polo


DEVELOPMENTAL BIOLOGY

Embryonic

Polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The embryonic transcripts are initially present throughout the syncytial embryo and come to occupy a cortical layer during the blastoderm. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos, unlike the association of transcripts with mitotic domains seen with the string gene. In the cell cycles of both syncytial and cellularized embryos, the Polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic (Llamazares, 1991).

The MPM2 antibody is a valuable tool for studying the regulation of mitotic events since it specifically recognizes a subset of mitosis-specific phosphoproteins. Some MPM2 epitopes are phosphorylated by p34(cdc2). However, recent results suggest that the newly emerging family of polo-like kinases (Plks) may also act as MPM2 kinases. The Drosophila Polo protein is required for the phosphorylation of MPM2 reactive epitopes. The level of MPM2 immunoreactivity is directly correlated to the severity of the polo mutant alleles. In cells carrying a hypomorphic allele, the centrosomes of abnormal cells are small and fail to efficiently recruit MPM2 epitopes. In neuroblasts homozygous for a severe loss-of-function allele, the mitotic index is low and the MPM2 labelling is severely reduced or absent. Rephosphorylation of MPM2 epitopes in detergent-extracted Schneider cells requires either Polo stably bound to the cytoskeletons or Polo present in soluble extracts. These results suggest that Polo is required for the phosphorylation of MPM2 epitopes in Drosophila, at the centrosomes, centromeres and the mitotic spindle, and thus might be involved in co-ordinating the mitotic changes of cellular architecture with the activity of Cdc2 and cyclin B, the catalytic and regulatory subunits of the maturation promoting factor which regulates mitotic progression (Logarinho, 1998).

Polo displays a dynamic localization pattern during mitosis that parallels the localization pattern of the MPM2 phosphoepitopes, since the same pattern is found in the centrosome and centromere from early prophase until late anaphase, the microtubule-overlapping region during anaphase, and the region on either side of the midbody during telophase. Centromere localization is not dependent on microtubules since it is retained in colchicine-arrested cells and is present in isolated chromosomes. At interphase, the Polo protein remains primarily cytoplasmic and no nuclear labelling appears. At prophase, there is strong nuclear staining, and it is possible to distinguish a prominent association of Polo with the separating centrosomes. Anti-Polo antibody labelling is also visible over certain areas of the condensing chromosomes. Cells in prometaphase reveal a punctuate staining pattern clearly associated with the primary constriction of most chromosomes. By late prometaphase, the centrosomes and centromeres remain strongly labelled and some spindle staining appears. Metaphase cells show the most prominent centromere labelling to be organized in pairs, running across the metaphase plate, with the two centrosomes stained at each side of the metaphase plate. Polo is also found associated with the spindle area. By early anaphase, the centromeres are strongly labelled. In mid-anaphase, Polo starts accumulating in the equatorial region of overlapping microtubules, centromeres are still stained and centrosomes remain labelled until early telophase. By late anaphase, Polo is no longer detected at the centromeres but persists in the region corresponding to future cleavage plane. During late telophase, the anti-Polo antibody staining concentrates close to the midbody in the postmitotic bridges connecting the dividing cells; the cytoplasmic staining becomes more evident and nuclear labelling is undetectable (Logarinho, 1998).

The cell-cycle distribution of Polo kinase has also been studied in wild-type third instar larval brains. The anti-Polo antibody staining pattern obtained in wild-type neuroblasts is identical to the one observed in Kc cells. However, certain details of Polo localization become much clearer in this cell type. At early prophase, Polo can be seen in association with the centromeric regions of prophase chromosomes. At metaphase, Polo is also associated with the spindle area. Double immunostaining was performed for Polo and the centrosome component Centrosomin (Cnn) in order to confirm Polo localization at the centrosomes. During anaphase, discrete dots can be seen at the centromeric regions of the migrating chromatids, suggesting that Polo might actually localize to the kinetochore regions, which are known to lead chromatid migration to the poles. At late anaphase, Polo is clearly localized to the overlapping zone of polar microtubules. These results show that Polo associates with multiple subcellular components during mitosis, including centrosomes, centromeres, spindle microtubules and the postmitotic bridges. Polo remains at the centromere of mitotic chromosomes in the absence of spindle microtubules (Logarinho, 1998).

Is Polo retained at the centromeric region of Kc cell chromosomes after microtubule depolymerization? In cells arrested in prometaphase with colchicine and stained with the anti-Polo antibody, all centromeres of the highly condensed chromosomes are labelled, and centrosome staining can also be detected. beta-tubulin staining of parallel preparations reveals that no microtubules are present after the colchicine treatment. Furthermore, analysis by serial optical sectioning of isolated mitotic chromosomes from Kc cells stained with the anti-Polo antibody shows specific labelling localized to the primary constriction on the surface of all chromatids. However, there is also some faint but very consistent signal at the centromeric pairing domain. Although the existence of a trilaminar kinetochore has not yet been demonstrated in Drosophila mitotic chromosomes, the product of the zw10 gene has been reported as a centromere/kinetochore protein. Centromere staining of mitotic chromosomes with the 3F3/2 monoclonal antibody has also been described. In order to confirm Polo localization to this chromosome domain, isolated mitotic chromosomes, Kc cells and neuroblasts were simultaneously labelled with the anti-Polo antibody and the crude rabbit anti-ZW10 serum. The results indicate that Polo co-localizes partially with ZW10. While ZW10 staining extends somewhat away from the pairing domain of sister chromatids, Polo concentrates close to the surface of each chromatid and extends into the pairing domain. This partial overlap between Polo and ZW10 is consistently observed in different preparations. Taken together, these results show that Polo is localized to the centromeres of mitotic chromosomes, overlapping with the kinetochore protein ZW10 and extending into the pairing domain of sister chromatids (Logarinho, 1998).

MPM2 phosphoepitopes are known to be concentrated in several mitotic subcellular structures such as centrosomes, kinetochores, spindle fibers, the chromosomal axis and the midbody. Since immunolocalization results show Polo association with multiple subcellular components during mitosis, a possible overall co-localization between Polo and MPM2 phosphoepitopes was examined. In Kc cells, MPM2 staining is detected over the chromosomes and cytoplasm of mitotic cells, in contrast to interphase cells, which exhibit little or no staining. During the embryonic syncitial cycles, MPM2 antigens are observed within the spindle compartment at all stages of nuclear division. Upon cellularization, the MPM2 epitopes are seen predominantly in mitotic cells. MPM2 staining is detectable in both interphase cells and mitotic cells. The intensity of the staining is significantly less in interphase cells and is restricted to discrete spots within the nucleus. In prophase cells, the staining intensity within the nucleus is drastically increased, the condensing chromosomes contain small bright spots, and the centrosomes are also labelled. By prometaphase, strong labelling is observed in the centrosomes and over the primary constriction of most chromosomes. Spindle pole, centromere and spindle fiber labelling at metaphase can be seen over a diffuse cytoplasmic staining, but the astral microtubules are not reactive. The staining intensity decreases during early anaphase; centromere labelling is still evident, but it is no longer apparent by late anaphase. The immunoreactivity detected in the spindle midzone at late anaphase becomes concentrated on either side of the midbody at telophase. The centrosome labelling disappears during telophase. The distribution pattern of the MPM2 phosphoepitopes just described is also observed in wild-type third instar larval neuroblasts. These results indicate a very similar dynamic distribution pattern of the mitotic kinase Polo and the mitosis-specific MPM2 phosphoproteins during the cell cycle (Logarinho, 1998).

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. This study assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, these results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, defects were observed in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells (Pearson, 2006).

