polo

Protein Interactions

The polo gene product immunoprecipitated from extracts of single Drosophila embryos can phosphorylate casein in vitro. The kinase activity peaks cyclically at late anaphase/telophase. This contrasts with the cyclical activity of cyclin B-associated p34cdc2 kinase, which is maximal upon entry into mitosis during the rapid cycles of mitosis in the syncytium (Fenton, 1993).

Polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. Wild type Polo protein migrates as a tight doublet of 67 kDA. By phosphatase treatment, which also inactivates the kinase, this is converted to a single band. Putative Polo substrates include proteins of sizes 220 kDa, 85 kDa and 54 KDa. Monoclonal antibody to ß-tubulin precipitates the phosphorylated 54 kDa protein, together with an associated 85 kDa protein also phosphorylated by Polo. Moreover, Polo binds to an 85 kDa protein that is enriched in microtubule preparations (Tavares, 1996).

Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. The septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV-KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. Polo kinase associates with PAV-KLP, with which it shows an overlapping pattern of subcellular localization during the mitotic cycle. This distribution is disrupted in pav mutants. It is suggested that PAV-KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins. PAV-KLP may be responsible for transporting Polo kinase from one set of centrosome-associated substrates to a second set of substrates in the midzone of the spindle as mitosis progresses (Adams, 1998).

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants abnormal spindle (asp) and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which gamma-tubulin has an abnormal distribution. Moreover, the synergism of polo1;aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with gamma-tubulin to organize microtubule asters. Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase (do Carmo Avides, 2001).

These findings offer a route to understanding the role of phosphorylation in regulating the behaviour of the centrosomal microtubule-organizing centre. Thus, of the three microtubule-associated Polo substrates in Drosophila embryos, one was identified as beta-tubulin and another is now shown to be Asp. Asp becomes an MPM2 epitope when phosphorylated by Polo in vitro, suggesting that its MPM2 reactivity in wild-type but not asp mutant embryos is likely to be a direct consequence of a Polo-mediated phosphorylation event. The phosphorylation of Asp by Cdk1 and MAP kinases might also have physiological significance and, in fact, a genetic interaction has been reported between asp and rolled, a MAP kinase mutant, in Drosophila. Asp and Polo immunoprecipitate together and seem to be components of a complex that sediments at 25-35 S and has an estimated Mr of 3,000K by gel filtration. The size of the complex suggests that it either contains multimers of Asp and/or Polo or has additional components. It is not know where in the cell this complex might be located but it is known that both Asp and Polo localize to the centrosomes. Polo is likely to be associated with many proteins as it is dispersed both on sucrose gradients and following gel filtration. Polo-like kinases can associate with Cdc25 and the MKLP1/PAV-KLP motor protein. The position at which Cdc25 fractionates by these methods has not been checked, although it is known that PAV-KLP sediments at a position distinct from Asp, suggesting that it is part of a different complex (do Carmo Avides, 2001).

Although Polo, Cdk1 and MAP kinases all phosphorylate Asp in its amino-terminal region, this phosphorylation does not seem to be required for the binding of this segment of Asp to microtubules. Moreover, since Asp is found at spindle poles in polo mutants, it would seem not to require phosphorylation, at least by Polo kinase, for this aspect of its localization. Nevertheless, phosphorylated Asp protein is required for cytoplasmic extracts from polo¹-derived embryos to be able to restore microtubule-nucleating activity to preparations of salt-stripped centrosomes. This provides an explanation of why mutations in both polo and asp result in broad spindle poles at which the gamma-tubulin ring complex is not focused, and why polo:asp double mutants have a synergistic interaction. It is proposed that, upon association of Asp and Polo with the centrosome at the onset of prophase, Polo phosphorylates Asp and thereby stimulates its activity to organize microtubules into asters that will later form the spindle poles (do Carmo Avides, 2001).

Polo kinase regulates the Drosophila centromere cohesion protein MEI-S332

Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein Mei-s332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. Mei-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. A mechanism has been elucidated for controlling release of Mei-S332 from centromeres via phosphorylation by POLO kinase. Polo antagonizes Mei-S332 cohesive function and full Polo activity is needed to remove Mei-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. Polo phosphorylates Mei-S332 in vitro; Polo and Mei-S332 bind each other, and mutation of Polo binding sites prevents Mei-S332 dissociation from centromeres (Clarke, 2005).

The results demonstrate that Polo regulates Mei-S332 localization and aspects of its function. polo mutants dominantly suppress the mei-S332⁸ nondisjunction phenotype and wild-type Mei-S332 is retained at centromeres past the metaphase II/anaphase II transition in these polo mutants. Mei-S332 appears to be phosphorylated in mitosis at the metaphase/anaphase transition, and in polo mutants, Mei-S332 persists on centromeres into interphase, consistent with phosphorylation being a signal for Mei-S332 to delocalize. Polo kinase binds to Mei-S332, and this is partly dependent on two PBD binding site motifs. Furthermore, in vitro phosphorylation of Mei-S332 is dependent on at least one motif and dependent on Polo. These two PBD binding site motifs in vivo are likely required for chromosomal dissociation of Mei-S332, similar to the effects observed in polo mutants. Together, these data point to Polo as a key regulator of Mei-S332 centromere localization in both mitosis and meiosis (Clarke, 2005).

Polo function is required for Mei-S332 delocalization from centromeres in mitosis and meiosis, but the ability of polo mutants to dominantly suppress mei-S332 mutants additionally shows that Polo antagonizes Mei-S332 function. This is important because the results indicate that cohesion can be released even if Mei-S332 remains localized in polo mutants. Mei-S332 remains on centromeres after the metaphase II/anaphase II transition in polo/+ mutants, yet reasonably normal disjunction of chromosomes occurs during meiosis II in these heterozygotes. In mitosis, Mei-S332 can remain on the centromeres of the fourth chromosomes in polo mutants even when sister chromatid cohesion is released and they segregate to the poles. Thus, Polo phosphorylation is necessary for delocalization of Mei-S332, but there must exist a mechanism to inactivate Mei-S332 to release cohesion that is independent of Mei-S332 dissociation. This pathway is not entirely dependent on Polo, although Polo may contribute (Clarke, 2005).

