In the course of screening a collection of P-element insertion mutants for those showing mitotic defects, a gene was discovered that has been called mákos (mks). This term was adopted because the cells of the larval central nervous system in the mutant exhibit a high mitotic index and contain highly condensed mitotic chromosomes that resembled the poppy seeds in the Hungarian cake of this name. Both the original mutant allele, mks1, and a second allele mks2 that was identified in the Bloomington stock collection show recessive pharate adult lethal and semilethal female sterile phenotypes, respectively. Analysis of the extent of hypercondensation of mitotic chromosomes in mks1 shows that although the arms of mitotic chromosomes in wild-type cells are clearly evident in both metaphase and anaphase, it is difficult to discern the arms of the dot-like chromosomes in mks. The high proportion of cells in a metaphase-like state and the low frequency of anaphases suggests that mks cells arrest or delay in metaphase. When anaphase-like cells were observed, they were frequently disorganized, with chromosomes lagging along the spindle. A low frequency of polyploid cells (~3% of mitoses) is observed. The extent of chromosome condensation indicates that cells have been delayed in metaphase for a period of time. It suggested a role for the mákos gene product either in some aspect of spindle structure (failure of which would trigger the spindle integrity checkpoint) or in the mechanism regulating the metaphase-anaphase transition itself (Deak, 2003).
A major role of the APC/C is to mediate the metaphase-anaphase transition by initiating events that lead to the separation of sister chromatids. mks cells were examined to determine which, if any, of these events had taken place. Surprisingly, careful observation of orcein stained preparations of mitotic cells in mks larval brains revealed that the sister chromatids of at least one chromosome had separated in 20% of all mitotic figures. To confirm that sister chromatid separation could occur in the mks mutant, the spatial distribution was studied of the dodecasatellite, a repetitive sequence element associated with the pericentromeric heterochromatin of chromosome 3. In situ hybridization of the dodecasatellite probe on wild-type larval brain cells reveals these sequences at two sites on 4N mitotic chromosomes corresponding to the connected sister centromeres of the homologous third chromosome pair. In interphase cells, the signal was often seen as two closely paired dots. In mks cells, cells were found containing a 4N complement of chromosomes in which there were four sites of hybridization indicating that sister chromatids had separated in 19% of mitotic figures. This compared with a frequency of 0.4% metaphase figures showing clear separation of chromatids to give four sites of hybridization in wild-type cells. That the greater majority of cells that show four dots of hybridization are diploid cells with separated chromatids rather than tetraploid cells whose chromosomes have connected sister chromatids is confirmed from the frequency of their occurrence. It was possible to estimate the frequency of tetraploid cells both by counting the numbers of condensed chromosomes in squashed preparations or by counting the number of centromeres (revealed by staining for the Prod antigen on chromosomes 2 and 3) or kinetochores [revealed by staining for the Bub1 antigen. The proportion of polyploid cells (3-4%) was consistent using any of these criteria and was much lower than the proportion of cells showing four sites of dodecasatellite hybridization that was taken as indicative of sister separation (Deak, 2003).
Whole-mount preparations of mks larval neuroblasts were examined by immunofluorescence microscopy to determine whether it was possible to observe the separation of sister chromatids on the mitotic spindle. To this end, immunostaining was carried out to reveal the distribution of the product of the gene proliferation disrupter (prod), a pericentromeric protein found on chromosomes 2 and 3. In wild-type neuroblasts at metaphase, the Prod protein can be seen to be present in four punctate regions of staining corresponding to the adjoined centromeric regions of these two pairs of chromosomes. In mitotic mks cells, the chromosomes gave a broad band of DNA staining on the metaphase plate. However, in all mks cells, the centromeric regions of the two pairs of major autosomes revealed by Prod staining were present as a series of four separate pairs of dots indicative of chromatid separation. A consistent small proportion of such mks cells contained a replicated tetraploid set of chromosomes as expected from observations of the stained squashed preparations. Such cells showed 16 dots of Prod staining, also indicating separation of the centromeric regions in these tetraploid cells. Thus, these observations extended the conclusion from examining the distribution of dodecasatellite that, although APC/C function is likely to be compromised in the mks mutant, the centromeric regions of sister chromatids appeared to have separated in all metaphase-like cells and there had been separation of chromatid arms in some 20% of cells (Deak, 2003).