The association of Polo kinase with the actinomyosin cytoskeleton in the syncytial Drosophila embryo raised the question of whether Polo might have a role in localizing components of the actinomyosin cytoskeleton that was independent from its role in the formation of the central spindle. To address this question directly, attempts were made to determine if the loss of Polo kinase activity was associated with abnormalities in actinomyosin dynamics. First, the effects of Polo depletion on the localization of non-muscle myosin II or its regulatory light chain were examined in Drosophila cells. In the few S2 cells depleted of Polo kinase by RNAi that attempted cytokinesis, the extent of localization of myosin at the midpart of the cell was reduced proportionally by the amount of Polo remaining on the central spindle. However, in these cases, of the extent to which the distribution of myosin was a secondary effect to the disruption of the central spindle could not be known with certainty (Pearson, 2006).

Therefore it was whether reduced Polo kinase activity might affect the distribution of non-muscle myosin II earlier in the mitotic cycle when it becomes redistributed from the cytoplasm to the cortex before becoming concentrated at the equator of the dividing cell at anaphase-telophase. The distribution of non-muscle myosin II was examined in larval neuroblasts homozygous for polo1 using its regulatory light chain, Spaghetti-squash, in fusion with GFP (Sqh-GFP) as a marker. As expected, it was found that, in wild-type neuroblasts, Sqh-GFP underwent a cortical redistribution at the metaphase-anaphase transition. In contrast, at the metaphase-anaphase transition, polo1 homozygotes had considerably reduced cortical levels of Sqh-GFP, which was instead dispersed throughout the cytoplasm. Protein levels of Sqh-GFP in homozygous polo1 mutants were not affected (Pearson, 2006).

Although a reduction in cortical myosin was difficult to detect by immunostaining, depletion of Polo by RNAi in S2 cells resulted in the frequent formation of what was termed 'lopsided'. This observation is interesting in light of the recent findings that cortical myosin has a role in centrosome separation and spindle positioning and that the major defects observed in Myosin-depleted S2 cells were lopsided spindles. Similar results were also found in mammalian cells treated with myosin inhibitors. Polo-depleted cells showed an increased frequency of lopsided spindles when compared to control cells. Taken together, these results suggest that Polo-depleted cells are also defective in the recruitment of Myosin II to the cell cortex in the early stages of mitosis (Pearson, 2006).

Larval

Transcripts are present in late larval and early pupal stages and are specifically concentrated in dividing cells in larval discs and brain. The signal is particularly strong in the proliferating centers of the optic lobe (Llamazares, 1991).

Adult

Transcripts are abundant in adult females (Llamazares, 1991).

Effects of Mutation or Deletion

Mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed. There accumulate normal bipolar spindles with polyploid complements of chromosomes, bipolar spindles in which one pole can be unusually broad, and monopolar spindles. Although the extent of chromosome condensation is more like that normally seen in metaphase, the distribution is typical of prophase, the chromosomes not being aligned on a metaphase plate. Thus it appears that chromosome condensation continues to occur even though other aspects of mitosis are delayed (Llamazares, 1991).

Genetic interactions are described between mutations in (merry-go-round) (mgr), abnormal spindle (asp), and polo, genes required for the correct behavior of the spindle poles in Drosophila. The phenotype of a polo1 mgr double mutant is more similar to mgr than polo1, but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The aspE3;mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. Whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination, is discussed. A polo1;aspE3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo1 phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. It is speculated that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo (Gonzalez, 1998).

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Until now, a requirement for polo in regulating anaphase-promoting complex (APC) in Drosophila has not been apparent from the phenotypes of the alleles that have been studied. polo mutants, for example, are able to progress through development, since a function of the weakly hypomorphic protein is sufficiently supplemented by maternally provided wild-type protein. Consequently, it has been difficult to assess fully the functions of Polo kinase in polo somatic cells which are capable of progression through multiple cell cycles. Two strongly hypomorphic alleles, polo9 and polo10, block the proliferation of diploid tissues. Cells of these mutants appear poised to initiate anaphase and sister kinetochores are pulled apart. Chromatids undergoing partial separation can remain attached through their telomeres and do not migrate to the spindle poles. These strongly hypomorphic mutations of polo arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20%-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the Abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. Roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint are discussed (Donaldson, 2001).

Previous studies on the polo allele have revealed requirements for its protein kinase for the function of microtubule organizing centers at several stages of development, and in both chromosome segregation and cytokinesis in male meiosis. The strongly hypomorphic mutant alleles polo9 and polo10 suggest a new requirement for Polo kinase in the metaphase-anaphase transition, but also give additional insight into the requirements for Polo kinase to organize microtubule nucleating centers. It is known that both the gamma-tubulin ring complex (gamma-TuRC) and Asp are required for the integrity of mitotic microtubule organizing centers, and yet neither appears to be required for localization of the other at the spindle pole. gamma-Tubulin is found at the spindle poles in asp mutants, and Asp, which only associates with spindle poles during mitosis, still localizes to either the poles or the unfocused minus ends of microtubules in mutants of the dd4 gene that encodes the 91-kD component of the gamma-TuRC. The effect of the polo9/10 mutations on the constitution of centrosomes for the limited number of antigens that were studied is in fact remarkably similar to that seen when the gamma-TuRC is disrupted in dd4 mutants. In dd4 cells, gamma-tubulin is dispersed throughout the cytoplasm and a large proportion of the CP190 antigen shows a punctate distribution around the condensed chromosomes in much the same way as in polo9/10 mutants. Moreover, in addition to Asp, Centrosomin is present at the focused spindle poles both in dd4 and polo9/10 mutants. It is suggested that these common aspects of phenotype result from a common primary failure to correctly assemble and/or localize the gamma-TuRC at the centrosome. This would be consistent with the observation that gamma-tubulin is recruited to the centrosome at mitosis and the finding that centrosomes fail to grow and effectively nucleate microtubules in human cells injected with antibodies to Polo kinase. However, it is of interest that although centrosomes appear to lack gamma-tubulin and do not nucleate astral microtubules, they are nevertheless capable of assembling a spindle and of establishing a metaphase-like array of chromosomes (Donaldson, 2001).

If plks play an essential role in the activation of cdk1, an arrest at the G2/M transition in polo9 and polo10 might have been expected. Since this is not observed, it suggests either that this function is not required at this stage of Drosophila development, or it is redundant, or it can be fulfilled by the little amount of detectable Polo protein. The phenotype of polo9/10 is more typical of cells unable to pass beyond the metaphase-anaphase checkpoint, since they have low levels of cyclin A and high levels of cyclin B. It is generally believed that the inhibitory signal of this checkpoint arises from unaligned chromosomes and requires a complex containing the Bub1 and 3 and Mad1, 2, and 3 proteins that associates with unattached kinetochores. Mad3 binds to and inactivates Cdc20p-Fizzy, a protein that directs the APC to a specific set of substrates. In yeast, one of the Cdc20-APC substrates is Pds1, whose proteolysis releases the separin Esp1, which in turn catalyses the degradation of Scc1p resulting in sister chromatid separation. In the presence of the Bub1 complex of checkpoint proteins, the APC cannot be activated and cells become arrested in metaphase. polo9/10 cells have Bub1 and the 3F3 epitope at their kinetochores, suggesting they are held in a state of checkpoint arrest by the Bub1 complex. This is confirmed by the observation that bub1 mutation suppresses the mitotic phenotype of polo10 and so releases the metaphase-like arrest (Donaldson, 2001).