The idea that Mei-S332 can remain localized to centromeres without cohesion between sister chromatids is supported by several examples. In double parked mutants, Mei-S332 localizes to unreplicated, single chromatids on which cohesion has never been established (Lee, 2004). This shows that the presence of sister chromatid cohesion is not a prerequisite for Mei-S332 localization to centromeres. Similarly, Mei-S332 localizes to single sister chromatids in ord mutants, in which sister chromatids separate prematurely early in meiosis I. Finally, Sgo1, a yeast homolog of Mei-S332, can localize to centromeres in early anaphase II when the 3′ UTR of Sgo1 is disrupted, yet no interference of the release of sister chromatid cohesion is observed (Rabitsch, 2004). This result suggests that Sgo1 can promote sister chromatid cohesion in meiosis and can subsequently be inactivated yet remain at centromeres (Clarke, 2005).

It is proposed that Mei-S332 centromere localization is regulated by phosphorylation. In this model, the assembly of Mei-S332 onto centromeres in prometaphase I is controlled by the action of an unknown phosphatase. Mei-S332 remains localized to centromeres until the metaphase II/anaphase II transition when Polo kinase binds to Mei-S332, via the phosphorylated T331 PBD binding site, and phosphorylates Mei-S332 elsewhere, initiating Mei-S332 dissociation from centromeres. The data suggest that both S234 and T331 contribute to Polo binding and to Mei-S332 centromere dissociation, but in vitro T331 plays the predominant role in Plx1-dependent phosphorylation. Further, it is suggested that Polo functions to antagonize Mei-S332 activity in meiosis, thereby affecting the release of sister chromatid cohesion. This may be either through phosphorylation of Mei-S332 or by affecting another component of sister chromatid segregation. Based on these results, it cannot be distinguished whether phosphorylation of Mei-S332 by Polo antagonizes Mei-S332 activity directly (Clarke, 2005).

It is proposed that Polo directly phosphorylates Mei-S332 because the proteins can bind each other. Importantly, this binding is reduced by disruption of the PBD binding site motifs, and these mutations abolish Plx1-dependent phosphorylation of Mei-S332 and prevent Mei-S332 from dissociating from centromeres in S2 cells. PBD binding sites are required to be phosphorylated in order for Polo to bind to its substrates. Given that Plx1-dependent phosphorylation of Mei-S332 was disrupted by mutating this site, it would seem that T331 and S234 need to be phosphorylated prior to Polo binding to Mei-S332, and then subsequent unknown sites on Mei-S332 can be phosphorylated by Polo kinase, thereby dissociating Mei-S332 from centromeres. In support of this idea, it has recently been proposed that once Polo binds to a substrate via an interaction with a PBD binding site, its kinase activity toward that substrate is stimulated (Clarke, 2005).

Which kinase is responsible for phosphorylating S234/T331 initially? Candidate kinases are a cyclin-dependent kinase, with specificity for sites with a proline in the +1 position as at the T331 and S234 sites, or a kinase such as Aurora B, which is localized to centromeres at the metaphase/anaphase transition in mitosis. The control of this precise phosphorylation event would effectively prevent Polo from phosphorylating Mei-S332 and releasing it from centromeres until the appropriate time. Correlating with this possibility, during both mitosis and meiosis, Polo is poised at centromeres yet does not act to remove Mei-S332 until the proper time. In mitosis, Mei-S332 becomes localized to centromeres in prometaphase, and Polo kinase is also localized to centromeres at this time. In meiosis, from metaphase I to metaphase II Polo localizes to centrosomes and centromeres, yet Mei-S332 is not released until metaphase II/anaphase II (Clarke, 2005).

Mei-S332 is the founding member of a family of proteins required for maintaining centromere cohesion between sister chromatids. This study has define Polo kinase as crucial for delocalization of Mei-S332. It was also shown that Polo antagonizes Mei-S332 activity. These results strongly indicate that Polo directly phosphorylates Mei-S332 and this leads to delocalization. It will be interesting to identify the anchor for Mei-S332 centromere binding and to decipher how phosphorylation of Mei-S332 affects this interaction. The results additionally uncover a mechanism distinct from delocalization to inactivate Mei-S332 (Clarke, 2005).

Interaction between Polo and BicD proteins links oocyte determination and meiosis control in Drosophila

Meiosis is a specialized cell cycle limited to the gametes in Metazoa. In Drosophila, oocyte determination and meiosis control are interdependent processes, and BicD appears to play a key role in both. However, the exact mechanism of how BicD-dependent polarized transport could influence meiosis and vice versa remains an open question. This article reports that the cell cycle regulatory kinase Polo binds to BicD protein during oogenesis. Polo is expressed in all cells during cyst formation before specifically localizing to the oocyte. This is the earliest known example of asymmetric localization of a cell-cycle regulator in this process. This localization is dependent on BicD and the Dynein complex. Loss- and gain-of-function experiments showed that Polo has two independent functions. On the one hand, it acts as a trigger for meiosis. On the other, it is independently required, in a cell-autonomous manner, for the activation of BicD-dependent transport. Moreover, Polo overexpression can rescue a hypomorphic mutation of BicD by restoring its localization and its function, suggesting that the requirement for Polo in polarized transport acts through regulation of BicD. Taken together, these data indicate the existence of a positive feedback loop between BicD and Polo, and it is proposed that this loop represents a functional link between oocyte specification and the control of meiosis (Mirouse, 2006).