Finally, it was of interest to see whether the separated centromeric regions were associated with spindle integrity checkpoint proteins. These are generally believed to associate with the kinetochores of chromosomes when the sisters are under unequal tension. Once chromosomes are aligned at metaphase and the sisters are under equal tension from both poles, the proteins are released allowing activation of the APC/C functions that mediate sister separation. Thus, localization of Bub1, a component of the spindle integrity checkpoint complex, would serve the two purposes of permitting visualization of kinetochores of non-aligned chromosomes and determining the state of the mitotic checkpoint when mks (APC/C) function was compromised. Again, it was found that chromatin in metaphase-like mutant mks cells is distributed in a broad band, and it is possible to recognize up to 16 dots of Bub1 staining, corresponding to each of the separated kinetochores of sister chromatids. This finding is similar to that reported for strong hypomorphic alleles of polo, which arrest in a metaphase-like state but with centromeric regions of the sisters separated and yet still with the Bub1 checkpoint protein associated with the kinetochore. This is unusual, in that all kinetochores might be expected to be under equal tension, and yet the checkpoint proteins would appear still to be present. Nevertheless, in those rare mks cells that undertake anaphase and show full separation of sister chromatids, Bub1 staining is not seen (Deak, 2003).
A second major function of the APC is to promote the degradation of mitotic cyclins, so the presence of A- and B-type cyclins in wild-type and mks neuroblasts was monitored by immunostaining. In wild-type cells, cyclin A is present at high levels at prometaphase. It is reduced at metaphase and is absent from cells in early anaphase. In mks neuroblasts, cyclin A is maintained at high levels in all cells arrested in a metaphase-like state. Cyclin B is present in all wild-type metaphase cells and absent from anaphase cells. It accumulates to high levels in all metaphase arrested mks cells. Thus, the metaphase arrest seen in mks cells differs from the checkpoint arrest seen in mutants such as dd4 or following depolymerization of microtubules with colchicine. In these circumstances, cyclin A is degraded whereas cyclin B accumulates to high levels. The high levels of both A- and B-type cyclins in mks mutants indicates that the Cdc27 component of the APC/C is required for their degradation (Deak, 2003).
Evidence from studies in several organisms indicates that the Polo-like kinases can regulate APC/C function. It was therefore asked whether mutations in polo would show genetic interactions with the mks1 mutant allele. To this end, double mutants were constructed between mks and hypomorphic alleles of polo. A comparison of viability shown by flies with these genotypes indicates that the lethal phase of mks1 is advanced to an earlier developmental stage when in combination with either a weak or strong hypomorphic allele of polo (polo1 or polo9 respectively). The stage of death is also earlier than with either polo allele alone (Deak, 2003).
The strongest enhancement of phenotype was seen in the mks1 polo1 combination, so squashed preparations of nervous systems from larvae of this genotype were examined. Such cells display highly condensed chromosomes and have a mitotic index comparable to those of mks1 larvae, although a proportion of the mitotic figures are circular, as is also seen in the polo1 single mutant. The ratio of metaphase:anaphase figures in the double mutant is similar to that seen in mks1 alone. When extracts of the central nervous system from polo1 larvae were examined for the presence of A- and B-type mitotic cyclins, elevated levels of cyclin B and reduced levels of cyclin A were found, typical of some delay at the spindle integrity checkpoint. By contrast, both mks1 and polo1 mks1 brains showed high levels of both A- and B-type cyclins, consistent with the block to APC/C function imposed by the mks1 mutation. Thus, in terms of the mitotic index, the metaphase:anaphase ratio, the condensation state of the chromosomes and the levels of mitotic cyclins, the phenotype of mks1 polo1 cells does not differ significantly from that of the mks1 single mutant (Deak, 2003).
When, however, the appearance of the arrested mitotic spindles was examined by immunostaining for features typical of each mutant, it was evident that centrosomal antigens showed differences in their patterns of distribution. The core centrosomal antigen Centrosomin (CNN) was present in distinct bodies at the spindle poles of the two single mutants and the double mutant combination. However, although both gamma-tubulin and the centrosomal antigen CP190 were present at the spindle poles of the individual mks1 or polo1 mutants, these proteins were both dispersed throughout mks1 polo1 mitotically arrested cells. This dispersal of gamma-tubulin and CP190 is also seen in strong polo hypomorphs such as polo9. Thus, rather unexpectedly, it was found that the polo1 phenotype with respect to spindle pole organization is enhanced by the mks1 mutation (Deak, 2003).