However, polo9/10 cells exhibit a paradox that sets them apart from other metaphase arrest mutants of Drosophila. Their chromosomes appear to have undergone alignment and anaphase movements activated because sister kinetochores appear to be not only attached through microtubules to the poles but also pulled apart by about 2-5 µm. This itself might have been expected to relieve the checkpoint arrest, and usually the 3F3 epitope is lost from chromosomes under tension. In contrast, Giemsa-stained squashed preparations reveal that only ~17% of sister chromatids remain separated in squashed preparations of polo9/10 brain cells made in the presence or absence of colchicine. This suggests that the majority of sister chromatids are still held together, explaining their failure to move to the spindle poles. In addition, some chromosomes appear connected in chains through telomeric linkages. Chaining of chromosomes in this way has been described previously in the mutants for the UbcD1 gene that encodes a class I ubiquitin-conjugating (E2) enzyme. This observation suggests that Polo kinase may in part play a role in regulating the proteolytic system responsible for breaking such linkages (Donaldson, 2001).

Centromere separation seen in intact polo9/10 cells must be accounted for by pole-directed forces of motor proteins on the chromatids. Although several explanations are possible to resolve the paradox of the polo phenotype, the simplest is that Polo kinase is actively required to relieve the function of the Bub1 checkpoint complex in the progression through mitosis. This would have some parallels with the finding that in budding yeast, CDC5 is essential to escape the metaphase arrest imposed by DNA damage surveillance mechanisms. This hypothesis implies that the polo arrest point lies on the cusp of the metaphase-anaphase transition. This is at a later stage than the requirement for the fizzy gene product function. In fizzy mutants, neither A- nor B-type cyclins are degraded and there is no separation of centromeric regions as seen in polo mitotic arrest. This incidentally contrasts with mutants for CDC20, the fizzy homolog of budding yeast where centromeric regions are observed to be separated. However, the transient separation of sister centromeres while chromosome arms still show cohesion does appear to reflect a general aspect of normal progression through metaphase in wild-type budding yeast. If similar events occur in metazoan cells, this aspect of normal progression through metaphase might be accentuated in polo cells that appear to have initiated anaphase by the criterion of the apparent poleward tension imposed at the centromeres, and yet are not released from 'checkpoint' arrest (Donaldson, 2001).

Polo kinase may also be required to promote complete separation of sister chromatids and for those specific aspects of APC activity that mediate cyclin B degradation. The latter would have some similarities to budding yeast where mutants in the plk gene CDC5 are reported to show no effect on Cdc20-APC function and so permit Pds1p to be degraded, in the absence of degradation of the mitotic cyclin Clb2p. Moreover, overexpression of Cdc5p results in proteolysis of Clb2p but not Pds1p, suggesting that the CDC5 plk promotes Hct1 (fizzy related)/APC functions to regulate cyclin B levels. In fact, the phenotype of polo9/10 mutants resembles that of mutants expressing nondegradable cyclin B in several respects. Expression of stable cyclin B (lacking its destruction box) from a GAL4 responsive promoter in Drosophila larval neuroblasts results in a mitotic arrest in which the greater proportion of cells arrest in anaphase. In embryonic cells, the phenotypes resulting from expression of stable forms of cyclins A, B1, and B3 support a sequential requirement for the three proteins: stable cyclin A leads to a metaphase delay; stable cyclin B an early anaphase arrest, and stable cyclin B3 to a late anaphase arrest. Sister chromatids clearly separate in those cells expressing stable cyclin B as indicated by in situ hybridization with a dodecasatellite probe to identify the centromeric region of the third chromosomes. A role for polo in promoting those aspects of APC activity that mediate cyclin B degradation would be consistent with the observations that mammalian Plk can phosphorylate and activate the APC in vitro. However, the ability of the polo10 bub1 double mutant to progress through mitosis would suggest that this APC function can also be activated at some level in the absence of polo function (Donaldson, 2001).

In budding yeast, there is now considerable evidence that Hct1p can be activated by the Cdc14p protein phosphatase. Cdc14p appears itself to be activated downstream of the GTP-bound active form of Tem1p. This pathway is held in check in response to spindle damage by a two component GTPase activating protein (GAP) formed between Bub2p and the Bfa1/Byr4 protein that promotes formation of the GDP-bound state of Tem1p which does not favor Cdc14 activation. The CDC5 polo-like kinase appears to regulate the phosphorylation of Bfa1/Byr4 in this process. In fission yeast, the equivalent pathway regulates the onset of the septation process and appears to be under the control of Plo1 kinase. It is of considerable interest to know the extent to which the pathway might be conserved and whether an analogous process regulates the onset of cytokinesis in metazoans. The requirement for polo for cytokinesis in Drosophila is obscured by earlier mitotic defects seen in somatic cells with all alleles. The exception is male meiosis, where it seems that spindle checkpoint pathways do not lead to metaphase arrest. However, cells from the polo9; bub1 double mutant do attain high levels of polyploidy, suggestive of a failure in cytokinesis once the metaphase arrest is bypassed. It will be a future challenge to determine the extent to which this mitotic exit network has been conserved in metazoans and its relationship to the regulation of the onset of anaphase (Donaldson, 2001 and references therein).

Progression through mitosis requires the ubiquitin-mediated proteolysis of several regulatory proteins. A large multisubunit complex known as the anaphase-promoting complex or cyclosome (APC/C) plays a key role as an E3 ubiquitin-protein ligase in this process. The APC/C adds chains of ubiquitin to substrate proteins, targeting them for proteolysis by the 26S proteasome. The gene makos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens gamma-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product (Deak, 2003).

The APC/C is activated upon entry into mitosis and its substrate specificity is thought to be regulated mainly by the transient association of co-factors such as Cdc20/Fizzy and Cdh1/Hct1/Fzr. APC/C function is further known to be regulated by phosphorylation, for which Cdk1/cyclin-B, Polo-like kinase 1 (Plk1) and cAMP-dependent protein kinases have all been implicated as playing major roles. Plk1 appears to facilitate APC/C activation by preferentially phosphorylating Cdc16 and Cdc23, and indeed its fission yeast counterpart, Plo1, binds specifically to the Cdc23 (APC8) subunit. Although Polo-like kinases have been reported to be required for APC/C activation, mks mutants have been found to enhance the mutant phenotype of an hypomorphic allele of polo with respect to its ability to orchestrate one known function of Polo-like kinases: the recruitment of gamma-tubulin and associated molecules to the centrosome (Deak, 2003 and references therein).

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).

The motivation for studying genetic interactions between mks and polo arose from evidence pointing to a role for Polo kinase in APC/C activation. Depleting Plx1 in extracts of Xenopus, blocks degradation of cyclin B. In budding yeast, the Polo-like kinase encoded by CDC5 has also been implicated in activation of the APC/C. Moreover, mammalian APC/C can be activated by Plk1 phosphorylation in vitro. The phosphorylation patterns of APC/C components have been carefully studied; Plk1 and Cdk1/cyclin-B have additive effects in phosphorylating and activating the APC/C; the former preferentially phosphorylates Cdc16 and Cdc23, and the latter preferentially phosphorylates Cdc27. The fission yeast Polo-like kinase Plo1 interacts physically with the Cut23 component of the APC/C. Nevertheless, it appears that the APC/C is active in the polo1 because cyclin A levels are reduced and cyclin B remains, as would be seen following checkpoint delay at metaphase. Only a slightly earlier lethal phase of mks was seen when in combination with different polo alleles. By contrast, the unexpected observation was made that the weakly hypomorphic phenotype of polo1 in larval neuroblasts is enhanced by mks in respect to its effects upon centrosome structure and function (Deak, 2003, and references therein).