This paper describes the localization of the Polo protein and its genetic control in the Drosophila germline during early oogenesis. Polo has a peculiar subcellular localization in cytoplasmic dots that do not correspond to any well-known structures of germline cysts or to microtubule minus-ends where BicD accumulates. Polo has previously been described as colocalizing with several subcellular structures depending on cell cycle phase, but none of these corresponds to the localization observed in this study. Similar cytoplasmic dots were observed in the primordial germline cells of the Drosophila embryo as soon as they were formed, suggesting that this unusual localization could be a specific feature of the germline (Mirouse, 2006).

From region 2a onward, Polo dots are present mostly in the cells containing SCs. This is the first report of a cell-cycle regulator whose localization is spatially and temporally correlated with meiotic progression during early oogenesis. Moreover this correlation is still conserved in mutants that affect polarized transport and the restriction and maintenance of meiosis. This indicates that Polo localization is dependent on polarized transport. One possibility is that Polo itself is directly transported to the oocyte. This hypothesis is reinforced by the physical interaction between BicD and Polo proteins, according to the proposed function of BicD as adapter for Dynein cargos. However, the BicD-dependent localization of Polo is not sufficient to explain its expression profile. Polo is strongly expressed in region 1 of the germarium, and the overall amount of the protein in the cyst progressively decreases, becoming undetectable after stage 2. This degradation seems to be compensated in meiotic cells and then in the oocyte by the polarized transport. The progressive degradation of Polo is also observed in egl and BicD null mutants. Degradation in association with a complete absence of Polo transport may explain why all the cells of a cyst enter into meiosis in these mutants (all the cells contain the same amount of Polo), and then exit meiosis simultaneously (none of the cells preferentially accumulates enough Polo). Alternatively, rather than by direct transport of Polo to the oocyte, its asymmetric distribution in the cyst could be due to a differential control of its stability between nurse cells and oocyte under the control of the BicD-dependent polarized transport (Mirouse, 2006).

BicD and egl null mutants showed a very similar phenotype, in which all 16 cells of a cyst first enter into meiosis but subsequently lose the synaptonemal complexes (SCs). This phenotype cannot be compared with null mutants of the dhc, since Dynein is required at earlier steps of cyst formation. The human homolog of BicD interacts directly with Dynamitin, and this interaction is thought to mediate the interaction of BicD with the Dynein complex. In contrast to BicD, Dynamitin is not involved in the initial restriction of meiosis, showing that the interaction of BicD with Dynamitin, and thus probably Dynein, is not required for the initial restriction of meiosis. In a similar way, LC8 (cut up) null mutants or egl mutants that specifically block the interaction between Egl and LC8 do not interfere with the initiation of meiosis in only four cells. Transport of the BicD protein between the cyst cells is apparently not required for this first step, sinc the BicDPA66 allele or drug-induced microtubule depolymerization does not affect this initial restriction, although BicD is diffuse throughout the entire cyst. Finally, a null mutant for the plakin shot, which has been proposed to be an essential upstream component of the Dynein function in centrosome migration, exhibits variable meiotic phenotypes but allows a normal initial restriction of meiosis to four cells. These data are consistent with a function of BicD and Egl independent of Dynein in the initial restriction of meiosis (Mirouse, 2006).

Polo is involved in many crucial steps of the cell cycle, including the G2/M transition of mitosis and meiosis processes. This study shows that hypomorphic polo alleles lead to a delay in meiotic entry and that Polo overexpression can trigger meiosis in more than four cells per cyst in region 2a. These phenotypes could be related to the function of Polo in the G2/M transition. In vertebrates, Polo is an activator of the String/CDC25 phosphatase, and it has also been proposed that Polo can repress the kinases Myt1 and Wee1. String is the main activator of the cyclinB/CDC2 complex, the activity of which triggers the G2/M transition, whereas Myt1 and Wee are repressors of this complex. However, the role of the cyclin B and CDC25 in meiosis in Drosophila oogenesis is not yet well understood because, for example, CDC25 seems to act as a negative regulator of meiotic oocyte cell fate. Further investigations will be needed to determine how Polo triggers meiotic entry during early oogenesis (Mirouse, 2006).

This study has shown that in mutants with partial loss of polo function, SCs start to disassemble in region 3 but are well formed again in stage 2/3 before disappearing in the following stages. One possible hypothesis to explain how meiosis is finally properly maintained in polo hypomorphic mutants is that the repression of cyclin E by Dacapo during stage 2/3 represses endoreplication, and thus allows meiotic progression. This is consistent with the finding that the specific localization of Dacapo to the oocyte and its requirement for meiosis maintenance begins only in region 3. Moreover, null mutations of dacapo do not lead to a fully penetrant 16-nurse-cell phenotype, confirming the existence of a partially redundant control. Therefore, it is proposed that the balance in favor of meiosis is initially due to the localized activation of meiosis by Polo, and later to the localized inhibition of endoreplication by Dacapo, and that both mechanisms partially overlap (Mirouse, 2006).

