The cell cycle distribution of SMC4 was determined by simultaneously immunostaining fixed S2 cells with the SMC4 antibody and with antibodies against Barren, one of the non-SMC condensin subunits. In interphase, both proteins are present in the cytoplasm (but were not apparently colocalized) and show low levels of diffuse nuclear staining, excluding the nucleolus. In prophase, increased nuclear labeling of both proteins shows accumulation with the condensing chromatin. In prometaphase, both proteins show discrete colocalization along the condensed chromosome arms, with more intense labeling of the kinetochore regions and the nucleolar organizing region (NOR) of the X chromosome. To visualize this more clearly, prometaphase cells stained for Drosophila SMC4 and MPM2 were used. In metaphase and early anaphase, when chromosomes are fully condensed, the labeling is more uniform throughout the core of the chromatids. During later stages of anaphase, both proteins are still associated with the distal ends of segregating chromatids but are excluded from the decondensing centromere-proximal regions. In telophase, both proteins show low levels of diffuse nuclear and cytoplasmic staining. These results show that a major pool of SMC4 and Barren undergoes significant relocalization from the cytoplasm to the nucleus during chromosome condensation and back to the cytoplasm as cells exit mitosis. A minor pool appears to be bound to chromatin independently of cell cycle stage. Similar results were obtained with third-instar larval neuroblasts (Steffensen, 2001).
Database searches using the SMC4 sequence identified one embryonic-lethal P element insertion allele, l(2)k08819, that had been previously named gluon. Subsequently, a late larval-lethal P element insertion allele, l(2)k06821, was identified. The alleles have been named glu1 and glu2, respectively. Molecular characterization of the glu1 excision allele glu88-82 was carried out by the sequence analysis of PCR products. In glu88-82 a large internal region of the P element is deleted (Steffensen, 2001).
Polyclonal antibodies against an internal fragment of the Drosophila SMC4 protein were generated and shown to specifically recognize the bacterially expressed polypeptide and a single band of approximately 190 kDa in extracts from third-instar larval brains. The antibodies were to determine the levels of expression during different developmental stages and in different tissues. Although SMC4 is present throughout development, it is most abundant in embryos, third-instar larval brains, and adult ovaries and testes (Steffensen, 2001).
Genetic analysis of the mutant alleles has shown that glu1/glu1 and glu88-82/glu88-82 are embryonic lethal; glu1/glu2 transheterozygotes die as late larvae, and glu2/glu2 homozygotes die as early pupa. These results suggest a gradual decrease in the level of the SMC4 protein in the different mutant alleles. Probing third-instar larval brain extracts with the affinity-purified SMC4 antibody confirmed these results. As compared to the wild-type control, SMC4 expression is moderately reduced in glu2/glu2 and significantly reduced in glu1/glu2. Thus, the P element insertion in glu1 probably corresponds to a severe hypomorph allele, while glu2 is a weak hypomorph. To confirm that the P insertions are responsible for the mitotic phenotypes and lethality, the complete cDNA was cloned into a p-UAS transformation vector and several stable transformants were obtained. Driven by a GAL4 insertion (M21277), cDNA expression in neuroblasts produces viable [pUAS-Gluon]; glu2/glu2; M21277 individuals showing no mitotic defects (Steffensen, 2001).
Brains were isolated from glu2/glu2 and glu1/glu2 third-instar larvae to determine mitotic parameters and phenotype. The mitotic index in wild-type neuroblasts is higher (1.11%) than that in either glu2/glu2 (0.87%) or glu1/glu2 (0.83%). Mitotic cells from mutant individuals show several defects as compared to wild-type controls. At prophase, when homologous chromosomes in Drosophila are known to pair, condensing chromatin appeared fuzzy. This diffuse appearance has been observed from prophase to anaphase and with different DNA dyes. In addition, many prometaphase and metaphase figures do not show well-defined sister chromatids. Most cells in anaphase show abnormally decondensed chromatin and contain chromatin bridges and some chromosome fragmentation. Chromatin bridges are also observed during telophase. These chromosome phenotypes are unlikely to be a secondary consequence of dying cells because immunofluorescence analysis with various antibodies against a number of cellular antigens, including checkpoint proteins and mitotic regulators, shows normal distribution (Steffensen, 2001).