The original polo1 allele encodes an apparently full-length protein that is incompletely phosphorylated and has low catalytic activity. Thus, polo1 is a weak hypomorph and, although embryos derived from homozygous polo1 mothers show defects in the recruitment of the centrosomal antigen CP190, those mothers would appear to have been able to develop to adulthood in part because of the lack of any obvious centrosomal defect in mitotically dividing cells of their larval central nervous systems. This is in contrast to brains of the strong hypomorphic alleles polo9 or polo10, in which neuroblasts arrest in a metaphase-like state, with spindles that have core centrosomal components, such as CNN, at their spindle poles but lack gamma-tubulin and CP190. This phenotype echoes the requirement for Polo kinase in recruiting gamma-tubulin to the centrosome upon mitotic entry following experiments in which antibodies against Plk1 were microinjected into HeLa cells (Deak, 2003 and references therein).

The finding that mks enhances the centrosomal phenotype of polo1 such that it resembles the stronger polo hypomorphs suggests that the APC/C is required either directly or indirectly to fully activate Polo kinase function. It is possible to gain a measure of the activity of Polo kinase by assessing the presence of the MPM2 epitope. Indeed, at least one Drosophila centrosomal protein (Asp) has been shown to acquire an MPM2 epitope following phosphorylation by Polo. The finding of the absence of MPM2 reactivity at the centrosome in mks1 polo1 mutants is therefore consistent with reduced Polo kinase activity. A direct functional interaction between the APC/C and Polo cannot be excluded because Polo kinase has been shown to bind the APC/C, at least in fission yeast, and both the APC/C and Polo kinase are present on the centrosome. However, the idea is favored that an early mitotic function of the APC/C might be required for the full activation of Polo kinase and the recruitment of the gamma tubulin ring complex (gamma-TuRC) to the centrosome. Indeed, cyclin A is normally degraded ahead of cyclin B and continues to be degraded when cells are arrested at the spindle checkpoint by microtubule depolymerizing drugs, but remains present in both mks1 and mks1 polo1 cells. Normally, the degradation of cyclin A takes place from the beginning of mitosis in an APC/C-dependent manner. Thus, it is postulated that because APC/C is normally active from the very beginning of mitosis, one of its functions might be to facilitate the full activation of Polo kinase at the centrosome by mediating the degradation either of cyclin A or some other crucial inhibitory molecule. This would constitute a new cell cycle autoregulatory loop whereby the early mitotic functions of the APC/C are required for the activation of an enzyme itself implicated in activating later functions of the APC/C (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).

Phenotypic interactions between stabilized Pimples and Polo

Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, it is shown in this study that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, PIM is shown to contain a KEN-box, which is required for mitotic degradation in addition to the D-box; sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals (Leismann, 2003).

Embryos homozygous for pim null mutations but equipped with a maternal pim+ contribution from pim heterozygous mothers progress normally through the initial embryonic cycles. Entry into mitosis 15 and progression to metaphase are still normal. Moreover, the transition from metaphase to anaphase is triggered as well, as evidenced by the degradation of the mitotic Cyclins A, B and B3. However, sister chromatid separation during mitosis 15 is completely inhibited. This block of sister chromatid separation after the exhaustion of the maternal pim+ contribution is almost completely prevented when pim embryos inherit a gpimdba-myc transgene. gpimdba-myc drives expression of Pim with C-terminal myc epitopes and a mutant D-box (AKPAGNLDA instead of KKPLGNLDN). Pimdba-myc is stable during mitosis according to confocal immunofluorescence microscopy, in contrast to Pim-myc with the wild-type D-box. gpimdba-myc expression is controlled by the normal pim regulatory region, and the resulting level of Pimdba-myc before mitosis 15 was found to be comparable to wild-type Pim levels. Because stabilized Pimdba-myc protein promotes sister chromatid separation in pim mutants, it appears that sister chromatid separation is not dependent on degradation of the Drosophila securin Pim. Analogous experiments with a gpimdba transgene driving expression of a D-box mutant Pim version without myc epitopes also revealed rescue of mitosis 15 in pim mutants, excluding the possibility that sister chromatid separation in the presence of stabilized Pimdba-myc occurs simply because C-terminal myc epitopes specifically abolish the inhibitory Pim function (Leismann, 2003).

Instead of being required during each mitosis, Pim degradation might be important to keep protein levels below a critical threshold. Moderate overexpression of wild-type pim (about fivefold) is sufficient to block sister chromatid separation. Moreover, although gpimdba rescues sister chromatid separation during mitosis 15 and 16 in pim mutants, it does not allow later divisions, perhaps because the levels of stabilized Pimdba have built up beyond the critical threshold (Leismann, 2003).

If degradation of the securin Pim was not an obligatory process required during each mitosis, separase bound to securin would be expected to have sufficient basal activity to allow sister chromatid separation. In this case, premature sister chromatid separation during interphase and early mitosis would have to be prevented by securin-independent regulation. Since securin-independent regulation at the level of Scc1 phosphorylation by Cdc5/Polo kinase has been described in yeast, whether a reduction in polo function enhances the effects of stabilized Pimdba was investigated. Within the CNS of polo-mutant embryos, many abnormal cells were observed with very large polyploid nuclei, when these embryos also carried gpimdba. Similar abnormal cells are almost never observed in either polo+ sibling embryos with gpimdba or in polo- sibling embryos without gpimdba. In the presence of stabilized Pimdba, therefore, the remaining level of maternal polo+ contribution is no longer sufficient to mask phenotypic abnormalities in polo-mutant embryos. Moreover, reduced polo+ function enhances the effects of stabilized Pimdba (Leismann, 2003).

In addition to Scc1 regulation by Cdc5/Polo kinase, vertebrate Cdk1 has been shown to regulate separase independently of securin (Stemmann, 2001). The effects of stabilized Cyclin A in Drosophila embryos are consistent with the finding that vertebrate Cdk1 phosphorylates and thereby inhibits separase. Mutant Cyclin A versions that cannot be degraded during mitosis delay progression through the embryonic cell divisions during metaphase before sister chromatid separation. Therefore, Drosophila Cyclin A-Cdk1 complexes might inhibit separase activity. Accordingly, the effects of stabilized Cyclin ADelta1-53 are expected to be enhanced by expression of stabilized Pimdba. Labeling with antibodies against tubulin and a DNA stain clearly reveal an increased number of metaphase figures in epidermal regions of embryos expressing both Cyclin ADelta1-53 and Pimdba, compared with embryos expressing only Cyclin ADelta1-53. The stabilized Cyclin ADelta1-53 therefore results in a more pronounced metaphase delay in the presence of the stabilized Pimdba (Leismann, 2003).

In principle, stabilized Cyclin A might delay cells in metaphase because it results in an inhibition of Pim degradation during mitosis. However, cells delayed in metaphase by stabilized Cyclin ADelta1-170 no longer contain Pim-myc according to immunolabeling experiments, whereas metaphase cells that do not express Cyclin ADelta1-170 are always positive for Pim-myc. It is concluded, therefore, that the metaphase delay induced by stabilized Cyclin A does not result from delayed Pim degradation (Leismann, 2003).

The phenotypic interactions between stabilized Pimdba and Polo or Cyclin A are consistent with the notion that separase complexed with non-degradable securin might have sufficient activity to allow sister chromatid separation and that the timing of this process is controlled by pathways other than securin degradation. However, the sister chromatid separation in Pimdba-expressing cells might also be supported by residual mitotic Pimdba degradation. A KEN motif, which is found close to the N-terminus in all of the securins, might allow some limited mitotic Pimdba degradation, escaping detection by confocal microscopy as applied in the previous experiments (Leismann, 2003).