It was also observed that Polo is required for the normal restriction of meiosis. Moreover, the defects in the restriction of meiosis caused by both loss and gain of polo function are correlated with defects in oocyte determination. Meiosis restriction and oocyte specification both depend on the Dynein complex and the BicD polarized transport system. Thus, it is assumed that these Polo phenotypes indicate that Polo is involved in polarized transport. This role may be indirect and thus reveals the influence of meiosis and cell-cycle control on oocyte differentiation. Such influence has been observed in situations where there is activation of the meiotic checkpoint due to a failure in DNA double-stand break repair. However, at least two results argue for a direct role of Polo in polarized transport, independently of its meiotic function. First, in mosaic germline cysts, nonmeiotic cells mutant for polo retain BicD protein. Thus, this phenotype cannot be due to the activation of the meiotic checkpoint. This strongly suggests that Polo is required in each cell of the cyst to initiate BicD-dependent transport to the presumptive oocyte. Second, the overexpression of Polo is able to restore the localization and therefore the function of BicDPA66 protein. Interestingly, this mutant allele is due to a single amino acid substitution (A40V) that leads to a hypophosphorylation of BicD, and genetic evidence indicates that this phosphorylation is crucial for BicD function. Polo overexpression might restore a functional level of BicDPA66 phosphorylation. Therefore, even if no significant changes were observed in the gel mobility of BicD in polo hypomorph mutants, it is tempting to propose that the function of Polo in the polarized transport could be to activate, directly or indirectly, BicD by phosphorylation (Mirouse, 2006).

Taken together, these results lead to a model that can explain a reciprocal requirement between the control of meiosis and oocyte specification. This model is based on four major points: (1) BicD is required for the Dynein-dependent polarized transport of oocyte determinants; (2) BicD is also required for the progressive localization of Polo to the oocyte; (3) Polo appears to trigger meiosis in the germarium; (4) Polo is required to activate the BicD and Dynein-dependent polarized transport. These findings suggest the existence of a positive feedback loop between Polo and BicD proteins, and therefore between oocyte specification and meiosis (Mirouse, 2006).

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. This study shows that Polo acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. These results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb (Wang, 2007).

Asymmetric localization of Numb depends on its adaptor protein Pon. The Pon localization domain (Pon-LD) is located at the carboxy terminus of the protein. The Ser 611 (S611) residue in this domain matches the consensus phosphorylation site for Polo. Because the localization of Pon is cell-cycle-dependent, tests were perfomred to see whether Polo can directly phosphorylate Pon. Pon-LD, but not Pon(S611A)-LD, in which S611 was mutated to Ala, was readily phosphorylated by mammalian Polo-like kinase 1 (Plk1) in vitro, demonstrating that Pon S611 is a Polo phosphorylation site (Wang, 2007).

To test whether Pon S611 is normally phosphorylated in vivo, an antibody was generated against S611-phosphorylated (p-S611) Pon. The specificity of this antibody was shown by its ability to recognize a glutathione S-transferase-Pon-LD fusion protein (GST-Pon-LD) only after the fusion protein was pre-phosphorylated by Plk1. It did not recognize GST-Pon(S611A)-LD in the same assay. Next, larval brain extracts prepared from wild type as well as heterozygotes [polo⁹(+/-) and polo¹⁰(+/-)], and homozygotes [polo⁹(-/-) and polo¹⁰(-/-)] of two different polo loss-of-function alleles were analysed by western blotting using this p-S611-specific antibody. p-S611-positive Pon was clearly detected in wild-type animals and in heterozygotes, but was barely detectable in homozygous mutant animals, demonstrating that Pon is phosphorylated at S611 in vivo in a Polo-dependent fashion (Wang, 2007).

Immunohistochemistry was used to verify S611 phosphorylation in vivo and to visualize phospho-Pon localization. p-S611-positive endogenous Pon was detected in wild-type larval brains as a crescent in metaphase neuroblasts, and was segregated to the ganglion mother cell (GMC, the daughter committed to the differentiation pathway) after division. In the polo⁹ mutant, however, p-S611-positive Pon was undetectable. The p-S611 antibody also reacted with Pon-LD, but not with Pon(S611A)-LD, in transgenic larval brain (Wang, 2007).

To test for a functional role of S611 phosphorylation, S611 was mutated to a non-phosphorylatable Ala residue (S611A) or to a phospho-mimetic Asp residue (S611D). Wild-type and phospho-mutant Pon-LD were fused to green fluorescent protein (GFP) and expressed in embryonic neuroblasts. Both GFP-Pon-LD and GFP-Pon(S611D)-LD showed the expected basal localization. In contrast, the localization of GFP-Pon(S611A)-LD was defective. At prometaphase and metaphase, it showed either uniformly cortical (80%) or basally enriched but apically detectable cortical (20%) distribution. At anaphase and telophase, however, it formed basal crescents in most neuroblasts. This 'telophase rescue' seemed to be partially mediated by endogenous Pon, because less rescue was observed in pon mutant neuroblasts, with 58 neuroblasts mis-segregating GFP-Pon(S611A)-LD at late anaphase/telophase. It is unlikely that the S611A mutation affects Pon localization by altering the charge or global conformation and folding of the protein, because mutation of an adjacent Ser residue (S616) or triple mutations at three potential atypical protein kinase C (aPKC) phosphorylation sites (S644A/S648A/S652A) had no effect on Pon-LD localization, suggesting that S611 represents a unique regulatory point in Pon localization (Wang, 2007).

To assess whether Polo has a role in neuroblast self-renewal and/or asymmetric division, central brain neuroblast numbers were quantified in two strong hypomorphic alleles, polo⁹ and polo¹⁰, using Deadpan (Dpn) and Miranda (Mira) as neuroblast markers. Wild-type larval central brains averaged 37 neuroblasts 24 h after larval hatching (ALH) and 10 neuroblasts 96 h ALH. polo⁹ larval central brains averaged 36 neuroblasts 24 h ALH. However, the number increased dramatically to 254 96 h ALH. Consistent with this increase in neuroblast number, the numbers of BrdU-labelled, CycE-positive or phospho-histone-H3-positive proliferating cells were also increased in polo⁹ mutant brains compared to wild type. Concomitantly, a dramatic reduction of differentiated cells expressing neuronal markers, Embryonic Lethal Abnormal Vision (Elav) or nuclear Prospero (Pros), was observed in polo⁹ mutant brains. A similar neuroblast overproliferation phenotype was observed in polo¹⁰ and in trans-heterozygotes between polo⁹ and a deficiency that deletes polo. A Polo-GFP genomic construct fully rescued this polo mutant phenotype, verifying that these defects are caused by polo loss-of-function. Excess self-renewal and proliferation at the expense of neuronal differentiation was also observed in MARCM (mosaic analysis with a repressible cell marker) clones derived from single polo⁹ mutant neuroblasts. These results indicate that Polo behaves like a tumour suppressor to inhibit neuroblast self-renewal and to promote differentiation. polo mutant GMCs may revert to neuroblast-like cells, as has been shown for brat (brain tumor) mutants (Wang, 2007).