Quantification of mitotic parameters in mutant cells shows that, of the cells in mitosis, the percentage of cells at different stages is similar to that for wild-type controls. These data indicate that the mutations do not cause a delay in mitotic progression. Most cells in prophase and metaphase have defects in condensation and/or segregation. These results indicate that the effects of the mutations manifest early in chromosome condensation and that they persist throughout mitosis. Also, a large proportion of the anaphase figures in glu2/glu2 (58%) and glu1/glu2 (82%) have chromatin bridges, indicating a direct correlation between condensation defects and the ability to segregate properly in anaphase. Additionally, the presence of chromatin bridges during telophase (54% and 81% respectively) shows that the abnormal chromatin does not resolve during segregation (Steffensen, 2001).
To study abnormal chromosome condensation in anaphases and telophases in more detail, wild-type and mutant cells were immunostained with an anti-phosphohistone H3 antibody. Histone H3 phosphorylation at Ser-10 is highly correlated with the state of chromosome condensation. Consistently, anaphase bridges that persist until telophase are always highly stained, and this result suggests that decondensation has not been completed (Steffensen, 2001).
An analysis of mitotic domains was performed in the glu88-82/glu88-82 excision derivative, since the phenotype appears to be more severe than either glu88-82/glu1 or glu1/glu1. Labeling of wild-type cells for DNA, phosphohistone H3, and tubulin shows the predicted patterns during mitosis. However, in mutant embryos, anaphases and telophases show chromatin bridges that are stained by the anti-phosphohistone H3 antibody. Quantification of the mitotic phenotypes in mutant embryos indicates that a number of cells arrest in metaphase and that the majority of cells reaching anaphase show chromatin bridges. The fact that embryos show metaphase arrest while larval neuroblasts do not may be because the embryonic alleles are more severe than the larval lethal allele, or it may imply different outcomes when the function of SMC4 is compromised. Thus, the mitotic phenotype caused by mutations in gluon shows that SMC4 is required for proper chromosome segregation in both embryonic and larval tissues (Steffensen, 2001).
Cytological analysis of mutant neuroblasts shows that many mutant cells contain anaphase bridges as well as DNA fragments. In order to determine the type of chromosomal aberrations present in these cells, in situ hybridization was performed with centromeric and telomeric probes. The results indicate that, of the anaphase or telophase cells containing chromatin bridges, only 27% showed centromeric signals in the bridges, while 43% showed a telomeric signal and the remaining 30% contained neither. These results indicate that in most cases centromeres segregate normally and that chromatin bridges are most frequently associated with telomeres or euchromatin (Steffensen, 2001).
Prophase and metaphase cells were observed with very irregular chromosome structure and condensation. These included chromosomes with abnormal condensation patterns and others that did not contain telomeric sequences. Other alterations/rearrangements included circular Y chromosomes, pericentromeric/centromeric fragments, and telomeric fragments. Metaphase chromosomes were also observed that appeared to be connected to chromatin masses. These observations suggest that chromosome fragmentation and chromosome rearrangements may involve different chromosome regions (Steffensen, 2001).
Mutant brains are generally smaller than wild-type brains of similar stages. Since the mitotic progression of mutant neuroblasts is not delayed and the mitotic indices are only moderately lower than those for wild-type controls, this difference could only be explained by the elevated loss of cells during development. Accordingly, glu2/glu2 and glu1/glu2 third-instar larval brains were stained for apoptotic cells. Wild-type have a low frequency of apoptotic cells (0.5%), while mutant brains contain a much higher number of apoptotic cells (2.5% in glu2/glu2 and 3.0% in glu1/glu2). Since chromosome breakage was detected in mutant cells, it appears most likely that the increased level of apoptosis is a consequence of irreversible chromosome damage (Steffensen, 2001).