Phosphorylation of a substrate by Polo-like kinases has been reported to confer immunoreactivity to the monoclonal antibody against mitotic phosphoepitopes MPM2. It is specifically known that Polo kinase can phosphorylate Asp, a Drosophila spindle pole component, to make it reactive to the MPM2 antibody and to activate its ability to nucleate asters of microtubules. When either polo1 or mks1 cells was examined with MPM2, the phosphoepitope was detected throughout the cell and also in distinct bodies corresponding to centrosomes and kinetochores. By contrast, the mitotically arrested mks1 polo1 cells lack the MPM2 epitope at their spindle poles. These observations are consistent with diminished levels of Polo kinase activity at the spindle poles in the double mutant. The continued presence of MPM2 epitopes elsewhere in the cell, including the kinetochores of the double mutant suggests either that Polo kinase activity is not affected at these other sites or that such sites result from phosphorylation catalysed by one of the other mitotic kinases known to generate MPM2 epitopes (Deak, 2003).
To confirm that the metaphase block in mks is a result of failure of APC/C function rather than a consequence of checkpoint activation, a double mutant was constructed between mks2 and bub1. Examination of the larval central nervous system from such double mutants showed cells arrested at metaphase at a comparable frequency to the mks2 mutant alone. Moreover, the high frequency of anaphases seen in the bub1 mutant as a result of the malfunctioning checkpoint is replaced in the double mutant by an elevated frequency of metaphase-like figures typical of mks. The appearance of mitotic figures in squashed preparations of mks2 bub1 brains is indistinguishable from that seen in the mks2 mutant alone. Thus, the mks1 arrest cannot be overcome by removing this checkpoint function (Deak, 2003).
An alternative checkpoint like function has been ascribed to CDC55 in budding yeast. CDC55 encodes the 55 kDa regulatory subunit of PP2A and has its counterpart in Drosophila in the gene twins/aar. Mutations in twins/aar result in an elevated proportion of anaphase figures that are highly disorganized and show many lagging and bridging chromatids. The protein phosphatase is likely to have pleiotropic function. The disorganized nature of these mutant anaphases suggests a role for this PP2A subunit in regulation of the metaphase-anaphase transition. However, the accumulation of anaphase figures might be due to a failure to exit mitosis because aar mutants have been shown to have reduced ability to dephosphorylate substrates of Cdk1. To determine whether mutations in tws/aar could overcome the metaphase arrest of mks1, the double mutant strain was constructed. Although there was no apparent shift in lethal phase in the mks tws618/5 double mutant, cytological analysis of neuroblasts showed an elevation of mitotic index and a dramatic reduction of metaphase figures replaced by an elevated frequency of anaphasetelophase figures equivalent to the tws618/5 mutant alone. Thus, the most striking feature of the double mutant is the ability to overcome the metaphase arrest caused by the mks mutation. Nevertheless, the degree of chromosome condensation in the double mutant is more comparable to mks, suggesting that the cells are delayed in a mitotic state, possibly through the prevention of cyclin B degradation. To confirm that cyclin proteolysis is prevented by the mks1 mutation in the double mutant, mutant neuroblasts were stained to reveal cyclin B and such cells were compared with each single mutant. Residual cyclin B can be seen associated with the abnormal anaphase figures in the tws618/5 mutant and in mks tws618/5 at a level not seen in wild-type anaphases. It would thus appear that mks tws618/5 cells are capable of entering anaphase without activation of the APC/C. However, under these conditions, anaphase is highly abnormal (Deak, 2003).