To determine whether the KEN motif of Pim functions as a degradation signal, the mitotic stability of a myc-tagged Pim version with a mutant KEN-box (Pimkena-myc with AAA instead of KEN) was analyzed. Pimkena-myc, and Pim-myc for control, were expressed in the anterior region of embryos during cycle 14. Immunolabeling at the stage of mitosis 14 indicate that Pimkena-myc is largely stable throughout mitosis, in contrast to Pim-myc, which is detected before but not after the metaphase-to-anaphase transition. Progression beyond the metaphase-to-anaphase transition was monitored by the labeling of DNA and Cyclin B, which is rapidly degraded when cells enter anaphase. These results show that the KEN-box is required and that the variant D-box (KKPLGNLDN), which is still present in Pimkena-myc, is not sufficient for normal mitotic Pim degradation (Leismann, 2003).

Overexpression of Pimkena-myc results in mitotic defects. Normal anaphase and telophase figures are not observed in Pimkena-myc-positive cells that have progressed beyond the metaphase-to-anaphase transition according to the absence of anti-Cyclin-B labeling. Instead of pairs of well-separated telophase daughter nuclei, which are readily observed in Cyclin-B-negative regions in the Pim-myc control experiments, Cyclin-B-negative regions of Pimkena-myc-expressing embryos display decondensing metaphase plates or chromatin bridges between partially separated nuclei. These abnormalities caused by Pimkena-myc are indistinguishable from those previously observed with Pimdba-myc, which has been shown to inhibit sister chromatid separation (Leismann, 2003).

Sister chromatid separation is also inhibited by strong overexpression of wild-type Pim-myc. By contrast, at low physiological expression levels, Pim-myc and, remarkably, also the stabilized versions Pimdba-myc and Pimkena-myc, can promote sister chromatid separation in pim mutants (Leismann, 2003).

To analyze the function of Pim with mutations in both D- and KEN-box, additional transgenes (g>stop>pimkenadba and g>stop>pimkenadba-myc) were constructed, allowing the expression of Pimkenadba or Pimkenadba-myc under the control of the normal pim regulatory region. To establish chromosomal insertions of these potentially detrimental transgenes, a stop cassette flanked by FLP recombinase target sites (>stop>) was inserted into the 5' untranslated region. This stop cassette was eventually excised by transmitting the established insertions via males expressing FLP recombinase specifically in spermatocytes. Expression of the paternally recombined transgenes (g>pimkenadba and g>pimkenadba-myc) started at the onset of zygotic expression during cycle 14 of embryogenesis. Expression of g>pimkenadba and g>pimkenadba-myc in pim-mutant embryos did not allow sister chromatid separation during mitosis 15. Instead of normal mitotic figures, which were readily apparent in pim+ sibling embryos, only decondensing metaphase plates were observed during exit from mitosis. Thus, pim-mutant embryos expressing g>pimkenadba and g>pimkenadba-myc display the same phenotype as pim mutants without transgene or with the non-recombined g>stop>pimkenadba transgene (Leismann, 2003).

Control experiments with g>stop>pim transgenes encoding wild-type Pim show that expression after stop-cassette removal is sufficient to promote normal sister chromatid separation in pim mutants. Moreover, additional control experiments show that the recombined g>pimkenadba-myc transgene is expressed as expected. Anti-myc immunoblotting clearly show expression, and co-immunoprecipitation experiments indicate that the Pimkenadba-myc protein associates efficiently with Separase (SSE) and Three rows (THR), a Drosophila protein known to form trimeric complexes with SSE and Pim. In addition, although g>pimkenadba-myc expression in pim+ sibling embryos has little effect during the initial embryonic cell divisions (mitosis 14-16), it results in a severe mutant phenotype in the CNS where additional cell divisions occur. Wild-type Pim therefore appears to protect cells from the effects of Pimkenadba-myc but only as long as the latter has not yet accumulated to high levels (Leismann, 2003).

In summary, the experiments with g>pimkenadba and g>pimkenadba-myc in pim mutants show that sister chromatid separation does not occur in the presence of physiological levels of the double mutants Pimkenadba and Pimkenadba-myc, in contrast to the findings with the single mutants Pimdba, Pimdba-myc and Pimkena-myc (Leismann, 2003).

It is concluded that mutations in either the D- or the KEN-box result in significant stabilization of Pim protein during mitosis. Neither the D- nor the KEN-box, therefore, are sufficient for normal degradation during the embryonic cell divisions in Drosophila. Similar observations have been described for human securin (Hagting, 2002; Zur, 2001). However, in contrast to Drosophila, mitotic degradation of human securin still occurs quite effectively when either only the D- or the KEN-box is intact. The D- and KEN-boxes of Drosophila Pim, therefore, might function less independently than the corresponding motifs in human securin. Eventually, the understanding of D- and KEN-box function will require structural analyses of their interactions with Fizzy/Cdc20 and Fizzy-related/Cdh1, which recruit proteins with these degradation signals to the APC/C. Fizzy and Fizzy-related are clearly both involved in Pim degradation, at least indirectly, since Pim is stabilized in both fizzy and fizzy-related mutants (Leismann, 2003).

Under the assumption that Pimkenadba and Pimkenadba-myc are still capable of providing the positive Pim function, these results with these stabilized mutants suggest that Pim must be degraded during each and every mitosis to allow sister chromatid separation. Although not detectable by confocal microscopy, the single mutants Pimdba and Pimkena might not be completely stable in mitosis. After low-level expression in pim-mutant embryos, residual mitotic degradation of single-mutant proteins might free some separase activity sufficient for sister chromatid separation. Similar results have been observed with the fission yeast securin Cut2, which is completely stabilized in a Xenopus extract destruction assay by mutations in either of the two D-boxes, and yet, low-level expression of single-but not double-mutant proteins is able to complement growth of cut2-ts strains at the restrictive temperature (Funabiki, 1997). It is emphasized that even in wild-type cells, mitotic Pim degradation appears to be far from complete, and it can be speculated that it is the Pim protein of a special pool of separase complexes that is more efficiently degraded, perhaps on kinetochores or during transport on spindles towards kinetochores. At high expression levels of Pim with or without single mutations, free excess of this securin might rapidly re-associate and inhibit the activated separase, resulting in the observed block of sister chromatid separation (Leismann, 2003).

These results also point to alternative pathways that might regulate separase activity and sister chromatid separation independently of Pim degradation. As in yeast, the success of mitosis in cells with reduced separase function is dependent on Polo kinase in Drosophila embryos. Moreover, since expression of mitotically stabilized Cyclin A versions result in a metaphase delay without inhibiting Pim degradation, Cyclin A appears to contribute independently of Pim to the inhibition of premature sister chromatid separation. Even though it remains to be analyzed whether Polo kinase and Cyclin A-Cdk1 act during Drosophila divisions as proposed for Polo homologs (Alexandru, 2001) and vertebrate Cyclin B-Cdk1 (Stemmann, 2001), these results indicate that separase and sister chromatid separation are unlikely to be regulated exclusively by securin degradation (Leismann, 2003).

Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators

Drosophila Subito is a kinesin 6 family member and ortholog of mitotic kinesin-like protein (MKLP2) in mammalian cells. Based on the previously established requirement for Subito in meiotic spindle formation and for MKLP2 in cytokinesis, the function of Subito in mitosis was investigated. During metaphase, Subito localizes to microtubules at the center of the mitotic spindle, probably interpolar microtubules that originate at the poles and overlap in antiparallel orientation. Consistent with this localization pattern, subito mutants improperly assembled microtubules at metaphase, causing activation of the spindle assembly checkpoint and lagging chromosomes at anaphase. These results are the first demonstration of a kinesin 6 family member with a function in mitotic spindle assembly, possibly involving the interpolar microtubules. However, the role of Subito during mitotic anaphase resembles other kinesin 6 family members. Subito localizes to the spindle midzone at anaphase and is required for the localization of Polo, Incenp and Aurora B. Genetic evidence suggested that the effects of subito mutants are attenuated as a result of redundant mechanisms for spindle assembly and cytokinesis. For example, subito double mutants with ncd, polo, Aurora B or Incenp mutations are synthetic lethal with severe defects in microtubule organization (Cesario, 2006).

