mitotic progression. The mitotic index was ~2.5-fold higher than controls, and the proportion of mitotic cells that were in anaphase or telophase was only 10–15% of that in wild type (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200310059/DC1
). Thus, gwl
mutant cells delay or arrest before anaphase onset. Almost all of the few residual anaphases/telophases in gwl
brains were aberrant, with lagging chromosomes or chromosome bridges. The spindles in gwl
mutant cells were nevertheless morphologically normal at all mitotic stages (Fig. S1).
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 ( 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.
Figure 3. Division of cultured neuroblasts in time-lapse microscopy. Time in h and min (h:min). (A) Wild-type neuroblasts. (B) gwl716 mutant neuroblast. First and third rows, histone H2Av-GFP signal; second and fourth rows, concurrent DIC images. In the (more ...)
To determine whether the delay in NEB might reflect mitotic spindle defects, we filmed mutant and control cells in the presence of colchicine (Table S3 and Fig. S2). Chromosome condensation took much longer in drug-treated gwl mutant cells than in similarly treated controls, and 30% of the mutant cells failed to condense their chromosomes fully or initiate NEB during periods exceeding 4 h. Thus, Greatwall is needed for proper chromosome condensation and timely NEB even in the absence of a mitotic spindle.
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 , 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, A–H). Third, the checkpoint protein Bub1 accumulates on gwl
mutant kinetochores, as is true in wild-type cells before anaphase onset (Fig. S4, I–T). 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 (), 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]; E–H). Greatwall-depleted cells also showed a 2–3-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).
Figure 4. Depletion of Greatwall by RNAi. Treatment of Drosophila tissue culture cells with gwl dsRNA causes aberrant chromosome morphology reminiscent of that produced by depletion of condensin subunits. (A and B) Karyotypes of control colchicine-treated tissue (more ...)
Greatwall belongs to the AGC family, a diverse group of serine/threonine kinases that phosphorylates targets surrounded by basic amino acids (Hanks and Hunter, 1995
; Morrison et al., 2000
). The kinase domain of Greatwall is split (), with ~500 amino acids separating subdomains VII and VIII (Hanks and Quinn, 1991
). Other insects and vertebrates, including humans, have proteins very closely related to Greatwall; the kinase domains of the fly and human Greatwall proteins share 59% overall amino acid sequence identity (). The homologies between Greatwall proteins extend beyond the kinase domain into the flanking blue and green regions shown in . The insertions of several hundred amino acids between subdomains VII and VIII are less conserved, but we can still detect limited homology between the insect and vertebrate insertions. The Greatwall proteins all form a single homology group: they are the closest relatives found in all pairwise searches between these species. Greatwall is more distantly related to several other proteins that also contain an interruption between kinase subdomains VII and VIII, including IRE and IREH1 in Arabidopsis
, CEK1 in Schizosaccharomyces pombe
, and RIM15 in Saccharomyces cerevisiae
Figure 5. Structure of Greatwall proteins. Conserved domains of the D. melanogaster and human Greatwall proteins, compared with the related kinases Rim15p/Cek1p and IRE. The kinase domain in all these proteins (red) is split by the insertion of unrelated amino (more ...)
The Greatwall protein localizes to the nucleus
To determine Greatwall's intracellular localization, we used anti-Greatwall antibodies as immunofluorescence probes (see Fig. S6 for the characterization of these antibodies). Greatwall accumulated in a punctate pattern in the nuclei of all interphase and prophase cells (Fig. S8). The distribution of the protein within nuclei does not match that of the DNA, but some Greatwall protein might still be chromosome associated. Greatwall was relatively evenly distributed throughout mitotic cells, with no obvious accumulation over the chromosomes or the spindle. The nuclear localization of Greatwall was verified by overexpressing GFP-tagged Greatwall in tissue culture cells (unpublished data).
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 metaphase–anaphase 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 (). However, because the undercondensed chromosomes seen in fixed, colchicine-treated brains () 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 ( 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.