During mitosis, a checkpoint mechanism delays metaphase-anaphase transition in the presence of unattached and/or unaligned chromosomes. This delay is achieved through inhibition of the anaphase promoting complex/cyclosome (APC/C) preventing sister chromatid separation and cyclin degradation. Bub3 is an essential protein required during normal mitotic progression to prevent premature sister chromatid separation, missegregation and aneuploidy. Bub3 is required during G2 and early stages of mitosis to promote normal mitotic entry. Loss of Bub3 function by mutation or RNAi depletion causes cells to progress slowly through prophase, a delay that appears to result from a failure to accumulate mitotic cyclins A and B. Defective accumulation of mitotic cyclins results from inappropriate APC/C activity, since mutations in the gene encoding the APC/C subunit Cdc27 partially rescue this phenotype. Furthermore, analysis of mitotic progression in cells carrying mutations for cdc27 and bub3 suggests the existence of differentially activated APC/C complexes. Altogether, these data support the hypothesis that the mitotic checkpoint protein Bub3 is also required to regulate entry and progression through early stages of mitosis (Lopez, 2005).
The data reveals that cell cycle progression after bub3 mutation or depletion is characterized by a high frequency of cells in prophase, suggesting a slower progression through the early stages of mitosis. Live analysis of Bub3-depleted cells confirmed these results. This delay in prophase appears to result from a defective accumulation of cyclins in both interphase and mitotic cells. Indeed, if cyclin B is stabilized in bub31 mutant cells by a mutation in the Drosophila gene for the APC/C subunit cdc27 or through expression of a stable form of cyclin B, the mitotic index is increased and bub31 mutant cells transit normally through early stages of mitosis. These results also suggest that the defective accumulation of cyclins in bub31 mutant cells is likely to be APC/C dependent, suggesting that Bub3 is able to regulate APC/C activity well before its established role in the mitotic checkpoint response during prometaphase (Lopez, 2005).
Recent results have suggested that more than one APC/C complex may be responsible for either sister chromatid separation or cyclin B destruction during mitosis (Huang, 2002). The results on the mitotic behaviour and cyclin B accumulation in single (bub31 and cdc27) and double (bub31; cdc27) mutant cells support this data and suggest that Bub3 might affect the activity of the different APC/C complexes: (1) it was shown that incubation of cdc27 mutant neuroblasts with colchicine leads to a mitotic arrest with unseparated sister chromatids and normal cyclin B levels, revealing a classical checkpoint response; (2) sister chromatid cohesion in cdc27 mutants can be abolished by a mutation in bub3, showing that the APC/C-dependent separation of sister chromatids does not require cdc27; (3) cyclin B can be degraded during G2 or mitosis in the absence of cdc27 when Bub3 is depleted or mutated. These observations are fully in accordance with previously published results showing that the APC/C subunits cdc16 and cdc27 have distinct locations during mitosis and that individual depletion of cdc16 or cdc27 proteins by RNAi leads to distinct mitotic phenotypes (Huang, 2002). Similarly, in yeast it has been shown that the APC/C subunit cdc27 is not required for the degradation of securin since overexpression of the cdk inhibitor Sic1 is sufficient to rescue the viability of cdc27 mutants (Thornton, 2003). Furthermore, mutations in the Drosophila homologue of APC5, another APC/C subunit, result in a phenotype similar in all respects to mutations in cdc27 including high levels of cyclin B and sister chromatid separation (Bentley, 2002). These data suggest that the activity of the different APC/C subunits may be required at different times during mitosis and at different locations within a cell, and may help to determine the specificity of the APC/C towards the substrates, thus reflecting differentially activated APC/C complexes (Lopez, 2005).
Overall, the results suggest that checkpoint proteins might be required to restrain APC/C activity at multiple times during entry and progression through mitosis, revealing that what has been previously called the spindle assembly checkpoint is indeed a much broader regulatory mechanism that monitors events both before and during mitosis (Lopez, 2005).
Zfrp8 is essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of γ-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring γ-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, it is proposed that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation (Minakhina, 2007).
The developmental mechanisms of human and Drosophila blood systems show remarkable parallels. In humans, several blood cell types with specific functions develop from the same pluripotent stem cells. In Drosophila, only a few specialized cell types exist, with functions similar to human cells. These are thought to originate from a common set of hematopoietic precursors. The development and specification of blood cells in humans and flies are controlled by conserved signaling pathways. Because of its relative simplicity, hematopoiesis in Drosophila is frequently used as a model to investigate the genetic control of hematopoiesis in flies and humans (Minakhina, 2007).