Subito is one of the two Drosophila kinesin 6 family members and probably the ortholog of MKLP2. In support of this classification, there are striking similarities between Subito and MKLP2. Both are required for localization of the passenger proteins to the midzone during anaphase. In addition, both Subito and MKLP2 interact with Polo kinase (or Plk1 in human) and are required for its localization to the midzone during anaphase. Plk1 phosphorylates MKLP2 at Ser528 and this phosphorylation promotes Plk1 binding to MKLP2. Plk1 phosphorylation negatively regulates MKLP2 microtubule bundling activity in vitro but is not required for the localization of MKLP2 to the midzone (Cesario, 2006).

Despite belonging to the same family, the two kinesin 6 family members probably have unique functions. The distinct phenotypes of sub and pav mutants indicate they have non-overlapping functions. Similarly, and despite having similar localization patterns, MKLP2 and MKLP1 have nonredundant functions in cytokinesis. MKLP2, but not MKLP1, has been shown to physically interact with Aurora B and Incenp. However, it has also been suggested that the MKLP2-dependent localization of Aurora B to the midzone is required for it to phosphorylate MKLP1. The importance of this phosphorylation on MKLP2 localization is unclear and the results are consistent with this indirect relationship between Subito and Pavarotti (Cesario, 2006).

It is possible that all members of the kinesin 6 group interact with antiparallel microtubules. Immunolocalization data is consistent with this because Subito is found on interpolar microtubules, which are characterized by an overlap of antiparallel microtubules in the midzone at mitotic anaphase in embryos, brains and testis. However, the localization of Subito to metaphase interpolar microtubules in the vicinity of the centromeres was a surprising finding. Although it is likely that Subito also associates with antiparallel microtubules at metaphase, the possibility that Subito interacts with the plus ends of the microtubules that interact with the kinetochores cannot be ruled. Surprisingly, a specific localization pattern of other kinesin 6 family members to metaphase microtubules has not been observed. This is not due to the absence of the appropriate substrate, since metaphase interpolar microtubules are present in most spindles. Either Subito is regulated differently than MKLP2, with an associated additional function in spindle assembly, or the localization pattern of MKLP2 at metaphase has not been informative with respect to its function (Cesario, 2006).

Since Subito is required to localize Polo, Aurora B and Incenp to the spindle midzone at anaphase, it is surprising that sub mutants are viable. Loss of MKLP2 causes cytokinesis defects. Drosophila mutants with strong defects in cytokinesis fall into the categories of male sterile, embryonic lethal (e.g. pav mutants) or pupal lethal. In fact, Incenp and polo mutants have embryonic lethal phenotypes that may be caused by a failure of cytokinesis. Unlike the loss of Incenp, Aurora B or Polo, sub mutants do not have any of these phenotypes and appear to complete cytokinesis most of the time in larval brains. In addition, because sub mutant males are fertile, and most mutants with strong defects in cytokinesis during spermatogenesis are male sterile, Subito does not appear to be essential for cytokinesis in the testis. A cytokinesis phenotype was also not evident in cultured Drosophila cells depleted of Subito by RNAi. These same studies did identify cytokinesis defects when Polo, Aurora B and Incenp were depleted. Thus, it seems likely that in some cell types, such as larval brains, the presence of Subito and the localization of the passenger proteins are not required for cytokinesis to occur (Cesario, 2006).

A close examination of sub mutants, however, revealed that anaphase did not proceed normally. In addition to the failure to accumulate Polo, Aurora B and Incenp in the midzone, the absence of Subito resulted in disorganized midzone microtubules at anaphase and a small increase in the frequency of polyploid cells. When the dosage of Incenp was reduced in sub mutants, the frequency of polyploidy was markedly increased. Therefore, Subito appears to have a similar function to MKLP2 in promoting cytokinesis, although there may be functional redundancy. Since the ability to complete cytokinesis in sub mutants depends on Incenp and Aurora B dosage, it is possible that unlocalized Incenp or Aurora B may promote cytokinesis. However, the observation that Incenp and Aurora B have a limited ability to spread along anaphase microtubules in the absence of Subito suggests an alternative; enough passenger protein activity may be present to promote cytokinesis. This model can account for the sensitivity of sub mutants to Incenp or Aurora B dosage because high levels of these proteins may be needed to promote cytokinesis if not concentrated in the midzone. It is also possible that anaphase may last longer and/or the microtubule organization improves with time in sub mutants. This would account for the relatively normal Fascetto localization and high success completing cytokinesis in sub mutants (Cesario, 2006).

Several lines of evidence suggest that Subito has a role in mitotic spindle assembly: (1) Subito initially localizes to interpolar microtubules at metaphase; (2) abnormally formed metaphase spindles were found in sub mutants more frequently than in the wild type; (3) sub mutant brains have an elevated mitotic index. Although the magnitude of the increase in sub mutants was lower than reported in some other mutants with spindle assembly defects, these mutants are lethal. Consistent with the conclusion that sub mutants have a defect in spindle assembly, the elevated mitotic index was dependent on BubR1, suggesting that the spindle assembly checkpoint is activated in the absence of Subito. (4) sub mutations exhibit synthetic lethality in combination with polo, Incenp and Aurora B mutations, and the cytological phenotype includes defects in spindle assembly and increased mitotic index. (5) RNAi of sub in Drosophila S2 cells results in frequent mitotic spindle abnormalities. These observations all point to a role for Subito in spindle assembly (Cesario, 2006).

The defects associated with sub mutants are less severe in mitotic cells than during female meiosis, possibly because of redundant spindle assembly pathways in mitosis. The double mutant studies suggest that the defects in spindle assembly or chromosome alignment in sub mutants are compensated for in two ways. First, the activation of the spindle assembly checkpoint allows defects in microtubule organization to be corrected. Second, the presence of redundant spindle assembly pathways allows microtubules to be assembled in the absence of sub. Double mutant studies support both of these mechanisms (Cesario, 2006).

The phenotype of the sub;polo16-1/+ double mutant is consistent with a redundant role for Subito in spindle assembly. Compared with the single mutants, the double mutants exhibit grossly abnormal metaphase and anaphase spindles. Similar to the results with sub, a role for Polo in spindle assembly has been shown through the analysis of polo hypomorphs that have an elevated mitotic index in larval brains, indicating that the spindle assembly checkpoint is activated. During metaphase, Polo localizes to the centromeres where it has a role in spindle formation but during anaphase it localizes to the spindle midzone where it has a role in cytokinesis. The very high mitotic index in the double mutants, however, suggests a more severe defect in spindle assembly than either single mutant. It is suggested that the abnormal spindle phenotype in sub/sub;polo/+ mutants arise from a combination of defects in two partially redundant spindle assembly pathways: improper assembly of kinetochore microtubules in polo/+ mutants and a reduction in assembling interpolar microtubules in sub mutants. Although polo mutants are recessive lethal, there is other evidence for dominant phenotypes, such as an elevated mitotic index in polo16-1/+ brains (Cesario, 2006).