The physiological role of Polo in regulating Pon localization and function was analyzed. Most larval neuroblasts were found at metaphase in polo⁹ mutant brains, and both Pon and Numb were uniformly distributed. In late anaphase/telophase neuroblasts, Pon and Numb were mis-segregated to both daughter cells. Defective Pon and Numb localization in the polo mutant is unlikely to be a secondary consequence of cell cycle arrest, because arrest of wild-type neuroblasts at metaphase with microtubule-depolymerizing drugs actually increased the number of cells possessing a Numb crescent (Wang, 2007).

To test whether Polo is specifically required for Pon/Numb localization, other apical and basal proteins were analyzed. In polo⁹ mutant neuroblasts, the basal localization of Brat and Pros was relatively normal. Moreover, double-labelling of the same mutant neuroblast showed that the localization of Mira, the adaptor protein for Pros and Brat, was normal, whereas Pon localization was abnormal. Introduction of a Polo-GFP transgene into polo⁹ effectively rescued the Pon localization and cell-cycle defects. Apical proteins such as Insc, Baz, Pins and Discs Large 1 (Dlg1) were localized normally in polo⁹ mutant neuroblasts. The only apical protein showing abnormal localization was aPKC, which became distributed uniformly on the cortex and showed cytoplasmic localization. During telophase, aPKC could be mis-segregated into both daughter cells (Wang, 2007).

Polo is localized to centrosomes and is required for centrosome organization and separation. Whether Polo has a role in orienting neuroblast mitotic spindles was tested. The tight coupling of spindle orientation with crescent formation seen in wild-type neuroblasts was disrupted in polo⁹ metaphase neuroblasts with two centrosomes. Therefore, Polo kinase is also required for coupling mitotic spindle orientation with cortical protein asymmetry. This spindle phenotype was fully rescued by the Polo-GFP transgene (Wang, 2007).

Next the role of Pon phosphorylation in mediating Numb localization was probed. Full-length Pon containing the S611A or S611D mutation was used to assess the effects of S611 phosphorylation. In pon mutant neuroblasts, Numb localization was defective. Introducing wild-type Pon restored Numb asymmetric localization at metaphase and later stages. Most pon mutant neuroblasts expressing Pon(S611D) also showed rescue. In contrast, pon mutant neuroblasts expressing Pon(S611A) showed largely abnormal Numb localization. Polo-mediated phosphorylation is therefore important for Pon to localize Numb. The function of Pon in neuroblast self-renewal was tested by generating pon MARCM clones. Interestingly, compared to wild-type clones, ectopic neuroblast self-renewal was observed in pon mutant clones (Wang, 2007).

This study has shown that polo mutations affect Numb and aPKC localization as well as spindle orientation -- processes known to affect neuroblast self-renewal to various degrees. Strikingly, overexpression of either Numb-GFP or Numb effectively suppressed the ectopic neuroblast self-renewal phenotype seen in the polo⁹ mutant. This effect was not caused by increased neuroblast apoptosis, and overexpression of Numb-GFP or Numb in a wild-type background did not affect the neuroblast number. These results indicate that Numb is a principal player downstream of Polo in regulating neuroblast self-renewal. Numb overexpression did not rescue the aPKC mislocalization and spindle misorientation phenotypes of polo mutants. These defects could also contribute to the neuroblast overgrowth phenotype of polo mutants, but their effects might have been masked by Numb overexpression. Consistent with this, introduction of Pon(S611D) into a polo mutant neuroblast did not significantly rescue the neuroblast overgrowth phenotype, despite the partial restoration of Numb localization. Because aPKC localization and spindle orientation defects were not rescued by Pon(S611D), these defects may account for the inability of Pon(S611D) to rescue the overgrowth phenotype of polo. aPKC has been shown to phosphorylate Numb. Delocalized aPKC at the basal side may be sufficient to inactivate endogenous Numb, but not overexpressed Numb, owing to titration by the overexpressed protein (Wang, 2007).

Numb was previously shown to inhibit neuroblast self-renewal by antagonizing Notch signalling. Reducing Notch significantly suppressed the neuroblast overgrowth phenotype of the polo⁹ mutant. Reducing Notch in a wild-type background also led to a partial loss of neuroblasts, consistent with Notch being required for progenitor self-renewal. It is envisioned that in polo or pon mutants, owing to the symmetric distribution of Numb, the GMCs receive insufficient Numb to inhibit Notch, thereby causing them to adopt a neuroblast-like fate. To test further the importance of Numb asymmetric localization in neuroblast self-renewal versus differentiation, the numb^S52F mutation, which apparently affects Numb asymmetric localization but not its stability or activity, was tested. In numb^S52F neuroblast clones, ectopic neuroblast self-renewal similar to that seen in polo or pon clones was observed. Thus, loss of Numb asymmetric localization is sufficient to cause neuroblast overgrowth (Wang, 2007).