In Drosophila, mature hemocytes arise from two distinct sources: the mature larval circulating hemocytes derive from the embryonic head mesoderm, whereas the lymph gland hemocytes are normally released into circulation at the onset of metamorphosis and perdure into the adult stage. As in vertebrate blood and vascular systems, the Drosophila lymph gland hemocytes and heart cells derive from a common progenitor, called the hemangioblast or cardiogenic mesoderm, which further splits into the lymph gland and cardiogenic progenitors (Mandal, 2004; Minakhina, 2007).
Among the earliest requirements for the specification of blood progenitors in mammals and Drosophila are the highly conserved, GATA zinc-finger transcription factors. The Drosophila GATA-factor Pannier (Pnr) is required for early specification of the hemangioblast/cardiogenic mesoderm. Another GATA-factor, Serpent (Srp), plays a central role in committing mesodermal precursors to the hemocyte fate (Minakhina, 2007).
By the end of embryogenesis, the lymph gland is fully formed and contains mostly pro-hemocytes. The third instar larval lymph gland contains a pair of primary and several secondary lobes. Each primary lobe is subdivided into (1) the medullary zone, populated by slowly proliferating pro-hemocytes; (2) the cortical zone, containing differentiated hemocytes; and (3) the posterior signaling center (PSC), first defined as a small group of cells expressing the Notch ligand Serrate (Ser). Under the control of the EBF-homolog (early B-cell factor) collier (col; knot), PSCs function as a hematopoietic niche to maintain a population of blood cell precursors. The blood cell precursors differentiate into three groups of hemocytes: plasmatocytes, crystal cells and lamellocytes. All three are released into the open circulating hemolymph during the onset of metamorphosis or as a part of an immune reaction. Differentiated plasmatocytes and crystal cells are found in both the cortical zone of the lymph gland and the larval hemolymph, but lamellocytes are rare (Minakhina, 2007).
Plasmatocytes, the predominant form of hemocytes in larvae, perform phagocytic functions and secrete extracellular matrix components and immune peptides similar to human white blood cells. Crystal cells are non-adhesive hemocytes responsible for melanization during wound healing and encapsulation of parasites. Crystal cell differentiation requires the cell-autonomous expression of the transcription factor Lozenge (Lz), homologous to the mammalian acute myeloid leukemia 1 protein (Aml1 or Runx1) (Minakhina, 2007).
Lamellocytes function in encapsulation. Their number is significantly increased at the onset of metamorphosis and in response to infection. Differentiation of lamellocytes is connected to two major pathways - the Drosophila Toll/NF-kappaB and the JAK/STAT - that regulate blood cells proliferation and activation during immune response. Constitutive activation of either pathway leads to overproliferation of circulating and lymph gland hemocytes, an increase in lamellocytes and activation of the cellular immune response (Minakhina, 2007).
A newly identified gene, Zfrp8, is essential for lymph gland growth and for the normal development of Drosophila larvae. Mutant larvae show hyperplasia of the hematopoietic organs. This phenotype is not linked to apoptosis but rather to an increase in cell proliferation. Mutant lymph glands also show a drastic increase in the number of lamellocytes (Minakhina, 2007).
These phenotypes are suppressed by mutations in the GATA factor gene pnr. Mutations in the two cell-cycle genes Cdc27 and l(1)dd4 [lethal (1) discs degenerate 4], have the opposite effect as they enhance the lymph gland overgrowth phenotype of Zfrp8/+. Cdc27 encodes a subunit of the APC complex, responsible for the turnover of cyclins, and l(1)dd4 encodes Dgrip91, a component of the centrosome involved in γ-Tubulin anchoring. In the Zfrp8 mutant lymph gland cells, both Cyclin B (CycB) and γ-Tubulin exhibit abnormal subcellular distribution, suggesting that Zfrp8 plays an important role in their regulation (Minakhina, 2007).
In the literature, the Zfrp8 vertebrate ortholog, PDCD2, is routinely referred to as an apoptotic gene solely because it was upregulated during steroid-induced programmed cell death in rat thymocytes. Subsequent studies, using different cells and assay conditions, found no connection between PDCD2 expression and programmed cell death (Minakhina, 2007 and references therein).
It is unlikely that a reduction in cell death is the cause of the lymph gland overgrowth observed in Zfrp8 mutant larvae. Very few or no apoptotic cells are detected in wild-type larval lymph glands. This study found a statistically insignificant increase in the number of apoptotic cells in Zfrp8 mutants. No other evidence of change in programmed cell death in Zfrp8 mutant animals, no increase in apoptotic gene expression, no change in caspase cleavage and no genetic interaction of Zfrp8 with known apoptotic genes were found (Minakhina, 2007).