The combination of these two spindle assembly defects in polo/+;sub/sub mutants might result in the severe spindle assembly phenotype and lethality in the double mutant. Similar conclusions apply for the interactions between sub and Incenp or Aurora B. Like Polo, the passenger proteins have an important role in spindle assembly. Indeed, the effects of all three mutants are strikingly similar, suggesting that Subito, Polo and the passenger proteins have important interactions during metaphase and anaphase. Interestingly, there is evidence of a direct interaction between Plk and Incenp in mammalian cells (Cesario, 2006).

Like its kinesin 6 homolog MKLP1, Subito is probably a plus-end-directed motor that crosslinks and slides interpolar antiparallel microtubules. The results suggest that this activity is important from metaphase through anaphase. Interestingly, the metaphase and anaphase interpolar microtubules have functional differences. Metaphase interpolar microtubules are observed in the absence of Subito whereas their anaphase counterparts depend on Subito. Another important difference is that Polo and the passenger proteins localize only to anaphase interpolar microtubules in the midzone. It has been suggested that the precocious appearance of anaphase-like interpolar microtubules is an important feature of acentrosomal meiotic spindle assembly in Drosophila oocytes. The passenger proteins Aurora B and Incenp localize to the interpolar microtubules at metaphase of meiosis I, rather than the centromeres, which is typical during mitotic metaphase. Therefore, the regulation of the passenger protein localization pattern is modified in oocytes to bypass the centromere localization that is characteristic of mitotic metaphase, resulting in precocious localization to interpolar microtubules (Cesario, 2006).

Despite these differences, the same biochemical activities of Subito could be used to organize both centrosomal mitotic and female acentrosomal meiotic spindles. In mitotic cells, kinetochores can initiate microtubule fiber formation, but these fibers are not directed toward either spindle pole. Failure to organize these fibers could result in disorganized and frayed spindles, as was observed in sub mutants. A function for Subito and interpolar microtubules could be to properly orient undirected kinetochore fibers. Interpolar microtubules could interact with and direct the organization of kinetochore microtubules via motors that bundle parallel microtubules. This mechanism has been proposed for organizing a bipolar spindle in the acentrosomal meiosis of Drosophila oocytes. With motor-driven sliding of antiparallel microtubules, this is an example of a centrosome-independent model for the spindle assembly pathway. This is consistent with previous conclusions that centrosome-independent mechanisms for spindle assembly are active in mitotic cells. Indeed, since bipolar spindles can form in the absence of centrosomes in neuroblasts and ganglion mother cells, it appears that centrosome-independent mechanisms for spindle assembly are active in the mitotic cells analyzed (Cesario, 2006).

Another possibility is that Subito functions as part of the centrosomal assembly pathway. For example, an array of interpolar microtubules could help channel centrosome microtubules towards the kinetochores. This activity could reduce the element of chance associated with making contacts between centrosome microtubules and kinetochores. It has also been proposed that centrosomal microtubules may capture the minus ends of kinetochore microtubules. An involvement of Subito in this process would be surprising, however, because the ability to bundle microtubules in parallel has not been described for a kinesin 6 family member. Nonetheless, if Subito was involved in the interactions of centrosomal and kinetochore microtubules, subsequent plus-end-directed movement would explain why Subito localization overlaps with centromeres. Whether or not these models are correct, the redundant nature of spindle assembly and function may explain why a role for kinesin 6 motor proteins in spindle assembly has not been described previously (Cesario, 2006).

Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase

Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. This study shows that Scant creates a hyperactive form of Greatwall (Gwl) with altered specificity in vitro, another protein kinase recently implicated in mitotic entry in Drosophila (Yu, 2004) and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo-Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. These results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle (Archambault, 2007).

Reversible protein phosphorylation and periodic protein destruction play major roles in regulating the eukaryotic cell division cycle. The major protein kinase that directs cell division is cyclin-dependent kinase 1 (Cdk1), the active component of Maturation Promoting Factor, first found to promote meiotic entry in amphibian oocytes. The cyclical inactivation of Cdk1 prior to mitotic exit is brought about in part through destruction of its cyclin partner. Two other protein kinase families, the Polo and Aurora families, are known to have critical functions in progression into and through M phase (mitosis and cytokinesis) and functionally interact with each other and also with Cdk1 to mediate their functions (Archambault, 2007).

Polo, originally discovered in Drosophila, exemplifies an evolutionarily conserved mitotic protein kinase. Polo, as well as its close orthologs, has been shown to function in multiple events essential for cell division. Polo was initially found to be essential for centrosome maturation and separation. It promotes recruitment of the γ-tubulin ring complex and phosphorylates Asp to facilitate nucleation of an increased number of dynamic microtubules on mitotic entry. At the G2/M transition, Polo (Polo-like kinase 1 in vertebrates) phosphorylates and activates the Cdc25 phosphatase responsible for removing inhibitory phosphates on Cdk1; this promotes mitotic entry. It also functions at the kinetochore-microtubule interface to monitor tension; the 3F3/2 phospho-epitope seen on kinetochores in the absence of tension is a consequence of Plk1/Plx1 kinase activity in vertebrates. Removal of cohesins from chromosomal arms in mitosis and meiosis also requires phosphorylation of cohesin subunits by Polo kinases. In Drosophila meiosis II, Polo phosphorylates and inactivates the centromeric cohesion protector protein Mei-S332. In addition, Polo is required for cytokinesis. The growing list of Polo kinase substrates is evidence of its role in multiple mitotic events (Archambault, 2007).

It is clear that protein kinases such as Cdk1 and Polo are only part of a large network of protein kinases that regulate cell cycle progression, many of which are as yet poorly characterized. A genome-wide survey found that up to one-third of the protein kinome of Drosophila has some cell cycle role. Depletion of the Gwl kinase from S2 cells by RNA interference (RNAi) led to a mitotic delay characterized by formation of long spindles and scattered chromosomes (Yu, 2004). Yu also found a mitotic role for Gwl kinase by characterizing missense hypomorphic mutants. Reduced gwl function results in mitotic defects in larval neuroblasts and tissue culture cells, including delay between late G2 and anaphase onset and chromosome condensation defects. Gwl has close homologs across eukaryotes and more distant homologs in budding and fission yeasts. Indeed, Yu recently reported a function for Xenopus Gwl in mitotic entry, as part of the Cdc2/Cdk1 activation loop in oocyte extracts. In that system, Xenopus Gwl is directly activated by cyclin B-Cdc2 and is in turn needed to promote full activation of cyclin B-Cdc2, although the direct target(s) mediating this action is (are) still unknown and indeed no substrates of Gwl are yet known. The primary sequence of Gwl shows that the regions most homologous to other kinases are split by a long intervening sequence of unknown function. Despite this recent progress, nothing is known about how activity of this crucial kinase is coordinated with the multiple events of cell cycle progression. Moreover, it is not known how Gwl contributes to the different types of mitotic and meiotic cell cycles of a metazoan (Archambault, 2007).

Elucidation of protein function may be aided through the generation of multiple mutant alleles that can reveal separate functions of individual proteins in multiple cellular processes. Drosophila offers the possibility of such studies and, moreover, allows the study of protein function in different types of cell cycle during its development. This principle was applied to study the gene defined by Scant (Scott of the Antarctic), a gain-of-function, dominant enhancer of maternal-effect embryonic defects of polo mutants. Syncytial embryos derived from females heterozygous for both Scant and polo develop mitotic defects in which a centrosome disassociates from one pole. This study reports that the Scant mutation is an allele of gwl that introduces a K97M amino-acid substitution into the Gwl protein; this results in a hyperactive kinase with altered specificity in vitro. These results indicate an antagonistic relationship between Gwl and Polo and suggest that their activity has to be coordinated for proper embryonic mitotic function. An allelic series of new gwl mutations reveals multiple functions for the Gwl kinase in both mitosis and female meiosis. These display somatic developmental defects accompanied by chromosome condensation and segregation defects in larval neuroblasts. gwl+ encodes two isoforms, only one of which is expressed during vitellogenesis. An allele that specifically prevents the expression of this isoform reveals requirements for Gwl in meiosis and in the maternal contribution to the egg (Archambault, 2007).