These results indicate that Polo controls the self-renewal versus differentiation decision of neural progenitors by regulating the localization and activity of Numb and the orientation of mitotic spindles. Polo regulates the localization of Numb by means of Pon. Although immunofluorescence shows that Polo is primarily localized to the centrosomes, a cytosolic pool of Polo probably exists because Polo exhibits dynamic in vivo association with the mitotic apparatus and many non-centrosomal Polo substrates have been identified. Cytosolic localization of the centrosomal kinase Aurora-A has been demonstrated. How Polo regulates the localization of aPKC, the activity of Numb and the orientation of mitotic spindles awaits further investigation. In addition to the Numb/Notch pathway, other factors such as Pros and Brat are necessary for preventing GMCs from undergoing self-renewing divisions. Because these factors are segregated normally in polo neuroblasts, it seems that they are not sufficient to prevent progenitor self-renewal or that activation of Notch is able to override their effects. Intriguingly, some Plks behave as tumour suppressors in mammals, and loss of Numb has also been implicated in the hyperactivation of Notch signalling in breast cancer cells. These results and future studies in models like Drosophila will provide mechanistic insights into these observed tumour-suppressor roles of Polo and Numb (Wang, 2007).

The inhibition of polo kinase by matrimony maintains G2 arrest in the meiotic cell cycle

Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. These data suggest a model in which the eventual activation of Cdc25 (Drosophila Twine) by an excess of Polo at stage 13 triggers NEB and entry into prometaphase (Xiang, 2007).

The mechanism of the lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB) (which is a characterization of many female meiotic systems) has remained a mystery. One can imagine that both the maintenance and the termination of this arrest might involve either or both of two mechanisms: the transcriptional or translational repression of a protein that induces NEB, and thus meiotic entry, or the presence of an inhibitory protein that precludes entry into the first meiotic division. Because Drosophila females exhibit a prolonged G2 arrest and are amenable to both genetic and cytological analyses, they provide an ideal system in which to study this problem (Xiang, 2007).

The ovaries of Drosophila females are composed of a bundle of ovarioles, each of which contains a number of oocytes arranged in order of their developmental stages. For the purposes of this study, the process of oogenesis may be said to consist of three separate sets of divisions: the initial stem cell divisions, which create primary cystoblasts; four incomplete cystoblast divisions, which create a 16-cell cyst that contains the oocyte; and the two meiotic divisions. Although a great deal is known regarding the mechanisms that control cystoblast divisions and oocyte differentiation, relatively little is known about the mechanisms by which the progression of meiosis is controlled (Xiang, 2007).

As is the case in many meiotic systems, female meiosis in Drosophila involves preprogrammed developmental pauses. The two most prominent pauses during Drosophila meiosis are an arrest that separates the end of pachytene at stages 5-6 from NEB at stage 13, and a second pause that begins with metaphase I arrest at stage 14 and continues until the egg passes through the oviduct. It is the release of this second preprogrammed arrest event that initiates anaphase I and allows the completion of meiosis I followed by meiosis II. The end of meiotic prophase by dissolution of the synaptonemal complex (SC) at stages 5-6 is separated from the beginning of the meiotic divisions, which is defined by NEB at stage 13, by approximately 40 h to allow for oocyte growth (Xiang, 2007).

Elucidating the mechanisms that arrest meiotic progression at the end of prophase, but then allow onset of NEB and the initiation of meiotic spindle formation some 40 h later, was of great interest. One intriguing possibility is that during this period of meiotic arrest, the oocyte actively blocks the function of cell cycle regulatory proteins such as cyclin dependent kinase 1 (Cdk1), the phosphatase Cdc25, and Polo kinase (Polo), all of which promote meiotic progression just as they do during mitotic growth. Recently, Polo was shown to be expressed in the germarium and required for the proper entry of Drosophila oocytes into meiotic prophase, as defined by the assembly of the SC. Decreased levels of Polo resulted in delayed entry into meiotic prophase, whereas overexpression of Polo caused a dramatic increase in the number of cystocyte cells entering meiotic prophase, indicating that Polo is involved both in the initiation of SC formation and in the restriction of meiosis to the oocyte. How then is Polo, which is known to play multiple roles in promoting meiotic and mitotic progression, prevented from compelling the differentiated oocyte to proceed further into meiosis? One component of this regulation may well lie in the fact that Polo is not expressed during much of oogenesis. Polo is clearly visible in the germarium but is then absent until stage 11, when it begins to accumulate to high levels in the oocyte. A second component of Polo regulation is mediated by binding to the protein product of the matrimony (mtrm) gene, which occurs from stage 11 until the onset of NEB at stage 13. This binding serves to inhibit Polo in the early stages of its expression, and thus prevents precocious nuclear envelope breakdown (Xiang, 2007).

The mtrm gene was first identified in a deficiency screen for loci that were required in two doses for faithful meiotic chromosome segregation (Harris, 2003). mtrm/+ heterozygotes display a significant defect in achiasmate segregation (the meiotic process that ensures the segregation of those homologs that, for various reasons, fail to undergo crossingover). As a result of this defect, mtrm/+ heterozygotes exhibit high levels of achiasmate nondisjunction. As homozygotes, mtrm mutants are fully viable but exhibit complete female sterility. This study shows that the Mtrm protein prevents precocious NEB. Indeed, the effects of reducing the dose of mtrm on meiotic progression and on chromosome segregation are easily explained as the consequence of precocious NEB at stages 11 or 12, and can be suppressed by simultaneously reducing the copy number of polo⁺. In addition, the effects of heterozygosity for loss-of-function alleles of mtrm can be phenocopied by increasing the copy number of polo⁺. These genetic interactions suggest that Mtrm negatively regulates Polo in vivo (Xiang, 2007).

Interestingly, Mtrm was shown to interact physically with Polo by a global yeast two-hybrid study. This yeast two-hybrid finding reflects a true physical interaction in vivo by both coimmunoprecipitation studies and by multidimensional protein identification technology (MudPIT) mass spectrometry experiments, which indicate that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Moreover, ablating one of the two putative Polo binding sites on Mtrm by mutation prevents the physical interaction between Polo and Mtrm and renders the mutated Mtrm protein functionless. This experiment, along with genetic interaction studies, provides compelling evidence that the function of the binding of Mtrm to Polo is to inhibit Polo, and not vice versa (Xiang, 2007).