The results are consistent with an increase in cell division in Zfrp8 mutants throughout development. This conclusion is supported by the observation that Zfrp8 lymph glands are already twice the size of their normal counterparts in late-stage embryos, and that the number of cells in mitosis is about 30% higher in the mutant glands than in wild type (Minakhina, 2007).
Detailed analysis of Zfrp8 lymph glands shows that its phenotype is different from that of Drosophila hematopoietic/immunity mutants. Unlike hematopoietic/immunity mutants, the increase in lymph gland cell numbers is much larger than the increase in circulating hemocytes. Furthermore, the blood cell overproliferation in Zfrp8-null mutants is not accompanied by constitutive activation of immunity. Zfrp8 larvae show normal induction of immune peptide genes in response to bacterial challenge and normal wound clogging and wound melanization. That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (Minakhina, 2007).
In normal lymph glands, plasmatocytes are found mostly in the cortical region and very few lamellocytes are detected. The PSC is formed at the base of each primary lobe. The presence of additional PSCs in mutant lymph glands might indicate that additional primary lobes are formed by the large number of cells (Minakhina, 2007).
PSCs are essential for maintaining the undifferentiated hemocyte population in the medullary zone and that they control lamellocyte differentiation during parasitic infection. Lack of the transcription factor collier, essential for PSC maintenance, leads to a decrease in the pro-hemocyte population and abolishes lamellocyte differentiation. Loss of Zfrp8 leads to the opposite phenotype - an increase in pro-hemocyte proliferation, beginning during embryogenesis, and an increased number of cells acquiring the lamellocyte fate. Expansion of the PSCs alone does not account for this phenotype. Ectopic expression of the homeotic gene Antennapedia results in expansion of the PSCs, and a concomitant increase of the medullar zone, but not the gland overgrowth. Therefore, it is unlikely that Zfrp8 is directly involved in the establishment of PSCs (Minakhina, 2007).
The results link the Zfrp8 overgrowth phenotype to a defect in normal cell proliferation. In mutant lymph glands, the cell-cycle markers γ-Tubulin and CycB are misregulated. Zfrp8 genetically interacts with at least two genes functioning in the cell cycle, Cdc27 encoding a subunit of the anaphase-promoting complex (APC), and l(1)dd4 encoding the Drosophila gamma-ring protein Dgrip91 (Minakhina, 2007).
Dgrip91 and γ-Tubulin are components of the γ-TuRC microtubule-nucleating complex anchored to centrosomes. Beyond the conventional role in microtubule organization, centrosomes also serve as a scaffold for anchoring a number of cell-cycle regulators. For instance, centrosome-association of Cdc27 and CycB proteins plays an important role in CycB activation, degradation and entrance into mitosis (Minakhina, 2007).
The link between the phenotypes described above and Zfrp8 function became clear when it was discovered that a proportion of Zfrp8 protein localizes adjacent to the centrosome in wild-type tissue. This subcellular localization is consistent with a function of Zfrp8 in centrosome organization and in the anchoring of proteins such as γ-Tubulin and CycB to this organelle (Minakhina, 2007).
Zfrp8 might also affect the expression of bona fide cell-cycle regulators. The protein contains a zinc-finger domain, MYND, present in a number of transcriptional regulators, that fosters protein-protein interactions and recruits co-repressors. PDCD2/Zfrp8 is known to interact with the HCF-1 transcriptional regulator, which suggests that PDCD2/Zfrp8 might be involved in regulating the cell cycle at the transcriptional level (Minakhina, 2007).
Zfrp8 might have a dual function, through its association with the centrosome and as a transcriptional regulator of the cell cycle. Several transcriptional regulators have been found to localize to the centrosome, but their centrosomal function has not been documented (Minakhina, 2007).
Zfrp8 function is essential for the control of cell proliferation already in the embryo. With this being the case, it functions upstream from most of the conserved signaling pathways involved in fly hematopoiesis and immunity. Because of the similarity of the protein in flies and vertebrates, it is possible that PDCD2 has a similar function in vertebrate hematopoiesis (Minakhina, 2007).
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date revised: 10 April 2010
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