The cytological analysis of mutant cells suggests that chromosomes have abnormal condensation patterns. Two specific parameters of chromosome morphology have been analyzed in detail. The length of chromosome arms was measured to assess whether mutant cells are able to shorten the longitudinal axis of chromosomes, and the total number of chromosomes that had clearly defined sister chromatids was also quantified in order to assess whether proper sister chromatid resolution takes place. Mutant cells were arrested in prometaphase with colchicine for different periods of time, and they were hybridized with subtelomeric- and pericentromeric-specific probes to avoid measuring chromosomes that had lost distal fragments. Only chromosomes that had both pericentromeric and subtelomeric signals were used for the quantification. Representative samples of wild-type, glu2/glu2, or glu1/glu2 cells were taken that were hybridized with probes to chromosome II. Chromosome length was measured as the number of pixels (0.091 µm/pixel) from the center of the subtelomeric hybridization signal to the center of the pericentromeric signal for each arm, and the two values were added. Overall, the results show that mean chromosome length (chromosome II and III) for either mutant combination (glu2/glu2 and glu1/glu2) that had been incubated for different times in colchicine (0, 30, and 60 min) did not differ significantly from that of the wild-type controls. Accordingly, the dynamics of chromosome shortening in these experimental conditions were similar in both wild-type and mutant cells (Steffensen, 2001).
In order to evaluate if sister chromatid resolution is affected, the number of complete chromosomes that showed clearly resolved sister chromatids was counted. In the absence of colchicine incubation, the percentage of mitotic chromosomes in mutant cells showing sister chromatid resolution, glu2/glu2 (46.8%) and glu1/glu2 (32%), is significantly lower than in wild-type controls (75%). As expected from the severity of the alleles, this phenotype is stronger in glu1/glu2 cells. Incubation of either wild-type or mutant cells in colchicine for 30 min did not have any effect in terms of resolution. However, incubation for 60 min leads to a reduction in the percentage of chromosomes exhibiting visible sister chromatid resolution in both wild-type and mutant cells. This effect is probably a consequence of overshortening the longitudinal axis of chromosomes during mitotic arrest, which thus makes it difficult to distinguish sister chromatids. Nevertheless, the differences observed between mutant and wild-type chromosomes can never be abolished, and this finding suggests that chromatin of mutant cells is not able to compact properly to allow adequate sister chromatid resolution (Steffensen, 2001).
Since, similar to wild-type cells, chromosomes in mutant neuroblasts retain the ability to shorten their arms with normal kinetics, the question of whether these still contain detectable levels of other condensin subunits that might mediate this process was addressed. The anti-Barren antibody was used to determine the distribution of this condensin subunit in mutant cells, since it has a much higher titer than do SMC4 antibodies. This higher titer allows visualization of very low protein levels. Wild-type cells show Barren localizes throughout the chromosome axis with some accumulation at the centromere. Mutant cells that show chromosomes with largely resolved sister chromatids contain normal levels of Barren. However, most mutant cells that display abnormal chromatin compaction and partly resolved sister chromatids have reduced amounts of Barren, and this finding suggests that SMC4 may be essential for its proper localization. In these cells, the level of sister chromatid resolution is directly related to the intensity of Barren staining. This result suggests an inverse correlation between the level of condensin complex and the severity of the phenotype. Additionally, chromosomes in mutant cells frequently show most intense labeling of the centromeric region, with labeling intensity decreasing gradually to the telomeres. Finally, in mutant cells that contained metaphase chromosomes with clearly unresolved sister chromatids, the localization of Barren appears diffuse. This suggests that the sister chromatid cores do not individualize and that sister chromatid resolution probably requires stoichiometric amounts of the Barren subunit but likely also requires the entire condensin complex (Steffensen, 2001).
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date revised: 30 November 2010
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