The analysis of mtrm mutants allows us examination of the effects of premature Polo function during oogenesis. The evidence shows that in the absence of Mtrm, newly synthesized Polo is capable of inducing NEB from stage 11 onward. As a result of this precocious NEB, chromosomes are not properly compacted into a mature karyosome and they are released prematurely onto the meiotic spindle. In many cases, the centromeres of achiasmate bivalents subsequently fail to co-orient (Xiang, 2007).

The mtrm gene was first identified as a dosage-sensitive meiotic locus; heterozygosity for a loss-of-function allele of mtrm specifically induced the failed segregation of achiasmate homologs (Harris, 2003). The mtrm gene encodes a 217-amino acid protein with two Polo-Box Domain (PBD) binding sites (STP and SSP) and a C-terminal SAM/Pointed domain. The studies reported in this paper rely primarily on a null allele of mtrm (mtrm¹²⁶), which removes 80 bp of upstream sequence and the sequences encoding the first 41 amino acids of the Mtrm protein (Xiang, 2007).

Western blot analysis using an antibody to Mtrm reveals that Mtrm can be detected only in ovaries. This is consistent with a previous report by Arbeitman (2002), which showed that the expression profile of the mtrm gene product was strictly maternal and that its expression was reduced greater than 10-fold over 0-6.5 h of embryonic development. The specificity of this antibody is demonstrated by the fact that no signal was detected by either Western blotting or by immunofluorescence of ovarioles homozygous for the mtrm¹²⁶ mutant. Immunofluorescence studies using the same antibody reveal that Mtrm is expressed as a diffuse nuclear protein in the oocytes and nurse cells beginning at stage 4-5. The Mtrm signal was not restricted to the karyosome itself; but rather Mtrm seems to fill the space in the entire nucleus. Although Mtrm is restricted to the nucleus until approximately stage 10, it localizes throughout the oocyte in later stages. Mtrm brightly stains both the oocyte nucleus and cytoplasm between stage 11 and stage 12, but staining is greatly reduced at stage 13, the stage at which NEB occurs (Xiang, 2007).

The data presented in this study that Mtrm serves to inactivate newly synthesized Polo during the period of meiotic progression that precedes NEB. An excess of functional (unbound) Polo, produced by reducing the amount of available Mtrm, causes the early onset of NEB. This early entry into prometaphase releases an immature karyosome into the cytoplasm, which then fails to properly align the centromeres of achiasmate chromosomes on the prometaphase spindle. These observations raise a number of questions ranging from the role of Polo in mediating the G2/M transition in oogenesis to the role of the karyosome structure in facilitating the proper segregation of achiasmate chromosomes (Xiang, 2007).

The trigger for the G2/M transition is activation of Cdk1 by Cdc25, and multiple lines of evidence suggest that Polo can activate Cdc25. First, in Caenorhabditis elegans, RNAi experiments demonstrate that ablation of Polo prevents NEB. Second, the Xenopus Polo homolog Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25. Additionally, microinjection of Plx1 accelerates the activation of both Cdc25 and cyclinB-Cdk1. Moreover, microinjection of either an antibody to Plx1 or kinase-dead mutant of Plx1 inhibits the activation of Cdc25 and its target cyclinB-Cdk1. Another demonstrated that injection of a constitutively active form of Plx1 accelerated Cdc25 activation. These studies support "the concept that Plx1 is the 'trigger' kinase for the activation of Cdc25 during the G2/M transition." Finally, a small molecule inhibitor of Polo kinase (BI 2536) also results in extension of prophase (Lenart, 2007). These data are consistent with the view that the presence of functional (unbound) Polo plays a critical role in ending the extended G2 that is characteristic of oogenesis in most animals (Xiang, 2007).

In light of these data, it is tempting to suggest that in wild-type Drosophila oocytes, the large quantity of Mtrm deposited into the oocyte from stage 10 onward inhibits the Polo that is either newly synthesized or transported into the oocyte during stages 11-12. However, at stage 13, an excess of functional Polo is created when the number of Polo proteins exceeds the available amount of inhibitory Mtrm proteins. This unencumbered and thus functional Polo then serves to activate Cdc25, initiating the chain of events that leads to NEB and the initiation of prometaphase. In the absence of a sufficient amount of Mtrm, an excess of Polo causes the precocious activation of Cdc25, and thus an early G2/M transition. A model describing this hypothesis is presented. Based on this model, one can visualize that decreasing the dose of Mtrm or increasing the dose of Polo will hasten NEB, whereas simultaneous reduction in the dose of both proteins should allow for proper timing of NEB (Xiang, 2007).

Mtrm's first PBD binding site (T40) is required for its interaction with Polo. Mtrm T40 has to be first phosphorylated by a priming kinase, such as one of the Cdks or MAPKs, and was indeed detected as phosphorylated in the mass spectrometry dataset. The NetPhosK algorithm predicts T40 to be a Cdk5 site, and the serines immediately distal to T40 (S48 and S52), which were also detected as phosphorylated are sites for proline-directed kinases such as Cdk or MAPK sites as well. The other prominent phosphorylation event occurs at S137, which could be a Polo phosphorylation site since it falls within a Polo consensus (D/E-X-S/T-Ø-X-D/E). Although the combined sequence coverage for Mtrm was 44%, indicating that some phosphorylated sites might have been missed, Mtrm S137 is a suitable binding site for activated Polo, in agreement with the processive phosphorylation model. At this point of our studies, Mtrm T40 priming kinase or the kinase responsible for Polo activating phosphorylation on T182 has not been identified (Xiang, 2007).