Suppression of Scant identifies Endos as a substrate of Greatwall Kinase and a negative regulator of Protein Phosphatase 2A in mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. This study shows that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. It was also shown that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. The involvement of Polo kinase in such a regulatory loop is discussed (Rangone, 2011).

This study identified endos mutations as heterozygous suppressors of the dominant mutant phenotype of polo1 gwlScant. This suggests that Greatwall and Endos promote the same mitotic pathway. In accord with this it was found that the consequences of loss of gwl and of endos function in mitosis are very similar. This study found that larval neuroblasts from homozygous endos mutants show poorly condensed chromosomes and anaphase bridging, a phenotype very similar to recessive gwl mutants. In cultured Drosophila cells, depletion of endos interferes with proper mitotic exit and allows cells to accumulate that have elongated spindles but have not undertaken chromatid separation or Cyclin B destruction. This is similar to the removal of Gwl from CSF Xenopus extracts; there, an unusual mitotic exit occurs in which cyclins remained undegraded but Cyclin-dependent kinase 1 (Cdk1) is inactivated by phosphorylation at Thr14 and Tyr15 (Rangone, 2011).

Three lines of genetic evidence indicate that Greatwall and Endos are required to down-regulate the function of B55/Twins-bound PP2A. Lowering the dosage of either the catalytic C subunit or the B55/Twins regulatory subunit of PP2A enhances the maternal dominant effect of polo1 gwlScant and this is suppressed by lowering the dosage of endos. Secondly, opposing roles for Endos and PP2A in regulating Polo kinase function are seen in the absence of the gwlScant mutation; the low fertility of twins/polo trans-heterozygous females is also dramatically suppressed by one mutant copy of endos. Thirdly, the Endos depletion phenotype in cultured cells is suppressed by simultaneous depletion of either the catalytic C subunit, the structural A subunit, or the B55/Twins regulatory subunit of PP2A but notably not by co-depletion of the three other regulatory B subunits. Together these interactions suggest that Greatwall activates Endos leading to the inhibition of PP2A-B55/Twins. This is in accord with recent studies in Xenopus showing that inhibition or depletion of PP2A-B55 from mitotic extracts rescues the inability of Gwl-depleted extracts to enter M phase and also with two recent biochemical studies that show that the Xenopus counterpart of Gwl kinase can phosphophorylate two related members of the cAMP-regulated phosphoprotein family, Ensa (the Endos counterpart) or Arpp19, to make these molecules highly effective inhibitors of PP2A. Endos is the unique cAMP-regulated phosphoprotein family member in Drosophila. Indeed, such is the degree of conservation that Drosophila Gwl kinase phosphorylates Endos only at Serine 68, a site essential for Endos function; this is the exact counterpart of the Serine 67 site in Xenopus. Studies in Drosophila, Xenopus and human cells indicate that PP2A is a major protein phosphatase acting to dephosphorylate Cdk1 substrates. Thus gwl or endos reduced-function mutants should have increased activity of PP2A and therefore accumulate dephosphorylated Cdk1 substrates. Failure of Cdk1 substrates to become maximally phosphorylated in spite of high levels of Cyclin B accumulation would account for the prolonged prometaphase-like state and the eventual development of elongated spindles without having appeared to activate the anaphase-promoting complex in these mutantsThis leads to a model in which Greatwall kinase is active in mitosis in order to convert Endos into an inhibitor of PP2A-Twins/B55, which is then inactived upon mitotic exit to permit the dephosphorylation of Cdk1 substrates by this phosphatase (Rangone, 2011).

The above simple model is, however, confounded by genetic interactions suggesting that the gain-of-function mutation gwlScant negatively regulates the function of the mitotic kinase Polo or one of its downstream targets. Such evidence comes largely from the search for suppressors of polo11 gwlScant that identified mutations in two broad categories: (1) those that decrease the effect of Gwl or its downstream targets as exemplified by endos mutations and reversion of gwlScant to recessive mutant alleles; (2) those that increase the activity of Polo kinase such as the polo+ duplications that were obtained. Moreover, the degree of sterility (adult progeny per female) and frequency of embryonic centrosome loss co-vary with strength of polo allele. polo1, a hypomorphic allele with sufficient residual Polo function to be homozygous viable, is slightly fertile heterozygous with Scant and its embryos are only moderately defective, whereas polo11, a lethal amorphic mutation, is completely sterile heterozygous with Scant and its embryos are much more defective. Furthermore, over-expressing Map205 (a known binding partner of Polo which sequesters the kinase on microtubules) in ovaries of polo11/+ mothers mimics Scant regarding the centrosome detachment phenotype, and more defective nuclei are seen when the transgene carries a mutation preventing Polo release (Rangone, 2011).

Together these results suggest that the specific defect in Scant polo-derived embryos, detachment of centrosomes from the nuclear envelope, is a consequence of the reduction of the level of functional Polo below a critical threshold. Indeed this is the only phenotype that could be attributed to the Scant allele of gwl and its sensitivity to the gene dosage of polo suggests that this function requires the highest level of Polo kinase activity in comparison to all of Polo's other roles. It is important to note that centrosome detachment is an interphase phenotype. It occurs after the centrosomes have separated, which in wild type is during telophase in anticipation of the next round of mitosis in the rapidly alternating S and M phases of the syncytial Drosophila embryo. In the normal mitotic cycle, Greatwall kinase would not be active at this stage. Thus the functional complex of PP2A and its B55/Twins regulatory subunit seems to be required to positively regulate Polo activity or a process controlled by Polo between the exit from one mitotic cycle and entry into the next. This accounts for the finding that mutations in the PP2A subunit genes, mts and twins, enhance sterility when transheterozygous with polo11, and that this sterility is in turn relieved by heterozygous endos mutations. Although it is possible that PP2A removes an inhibitory phosphorylation from Polo, this seems unlikely because no such phosphorylation has been identified to date. Thus the alternative is favored, that PP2A acts to stimulate a process promoted by Polo and a dephosphorylated partner. Indeed it is known that Polo interacts with phosphorylated partners after mitotic entry and with dephosphorylated partners from late anaphase onwards (Rangone, 2011).

Loss of Polo ameliorates APP-induced Alzheimer's disease-like symptoms in Drosophila

Loss of Polo ameliorates APP-induced Alzheimer's disease-like symptoms in Drosophila

The amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer's disease (AD). Despite extensive studies, little is known about the regulation of APP's functions in vivo. This study report that expression of human APP in Drosophila, in the same temporal-spatial pattern as its homolog APPL, induced morphological defects in wings and larval NMJ, larva and adult locomotion dysfunctions, male choice disorder and lifespan shortening. To identify additional genes that modulate APP functions, a genetic screen was performed, and it was found that loss of Polo, a key regulator of cell cycle, partially suppressed APP-induced morphological and behavioral defects in larval and adult stages. Finally, eye-specific expression of APP induced retina degeneration and cell cycle re-entry; both phenotypes were mildly ameliorated by loss of Polo. These results suggest Polo is an important in vivo regulator of the pathological functions of APP, and provide insight into the role of cell cycle re-entry in AD pathogenesis (Peng, 2015).

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polo: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 5 August 2016
 

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