The finding that Polo not only is able to bind to Mtrm in vivo in a 1:1 ratio, but also is fully phosphorylated on T182 in its activation loop suggests a method by which Mtrm serves to inhibit Polo. In general, enzymes are usually not recovered from affinity purifications at levels similar to their targets. They do not form stable complexes, but rather form transient interactions with their substrates, which is how efficient catalysis is achieved. In this instance, Mtrm is able to sequester activated Polo away in a stable binary complex over a long period of time. It is only when this equilibrium is disturbed at the onset of stage 13 by the production of an excess of Polo or by degradation of Mtrm that Polo can be released. The molecular determinants of the Mtrm::Polo sequestration event are not clear, but it would be interesting to test whether the serines found phosphorylated in the vicinity of Mtrm PBD binding sites play a role in locking the binary complex into place (Xiang, 2007).

The data demonstrate that a reduction in the levels of Mtrm results in the release of an incompletely compacted karyosome that rapidly dissolves into individual bivalents during the early stages of spindle formation. For chiasmate bivalents, this is apparently not a problem, because they still co-orient correctly. However, the nonexchange bivalents frequently fail to co-orient properly, such that both homologs are oriented toward the same pole (but often occupy two different arcs of the spindle). This initial failure of proper co-orientation leads to high frequencies of nondisjunction as demonstrated by the genetic studies and analysis of metaphase I images (Xiang, 2007).

Although achiasmate homologs are properly co-oriented in wild-type oocytes, it has been noted that such homologs can often vacillate between the poles such that two achiasmate homologs are often found on the same arc of the same half-spindle during mid to late prometaphase (Gilliland, 2007). These chromosomes are often observed to be physically associated. This situation is quite different from the defect observed in mtrm/+ heterozygotes, where the homologs are neither physically associated nor on the same arc of the spindle (Xiang, 2007).

It is tempting to suggest that the chromosome segregation defects observed in mtrm/+ heterozygotes are simply the result of precocious release of an incompletely re-compacted karyosome. According to this explanation, the defects observed in meiotic chromosome segregation are solely the consequence of premature NEB. (Implicit in this model is the assumption that it is the events that occur during karyosome re-compaction, at stages 11 and 12, that serve to initially bi-orient achiasmate chromosomes, and there is no direct evidence to support such a hypothesis) (Xiang, 2007).

Alternatively, Polo plays multiple roles in the meiotic process (Lee, 2003a; Lee, 2003b), and it is possible that the chromosome segregation defects we see represent effects of excess Polo that are un-related to the precocious breakdown of the nuclear envelope. Such a view is supported by two observations. First, the bivalent individualization observed after NEB in mtrm/+ oocytes does not disrupt FM7-X pairings. Second, although heterozygosity for twine in mtrm¹²⁶/+ heterozygotes suppresses the frequency of precocious NEB from 42%, two alleles of twine tested (twe¹ and twe^k08310) failed to suppress the levels of meiotic nondisjunction observed in FM7/X; mtrm¹²⁶/+ heterozygotes. These data suggest that the effects of excess Polo on nondisjunction may not be regulated via Cdc25/Twine, but rather by the effects of excess Polo on some other as-yet-unidentified Polo target. This suggests that the effects of Mtrm on the level of Polo might affect multiple Polo-related processes (Xiang, 2007).

Support for such an idea that Mtrm can inhibit Polo-regulated proteins that are unrelated to NEB comes from the observation that the ectopic expression of Drosophila Mtrm in Schizosaccharomyces pombe blocks karyokinesis, producing long multi-septate cells with only one or two large nuclei. This phenotype is similar, if not identical to, that exhibited by mutants in the S. pombe Polo homolog plo1 (Plo1), which fail in later stages of mitosis due to the role of Plo1 in activating the septation initiation network to trigger cytokinesis and cell division. However, Plo1 also plays a role in bipolar spindle assembly that might also be inhibited in the Mtrm expressing cells, but this function of Plo1 is less well understood (Xiang, 2007).

Thus the possibility exists that the effect of mtrm mutants on meiotic chromosome segregation may well not be the direct consequence of early NEB, but rather may be due to the role of Polo in other meiotic activities, such as spindle formation or the combined effects of these defects with precocious NEB. Efforts to identify such processes and their components are underway (Xiang, 2007).

Finally, it should be noted that while Mtrm is the first known protein that is able to inactivate Polo by physical interaction with Polo itself; there is certainly additional mechanisms of Polo regulation. For example, mutants have been described in the gene that encodes Greatwall/Scant kinase, that have both late meiotic and mitotic defects. Although there is no evidence for a physical interaction between these two kinases, the authors speculate that the function of the Greatwall kinase serves to antagonize that of Polo. The Scant mutations create a hyperactive form of Greatwall, which might be expected to lower the dosage Polo, and thus perhaps partially suppress the defects observed in mtrm/+ heterozygotes. Indeed, exactly such a suppressive effect has been observed in Scant homozygotes (however, this suppression is much weaker than that obtained by heterozygosity for loss of function alleles of polo) (Xiang, 2007).

The data presented in this study demonstrate that Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest during meiosis. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase. Although the data do shed some light on the mechanism by which Mtrm inhibits Polo, it is not entirely clear whether Polo's ability to phosphorylate targets other than Cdc25 might be blocked by Mtrm::Polo binding. These issues will need to be addressed in the future studies. Finally, it is noted that although small molecule inhibitors of Polo have been identified, Mtrm represents the first case of a protein inhibitor of Polo. It would be most exciting to identify functional orthologs of Mtrm outside of the genus Drosophila. Perhaps that might best be accomplished through a screen for oocyte-specific Polo-interacting proteins (Xiang, 2007).

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